A Systematic Review of Cortisol Levels in Wild and Captive Atlantic Bottlenose Dolphin (Tursiops truncatus), Killer Whale, (Orcinus orca), and Beluga Whale (Delphinapterus leucas)

Item

Title

Eng A Systematic Review of Cortisol Levels in Wild and Captive Atlantic Bottlenose Dolphin (Tursiops truncatus), Killer Whale, (Orcinus orca), and Beluga Whale (Delphinapterus leucas)

Date

2013

Creator

Eng Proie, Shelby

Subject

Eng Environmental Studies

extracted text

A SYSTEMATIC REVIEW OF CORTISOL LEVELS IN WILD AND
CAPTIVE ATLANTIC BOTTLENOSE DOLPHIN (Tursiops truncatus),
KILLER WHALE, (Orcinus orca), AND BELUGA WHALE (Delphinapterus
leucas).

by
Shelby Proie

A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
June 2013


 

© 2013 by Shelby Proie. All rights reserved.


 

This Thesis for the Master of Environmental Studies Degree
by
Shelby Proie

has been approved for
The Evergreen State College
by
________________________
Kevin Francis
Member of the Faculty

________________________
Date


 

ABSTRACT

Systematic Review of Mean Cortisol Levels in Wild and Captive Atlantic
Bottlenose Dolphins (Tursiops truncatus), Killer Whales (Orcinus orca), and
Beluga Whales (Delphinapterus Leucas).

Shelby Proie

Cortisol levels can be used as a tool to measure stress in wild and captive cetaceans.
Cortisol is the primary glucocorticoid found in most mammals including humans and
cetaceans. This systematic review compiles all published studies conducted on cortisol
levels in wild and captive Atlantic bottlenose dolphin (Tursiops truncatus), killer whales
(Orcinus orca), and beluga whales (Delphinapterus leucas) and compares the reported
mean cortisol levels between 1) wild and captive members of the same species, 2)
references to sampling time, 3) wild and captive members of different species, and 4)
studies on captive Atlantic bottlenose dolphins throughout time. The results show that
sampling methodology affects mean cortisol levels in all three species. Cortisol levels
obtained in wild cetaceans reflect similar levels to samples collected in captive animals
that were sampled utilizing non-husbandry methodology. Samples obtained from both
wild and non-husbandry practices reflected significantly higher cortisol levels than those
sampled utilizing captive husbandry methodology. Additionally, cortisol samples of wild
Atlantic bottlenose dolphins may display elevated levels within an hour of chase
initiation, which challenges the claim that chase and capture sampling techniques produce
accurate baseline cortisol levels of this species in the wild. Finally, mean cortisol levels
of captive Atlantic bottlenose dolphins have not significantly decreased as captive care
has evolved over time. Future studies need be conducted to corroborate these results,
specifically studies that analyze fecal glucocorticoid concentrations so invasive sampling
methodology that may skew results is minimized.

Table of Contents
List of Figures…………………………………………………………………….iv
List of Tables……………………………………………………………………...v
Acknowledgments……………………………………………………………….vii
Chapter One: Introduction………………………………………………………...1
Purpose of Study…………………………………………………………..1
Hypotheses………………………………………………………………...2
Significance of Study……………………………………………………...5
Delimitations, Limitations, and Assumptions……………………………..7
Chapter Two: Literature Review………………………………………………....9
Stress………………………………………………………………………9
Cetacean Stress Physiology……………………………………………...12
Stress Hormones…………………………………………………………14
Catecholamines…………………………………………………..14
Mineralocorticoids……………………………………………….15
Glucocorticoids…………………………………………………..16
Stressors………………………………………………………………….21
Acoustic Stressors………………………………………………..22
Environmental Contaminants…………………………………….24
Food/Prey Scarcity……………………………………………….27
Fisheries Practices………………………………………………..29
Stress Hormone Sample Collection……………………………………...31
Saliva……………………………………………………..............33
Blow……………………………………………………………...36
Fecal……………………………………………………………...38
iv

 

Blood……………………………………………………………..41
Tissue…………………………………………………………….44
Environments…………………………………………………………….47
Natural/Wild……………………………………………………..47
Captive/Artificial………………………………………………...48
Importance of Cortisol Monitoring………………………………………49
Comparing Cortisol Levels in Wild and Captive Animals………………50
Comparing Cortisol Levels Across Taxa………………………………...51
Stress Hormone Analysis………………………………………………...56
Problems…………………………………………………………………58
Chapter Three: Methodology…………………………………………………….60
Data Collection…………………………………………………………..60
Conversion……………………………………………………………….64
Statistical Analysis……………………………………………………….65
Microsoft Excel…………………………………………………..66
Odontocete Physiology…………………………………………………..66
Stress in the Wild………………………………………………………...68
Stress in Captivity………………………………………………………..70
Atlantic Bottlenose Dolphin (Tursiops truncatus)……………………….74
Biology…………………………………………………………...75
Status……………………………………………………………..76
Wild Studies Examining Cortisol Levels ………………………..76
Ortiz & Worthy, 2000……………………………………78
Wild/Captive Studies Examining Cortisol Levels……………….79
St. Aubin et al., 1996…………………………………….79
iv

 

Thomson & Geraci, 1986………………………………...81
Captive Studies Examining Cortisol Levels……………………..83
Orlov et al., 1988………………………………………...84
Orlov et al., 1991………………………………………...85
Blasio et al.,2012…………………………………………86
Copland & Needham,1992……………………………….87
Houser et al., 2011……………………………………….87
Naka et al., 2007…………………………………………88
Noda et al., 2006…………………………………………89
Ortiz et. al., 2010…………………………………………90
Reidarson & McBain, 1999……………………………...91
Ridgeway et al., 2006…………………………………….92
Ridgeway et al., 2009…………………………………….93
Suzuki et al., 1998………………………………………..94
Medway et al.,1970………………………………………95
Pedernera-Romano et al., 2006…………………………..96
Suzuki & Komaba,2012………………………………….98
Killer Whale (Orcinus orca)……………………………………………..98
Biology………………………………………………………….100
Status……………………………………………………………101
Wild Studies Examining Cortisol Levels……………………….101
Ayres et al., 2012……………………………………….102
Captive Studies Examining Cortisol Levels……………………104
Lyamin et al., 2005……………………………………………..105
Suzuki et al., 1998………………………………………………106
iv

 

Suzuki et al., 2003………………………………………………107
Beluga Whale (Delphinapterus leucas)………………………………...108
Biology………………………………………………………….109
Status……………………………………………………………110
Wild Studies Examining Cortisol Levels……………………….110
St. Aubin & Geraci, 1989………………………………112
St. Aubin & Geraci, 1992………………………………113
St. Aubin et al., 2001…………………………………...115
Tryland et al., 2006……………………………………..115
Captive Studies Examining Cortisol Levels……………………116
Schmitt et al., 2010……………………………………..117
Spoon & Romano, 2012………………………………...118
Orlov et al., 1991……………………………………….120
Chapter Four: Results…………………………………………………………..122
Wild vs. Captive Mean Cortisol Levels Within Species………………..122
Atlantic Bottlenose Dolphin……………………………122
Cortisol Collection in Relation to Sampling Time……………………..126
Killer Whale…………………………………………….128
Beluga Whale…………………………………………...129
Wild vs. Captive Mean Cortisol Levels Among Species……….131
Wild & Captive Atlantic Bottlenose Dolphin and Beluga
Whale…………………………………………………...133
Captive Killer Whale and Atlantic Bottlenose Dolphin..133
Captive Beluga and Killer Whale………………………133
Trends in Mean Cortisol Levels Throughout Time in Captivity.134

iv

 

Atlantic Bottlenose Dolphin……………………………134
Chapter five: Discussion………………………………………………………..137
Analysis…………………………………………………………………137
Wild vs. Captive Mean Cortisol Levels Within Species………..138
Atlantic Bottlenose Dolphin……………………………139
Cortisol Collection Time in Wild Atlantic Bottlenose Dolphin..141
Killer Whale…………………………………………….143
Beluga Whale…………………………………………...144
Wild vs. Captive Mean Cortisol Levels Among Species……….145
Wild & Captive Atlantic Bottlenose Dolphins and Beluga
Whales…………………………………………………..146
Captive Killer Whales and Atlantic Bottlenose
Dolphins………………………………………………...147
Captive Beluga Whales and Killer Whales……………..148
Captive Killer Whales, Beluga Whales, and Atlantic
Bottlenose Dolphins…………………………………….149
Trends in Mean Cortisol Levels Throughout Time in
Captivity………………………………………………………...150
Findings in Comparison with Other Studies……………………………152
Complications in using Cortisol as a Primary Indicator of Stress……...153
Complications in Methodology…………………………………………158
Complications in Immunoassay Analysis………………………………163
Chapter Six: Conclusion………………………………………………………..166
Conclusion Drawn by Results…………………………………………..166
Recommendations for Further Research………………………………..168
Appendices……………………………………………………………………...171
Bibliography……………………………………………………………………176
iv

 

List of Figures

Figure 1, Mean Cortisol Levels in Tursiops truncatus…………………………124
Figure 2, Average Mean Cortisol Levels in Wild and Captive Tursiops
truncatus………………………………………………………………………..126
Figure 3, Mean Cortisol Levels in Tursiops truncatus based on Sampling
Methodology and Time…………………………………………………………127
Figure 4, Mean Cortisol Levels in Captive Orcinus orca………………………129
Figure 5, Mean Cortisol Levels in Wild and Captive Delphinapterus leucas….130
Figure 6, Average Mean Cortisol Levels in Wild and Captive Delphinapterus
leucas…………………………………………………………………………..131
Figure 7, Average Mean Cortisol Levels in Three Species of Wild and Captive
Odontocete……………………………………………………………………...132
Figure 8, Mean Cortisol Levels in Captive Tursiops truncatus from 19702012......................................................................................................................135
Figure 9, Trends in Mean Cortisol Levels in Captive Atlantic Bottlenose
Dolphins………………………………………………………………………...136
Figure 10, From Ortiz & Worthy (2000), Mean Cortisol Levels in Wild Atlantic
Bottlenose Dolphins Sampled within or after 36 Minutes of Chase Initiation…141
Figure 11, From St. Aubin et al. (1996), Mean Cortisol Levels in Wild Atlantic
Bottlenose Dolphins Sampled within an Hour of Capture……………………142

v

 

List of Tables

Table 1, Sample Methods Abbreviation Key……………………………………63
Table 2, Tursiops truncatus Studies, Basic information……………………..…..77
Table 3, Orcinus orca Cortisol Studies, Basic information…………………….105
Table 4, Delphinapterus leucas Cortisol Studies, Basic information..................111
Table 5, Immunoassay Information Table……………………………………...171
Table 6, Tursiops truncatus Cortisol Studies, Assay information…………...…171
Table 7, Orcinus orca Cortisol Studies, Assay information……………………172
Table 8, Delphinapterus leucas Cortisol Studies, Assay information………….172
Table 9, Confounding Variables Information Table……………………………173
Table 10, Tursiops truncatus Cortisol Studies, Confounding Variables...……..173
Table 11, Orcinus orca Cortisol Studies, Confounding Variables……………..174
Table 12, Delphinapterus leucas Cortisol Studies, Confounding Variables..….175

v

 

Acknowledgements

This topic was the entire reason I decided to attend graduate studies. Through my
interaction with wild and captive cetaceans, I wondered if anthropogenic stress
was a significant component of their existence. I was not able to answer that
question, but I now know the importance of establishing accurate methodology to
analyze this question in the future. The ability to gauge and monitor stress in wild
and captive cetaceans not only allow us to create a better environment for them to
live in, but us as well.
For all of those whom inspired me, especially: Lolita, Kibby, Delphi, JJ, the Key
West resident pod, Ric O’Barry, Howard Garrett, Sheri Sullinger, Naomi Rose,
Ingrid Visser, Lori Marino, Dr. Miwa Suzuki for sharing data and answering all of
my questions, and Kevin Francis for his advise and guidance. Lastly I wish to
thank my family, boyfriend Mikey, and close friends, especially Katie, for their
encouragement, which gave me the motivation to complete this achievement.

vii

 

CHAPTER 1
INTRODUCTION

A Systematic Review of Cortisol Levels in Common Bottlenose
Dolphin, Killer Whale, and Beluga Whale1

 

 

In the past decade the first species of cetacean to be driven to extinction,
due to anthropogenic activity was the Yangtze River dolphin (Lipotes exillifer)
(Morisaka et al. 2010). Currently the Vaquita (Phocoena sinus) and Maui’s
dolphin (Cephalorhynchus hectori maui), a subspecies of Hector’s dolphin, are
among the most endangered animals on the planet with a population of around
150 for the former and 50 for the latter (Jefferson, 2012). A likely factor in the
decrease of these species of cetacean along with many others is stress related
ailments caused by anthropogenic stressors (Morisaka et al. 2010). According to
Morisaka et al. (2010) the preservation of these animals can only be attempted
with a clear understanding of “physiology, psychology, and behavior.” Analyzing
stress hormones in cetaceans living in both wild and captive environments can
lead to better strategies to conserve their environment and establish better
individual care. Studies of stress levels in common and endangered cetaceans
may lead to better conservation strategies for all at-risk cetacean species in
environments facing increasing anthropogenic stressors.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1

 Definitions: In this study, wild will be synonymous with natural environment, captive will be synonymous with artificial


 

1

 

This thesis is related to stress levels of common bottlenose dolphins
(Tursiops truncatus), killer whales (Orcinus orca), and beluga whales
(Delphinapterus leucas) in wild and captive environments. The aim is to assess
stress levels between animals of the same species living in different environments,
cortisol levels in reference to sampling time, stress levels between species living
in different environments, and cortisol levels in response to evolved captive care.
These are extremely difficult questions to answer due to numerous factors
including convoluted sampling methodology and the large expense of sample
collection and analysis which results in a relatively small number of studies that
have been conducted on cortisol levels in wild and captive cetaceans. Studies that
have been conducted encounter confounding factors and methodological errors.
Because cetaceans live most of their lives below the surface of water bodies,
accurate cortisol levels in wild cetaceans may be biased by having to capture or
restrain the animals in order to obtain a sample, thus exciting the stress response
in these animals (St. Aubin et al. 1996, Thomson & Geraci 1986). The premise of
this thesis is that a better understanding of cetacean stress can be accomplished by
systematic review of all the data presently available in one conclusive review.

Hypotheses

 

I have four specific research questions, which are followed by three null
and research hypotheses. The first of each category is the research question
followed by the research hypothesis (!! ) then null hypothesis (!! ). 1: Do captive
2

 

Atlantic bottlenose dolphin, killer whale, and beluga whale of the same species
exhibit higher mean levels of cortisol than their wild counterparts? !! : Captive
members of these species will exhibit higher mean levels of cortisol than their
wild counterparts due to constant exposure to anthropogenic stressors. !! : Mean
cortisol levels will not vary between wild and captive members of these species.
2: When do cortisol levels increase in wild cetaceans (using Tursiops
truncatus as a model) during sampling methodology? !! : When cortisol samples
are taken from wild Atlantic bottlenose dolphins within an hour of chase initiation
the samples will not display significantly elevated cortisol levels (see St. Aubin et
al. 1996 and Ortiz and Worthy, 2000). !! : There will be no difference in cortisol
levels sampled within one hour of chase initiation and after one hour of chase
initiation.
3: Do Atlantic bottlenose dolphin, killer whale, and beluga whale species
collectively differ between species in their circulating mean cortisol levels in wild
and captive environments? !! : Mean cortisol levels will vary between species and
environments. !! : Mean cortisol levels will not vary between species and
environment.
4: Have mean cortisol levels in captive Atlantic bottlenose dolphins
trended over time? !! : Mean cortisol levels will show a decreasing trend over
time in captive Atlantic bottlenose dolphins. !! : No trend will be detectable in
captive Atlantic bottlenose dolphins over time.

3

 

To answer these questions I assembled a compilation of published journal
articles along with unpublished data. With the data gathered from previous studies
I completed a systematic review of mean cortisol levels recorded in three species
of wild and captive cetacean: Atlantic bottlenose dolphin, killer whale, and beluga
whale. I anticipate that this research project will result in significant
understanding of how these three common species of cetacean cope with stress in
two different environments: natural (wild) and artificial (captive).
Captive studies of cortisol levels have been used to determine baseline,
resting, diurnal, and circulating levels for each species in a captive environment;
mean cortisol levels can also be used to observe if stress levels have decreased as
new husbandry practices have been implemented to improve the health and living
conditions of the cetaceans. The completed research on cortisol levels in wild
cetaceans is subject to confounding variables of stressful conditions due to the
collection method. Accurate studies of cortisol levels in wild cetaceans can
provide a baseline for captive animals, but collection methods in the wild may
produce skewed data. Collection methods that involve retrieving serum samples,
such as biopsy or chase, capture, restraint, are invasive and may excite the stress
response prior to or during sample collection. Data collected in these studies
could serve as a display of high-moderate stress in these cetaceans. Non-invasive
methods such as fecal collection can produce a more accurate analysis of normal
circulating cortisol levels.

How Analyzing Cortisol Levels can Impact Wild and Captive
Cetaceans
4

 

Humans have always had a connection with cetaceans. As research has
advanced our fascination and respect for these species has increased. These three
cetacean species are currently viewed as some of the most intelligent and socially
bonded animals on Earth. Delphinids, like bottlenose dolphin and killer whales
display self-awareness (Reiss & Marino, 2001), a quality only expressed by
humans and a few other species of primate. Anthropogenic stressors are
increasingly present in both natural and artificial environments. With current
research able to completely assess lifespans of cetaceans in captivity the ethics of
keeping these species in captivity has been questioned (Rose, 2011). Stress is an
important factor that impacts every facet of cetacean lives, much like humans.
Prolonged exposure to stressors, know as chronic stress, can cause
immunosuppression and reproductive problems (Curry, 1999), which are
commonly found in animals living in artificial, captive environments (Rose,
2011). The ability to analyze stress in cetaceans can help target what is inducing
in the stress response and help alleviate it with better best management and
husbandry practices (Morisaka et al., 2010).
Anthropogenic stressors are becoming more prevalent due to
overpopulation, overconsumption, and global climate change. Stress can cause
psychological and physiological problems. Highly intelligent and social animals
like cetaceans may be more vulnerable to the effects of stress than other species
due to their longevity and wide ranges (Curry, 1999). Prolonged exposure to
stressors, chronic stress, can cause fatal complications (Curry, 1999), which are
5

 

commonly found in animals living in artificial, captive environments (Marine
Mammal Inventory Reports, obtained from National Marine Fisheries Service via
Freedom Of Information Act). This study will serve as the first systematic review
of its kind to assess the stress levels through mean cortisol concentrations of wild
and captive common bottlenose dolphin, killer whales, and beluga whales by
combining and comparing all published data on record. The ability to analyze
stress in cetaceans can help target what is inducing the stress response and help
alleviate it with a better understanding for these organisms and their environment,
and in time may be applied to conserve endangered wild cetacean species and
their habitat.
My contribution to the field of cetacean endocrinology takes on the issues
associated with serum collection as an indicator of baseline stress levels in the
wild and compares it to non-invasive fecal cortisol collection. By comparing the
cortisol levels of animals that were assessed in an invasive manner to others that
were either collected voluntarily or with non-invasive fecal collection more
accurate baseline cortisol levels may be produced and less invasive methodology
may become the norm. It will demonstrate the importance of analyzing trends in
glucocorticoids between fecal and serum samples and show how that can be used
a good indicator of stress level (Wasser 2013, personal communication). It also
displays the importance of realizing and reducing confounding factors when at all
possible, by comparing mean cortisol levels, instead of individual samples
between studies, and the importance of conducting longer studies where circadian

6

 

rhythm and seasonality can be taken info effect to produce the most accurate
analysis.

Delimitations, Limitations, and Assumptions

Studies comparing cortisol levels in wild and captive terrestrial animals
have displayed problems with reproduction and consistently higher levels of
cortisol in captive animals (Terio et al., 2004, Rangel-Negrín et al., 2009). A
study on captive African elephants discovered that cortisol levels increased as
enclosure size decreased (Stead et al., 2000). Captive cheetahs have displayed
significantly higher levels of cortisol than their wild counterparts (Terio et al.,
2004). A study on Yucatan spider monkeys concluded that captive animals
display the higher levels of cortisol, while animals living in fragmented forest
display lower levels of cortisol than the captive animals, but higher levels than
animals living in un-fragmented forests (Rangel- Negrín, 2009). Recent studies
related to stress hormone analysis in wild and captive terrestrial animals have
employed fecal glucocorticoid (FGC) monitoring. This is a non-invasive way to
collect feces from wild and captive animals for FGC metabolite analysis.
Conversely, studies on marine mammals have displayed the opposite. In
studies conducted on harbor seals (Phoca vitulina) and harbor porpoises (Phocina
phocina) concentrations of cortisol were higher and occupied a more varying
range than captive animals (Gardiner & Hall, 1997, Buholzer et al., 2004). Other
studies have noted the invasive methodology (chase, capture, restraint) associated
7

 

with blood sample collection cannot be employed without eliciting a stress
response and influencing the results (Stead et al., 2000, Liar et al., 2009). To date
no studies have been completed utilizing FGC monitoring in a population of wild
and captive cetaceans of the same species for analysis.

Only one study conducted on stress hormone analysis of wild killer whales
was available for this systematic review, Ayres et al., (2012). The study utilized
fecal glucocorticoid (FGC) metabolites to assess glucocorticoid levels in wild
Southern Resident Killer Whales. FCG analysis is not comparable to serum,
plasma, or salivary cortisol measurements where the parent hormone cortisol is
present. Captive killer whale studies are present in this analysis, but the wild
study is only being used as a guide to encourage migration to non-invasive stress
hormone monitoring in wild and captive cetaceans.
One study (Spoon & Romano, 2012) uses estimated cortisol levels in the
captive beluga whales sampled obtained from a figure published in that study.
Estimations were made based on the samples shown in that graph. In total the
mean cortisol value estimated could be ±1 µg/dl. When those levels are applied to
the analysis statistical significance or lack thereof is not altered. Contact with the
living author was made via phone message on 3/15/2013. If the exact levels are
possible to obtain they will be implemented into the analysis.

8

 

CHAPTER 2
LITERATURE REVIEW
Stress

 

 

Stress can cause psychological and physiological problems in all animals.
Highly intelligent and social animals like cetaceans, may be more vulnerable to
the effects of stress than other species due to their longevity and wide ranges
(Curry, 1999). The concept of stress was recognized as far back as 450 BC
Hippocrates’s concept viewed health as a state of harmonious balance and disease
a disruption to that balance (Chrousos and Gold 1992). Harmonious balance is a
homeostasis, viewed as an ideal state of equilibrium within the body where all
systems are working and interacting in the correct way to fulfill the needs of the
body and the mind. Something that disrupts the homeostasis such as a stressor
can cause imbalance in the mind and body (Curry, 1999). It wasn’t until 1936
that the word stress was accepted in a biological context (St. Aubin & Dierauf,
2001). A current biological definition comes from Dr. Vahdettin Bayazit and
defines stress as “a physiologic response to events perceived as potentially or
actually threatening the integrity of the body” (Bayazit, 2009). Physical and
behavioral effects of stress in cetaceans are relatively limited, although changes in
adrenal and thyroid hormone levels have been documented since the 1970’s
(Curry, 1999). Cetaceans are exposed to a large number of environmental
(natural) and anthropogenic (unnatural) stressors throughout their lives, in the

9

 

wild and in captivity. Monitoring stress hormones in cetaceans may lead to better
conservation strategies and positive stewardship towards their habitat (Noren et
al. 2011).
Stress hormone monitoring in cetaceans is used to observe the animal’s
adaptation to environmental changes and physical stimuli (Schmitt et al., 2010).
It is valuable to monitor stress and its effects on an animal’s state of wellbeing
(Schmitt et al., 2010). Free-ranging, wild cetaceans can experience stress in
numerous ways such as noise, predation, fisheries, ecotourism, climate change,
declining food sources, diseases, social issues, pollution, and habitat degradation,
among others (Schmitt et al., 2010). Captive cetaceans can also face a variety of
stressors such as isolation, social instability, depression, lack of stimulation, food
quality, confined spaces, artificial water, unnatural noises, and human interaction
(Rose, 2011). These factors can cause acute and chronic physiological stress
responses. The acute stress response is short in duration and anxiety invoking,
making behavioral assessment an effective tool used to recognize it in animals
(St. Aubin & Dierauf, 2001). Capture myopathy and problems with
thermoregulation can be attributed to acute stress as well (Curry, 1999).
Frequent, intermittent, and/or repetitive stressors may induce the chronic
stress response (St. Aubin & Dierauf, 2001). Constant exposure to chronic
stressors can cause habituation, sensitization, and desensitization (Dantzer &
Mormede, 1995). Chronic stress can cause stress-induced pathologies along with
changes in immune system and impaired growth and reproductive functions
(Curry, 1999). Immunosuppression is often the greatest threat of exposure to
10

 

chronic stress in cetaceans (St. Aubin & Dierauf, 2001). Physiological conditions
have the ability to change concentrations of metabolites, minerals and enzymes in
the body (Tryland et al., 2009). The close relationship between the nervous and
immune system dictates the ability of a perceived physiological stressor to impact
an animal’s immune system (Spoon & Romano, 2012). Glucocorticoids
(cortisol) which increase in the presence of a stressor, impact many aspects of the
immune system including white blood cell production. Thomson & Geraci (1986)
discovered that lymphoctytes, the white blood cells that contain T, B, and NK
cells can decrease by 50% when dolphins are under a moderate stress response.
They also found that eosinophil granulytes, white blood cells that combat
parasites and infections, decreased immediately as a stressor was perceived and
continued to decline until the stressor was gone, not recovering to normal
numbers until the next day (Thomson & Geraci, 1986). Decreased levels of
lymphocytes and eosinophils, along with hyperglycemia are classic characteristic
of a classic stress leucogram, and have the ability to lower immunity especially
when facing chronic stress (St. Aubin & Dierauf, 2001).
Changes in levels of stress hormones and other hematologic parameters
are not the only effects cetacean experience when living with constant acute and
chronic stressors. Clark et al. (2006) found that adrenal mass increased in
beached bottlenose dolphins that were under acute and chronic stress. The adrenal
gland was on average 2 times larger in chronically stressed animals than acutely
stressed animals. It is apparent that exposure to chronic stressors impact
cetaceans in many different ways, and has the possibility of causing an
11

 

exaggerated stress response that can cause deterioration and lead to death (Cowan,
2000). The culmination of acute and chronic stressors can occur and intensify
over time (Curry, 1999). Because cetaceans are exposed to a large variety of
stressors, stress level monitoring is an important tool that may help humans create
better environments with lower levels of anthropogenic stressors.
Cetaceans are exposed to a large number of anthropogenic and
environmental stressors throughout their lives, in wild and captive environments,
which for this paper, will be interchanged with natural and artificial
environments. Assessing stress hormones in captive cetaceans may help
caretakers eliminate or manage potential stressors presented to the animal’s to
facilitate better health (St. Aubin & Dierauf, 2001). In the wild cetaceans are
regularly exposed to natural and anthropogenic stressors. Collecting data on
stress hormones may lead to important management decisions that could
contribute to lessening the effects of stress on cetaceans (St. Aubin & Dierauf,
2001). Although not all aspects of stress are negative and some studies even
suggest that the acute stress response could even be healthy in small quantities
(St. Aubin and Dierauf, 2001), continuous exposure to chronic stressors often
results in distress and can be fatal (Thomson & Geraci, 1986).

Cetacean Stress Physiology

The physiology of stress in cetaceans is similar to most other mammals
(Schmitt et al. 2010). Although it is composed of a series of related events, there

12

 

is variation between species and from individual to individual within species (St.
Aubin & Dierauf, 2001). The stress response is composed of a quick mode driven
by the medulla’s releases of catecholamines, which enacts the fight-or-flight
response and increases alertness known as the acute stress response.
Catecholamine release is followed by a delayed sustained response that is initiated
by the release of glucocorticoids, which coordinate brain and body function
(Wells, 2012). When exposed to a stressor the autonomic nervous system (ANS)
is triggered. The ANS is composed of two branches the sympathetic nervous
system, which is responsible for the fight-or-flight response and the
parasympathetic nervous system which, is designed to mitigate the stressor. The
sympathetic nervous system triggers the adrenal medulla to release
catecholamines, epinephrine and norepinephrine, into the blood (Martineau,
2007). Concurrently with the catecholamine release, the chronic stress response is
controlled by the hypothalamic-pituitary-adrenal axis that when faced with a
stressor will releases corticotrophin-releasing hormone (CRH). CRH then targets
the anterior pituitary to release adrenocorticotropin (ACTH), which triggers the
release of mineral- and glucocorticoids from the adrenal gland (Groschl, 2008).
These adrenal hormones create energy production, regulate the immune system,
increase metabolic processes and assist in osmoregulation, which can allow
adaptation to the stressor (Schmitt et al. 2010). Although activation of the HPA
axis is necessary to successfully respond and adapt to stress prolonged stimulation
of the HPA axis in cases of exposure to chronic stressors can be life threatening
(Wells, 2012). To analyze stress hormones in cetaceans the steroid hormones are

13

 

collected and analyzed using a variety of methods. Steroid hormones include
chemical compounds that are secreted by the adrenal cortex, which include
mineral- and glucocorticoids.

Stress Hormones


 

Stress hormones are released in the presence of an acute stressor and can

continue to be exhausted during chronic stress. A stressor increases physical
and/or psychological demands and to cope with it the body releases several
adaptive hormones: adrenocorticotropin by the anterior pituitary, gluco- and
mineralocorticoids by the adrenal cortex, epinephrine from the adrenal medulla,
and norepinephrine from the sympathetic nerves. Secondary endocrine
components include prolactin, growth hormone, thyroid hormones, and
vasopressin among other pituitary hormones (St. Aubin & Dierauf, 2001). These
hormones are involved in complex interactions, regulate each other and help the
body adapt to stressors (Axelrod & Resine, 1984).

Catecholamines


 

Catecholamines are the first line of defense and first responders to a

stressor. These agents are fast acting and necessary to increase vigilance,
alertness, arousal and attention when presented with a stressor. They involve
activation of the cardiovascular system and metabolism. Catecholamines are
typically produced during the acute stress response, but have also been reported to
14

 

increase during dives in marine mammals. The hypothalamic-pituitary-adrenal
axis is activated by a stressor, which triggers the release of neurotransmitters
(Axelrod & Resine, 1984). The well-known neurotransmitters dopamine,
norepinephrine, and epinephrine are all catecholamines. They activate the
amygdala, which triggers an emotional response. Catecholamines excite the HPA
axis that triggers the secretion of glucocorticoids and can suppress short-term
memory, concentration, and inhibition (Wells, 2012). Catecholamines are
induced rapidly and can subside quickly (St. Aubin & Dierauf, 2001). Numerous
studies on wild and captive cetaceans support the hypothesis that the same stress
response pathways that exist in humans are present in marine mammals.

Mineralocorticoids

Aldosterone is the principle mineralocorticoid in mammals. Zona
glomerulosa cells, which are prominent in marine mammals, produce aldosterone
(St. Aubin, 2001). In most animals aldosterone is not viewed as a stress hormone,
but it is active in the stress response of cetaceans (St. Aubin & Dierauf, 2001).
Aldosterone primarily functions to provide water conservation and electrolyte
balance including sodium transport and potassium excretion (Schmitt et al. 2010).
Ion regulation and changes in blood pressure activate aldosterone production and
releases it via the adrenal gland (Schmitt et al. 2010). ACHT production, which
occurs during the stress response, elicits an unusually high elevation of
aldosterone in bottlenose dolphins (Thomson & Geraci, 1986) and belugas (St.
Aubin & Geraci, 1989), when compared with other mammals (St. Aubin et al.
15

 

1996). Although levels of aldosterone regularly increase 4-5 times that of base
level readings during human induced stressors no significant changes in sodium,
potassium, or chloride have been documented (Schmitt et al. 2010). Similar
elevations in aldosterone correlated with capture and handling stress during
experiments in bottlenose dolphins (Thomson and Geraci, 1986; St Aubin et al.,
1996) and belugas (St. Aubin and Geraci, 1989). This discovery has lead to the
hypothesis of aldosterone functioning to maintain ion homeostasis during stress
and its function as a primary indicator of stress in cetaceans (Schmitt et al. 2010).
Aldosterone has increased during known stressful events such as capture and
handling in bottlenose dolphins (Thomson & Geraci, 1986; St. Aubin et al., 1996)
and belugas (St. Aubin & Geraci, 1989). In other species of odontocetes,
however, aldosterone has not been observed to rise in the presence of an acute
stressor (St. Aubin et al. 2013). This could be due to a state know as “aldosterone
escape” which can occur when aldosterone levels are elevated for a prolonged
amount of time (Turban et al. 2003).

Glucocorticoids

Glucocorticoids are comprised of corticosteroids including corticosterone
and cortisol. They are known in endocrinology for the role they play in the stress
response. Cortisol is the most prominent glucocorticoid in cetaceans according to
studies to date (St. Aubin & Dierauf, 2001). Cortisol is one of the initial adrenal
hormones to increase when introduced to acute and chronic stress (Möstl & Palme
2002). Cortisol can stay elevated for up to five hours after presented with a
16

 

stressor (St. Aubin & Geraci, 1989), and usually peaks between 1-2 hours (St.
Aubin et al., 2001). This increase makes cortisol one of the most reliable and
common stress hormones collected to analyze the stress response in cetaceans
(Schmitt et al. 2010). Cortisol is commonly used in stress analysis because it is
known to be indicative of the stress response in cetaceans. Glucocorticoids are
important to monitor because they have three functions in stress. According to
the Handbook of Marine Mammal Medicine, they alter metabolism to increase
energy; they permit catecholamines to act on metabolic pathways and blood
vasculature; and they provide adaptations to distress by minimizing
immunological reaction to prevent tissue damage (St. Aubin & Dierauf, 2001).
They can inhibit gonadotropin, growth hormone and thyroid stimulating hormone
secretion, which contribute to reproductive, growth, and thyroid function (Tsigos
& Chrousos, 2002). Cortisol is able to inhibit thyroid hormones like as thyroxine
and triidothyronine (!! and !! ). The inhibition of these hormones along with
elongated lag time of 6-8 hours for !! and more than 20 hours for !! explain why
these hormones aren’t used as primary indicators of stress on their own. Cortisol
functions under stress by providing the body with energy through
gluconeogenesis and can act as an anti-inflammatory agent and decrease white
blood cells, which may cause immunosuppression during times of prolonged
psychological and physical stress (Kravitz et al, 2005).
Although basal cortisol levels are usually lower in cetaceans than
terrestrial mammals (Martineau, 2007), cortisol levels are usually significantly
raised when the animal is in distress or highly stressed (St. Aubin et al. 2001). A
17

 

possible explanation to the low level of circulating cortisol in cetaceans has to do
with the hormone-binding capacity of the plasma. Studies on belugas and
bottlenose dolphins show that the bound fraction of cortisol represents 50% or
less of the total circulating hormone (St. Aubin, 2001). The high level of
circulating hormone may account for small increases of cortisol to translate into
more unbound hormone and greater availability to affect the animals. Although
this is debated, it is thought that when presented with a stressor cortisol levels are
elevated within thirty minutes (St. Aubin & Geraci, 1986). Prolonged exposure to
raised cortisol levels can have deleterious effects on reproductive function along
with the neurological and immune systems (Martineau, 2007).
Glucocorticoids influence immunological factors by causing an antiinflammatory reaction and suppressing the immune system to keep cells and cell
mediators in check. Some of the cell mediators stimulate CRF secretion, which
increases ACTH and cortisol and weaken the immune response (St. Aubin &
Dierauf, 2001). Leukocyte counts can be used to recognize stress in cetaceans.
The classic stress leukogram depicts characteristics of cellular blood changes that
are commonly present during the stress response (ie an increase in cortisol). A
classic stress leukogram consists of lower counts of eosinophil and lymphocytes
and increased circulating neutrophils, which causes an increase in white blood
cells (St. Aubin et al., 2013). This trend has been found in bottlenose dolphins and
belugas exposed to various stressors including transport (Medway et al., 1970;
Reidarson & McBain, 1999), capture (St. Aubin & Geraci, 1989;1992) or
injection with ACTH (Thomson & Geraci, 1986; St. Aubin & Geraci, 1989).
18

 

These hormonal changes in reaction to elevated glucocorticoid levels due to
exposure to stress may disrupt the ability to fight infection (St. Aubin & Dierauf,
2001).
Many aspects of the stress response can cause alterations in reproductive
functions by inhibiting the reproductive process. Elevations of glucocorticoids
may cause reproductive complications by inhibiting secretion of gonadotropinreleasing hormone, blocking the release of luteinizing hormone along with
follicle-stimulating hormone, and altering the gonadal response to LH and FSH
secretion (St. Aubin & Dierauf, 2001). Although studies have not been conducted
in most cetaceans to evaluate these claims it is know that TH levels can be
drastically altered by stress in belugas (St. Aubin & Geraci, 1989; 1992).
Cortisol is the primary glucocorticoid produced in cetaceans. Although it
varies between species roughly 95% of cortisol in the body binds to corticoidbinding globulin (90%) and albumin (5%). The remaining 5% are free for target
cells (Curry, 1999). Cortisol concentration is reported to be a good indicator of a
stress response in cetaceans because it is rapidly secreted during both acute and
chronic stress response (Naka et al., 2006; Suzuki et al., 1998). Cortisol levels are
significantly lower in cetaceans than humans. According to the National
Institutes of Health, humans who display a diurnal circadian rhythm in cortisol
secretion exhibit higher levels in the morning than evening. Normal cortisol levels
in humans range from 165.64-634.57 nmol/L depending on sampling time. In
captive bottlenose dolphins who are also thought to exhibit a diurnal circadian
cortisol secretion pattern, Suzuki et al. (1998) found the mean baseline cortisol
19

 

level in captive Atlantic bottlenose dolphins sampled in the AM to be around 11
nmol/L with a range of 5.5-19.3 nmol/L. This comparison displays that normal
AM circulating cortisol levels in humans are on average 33-126 times higher than
what is thought to be normal in bottlenose dolphins.
The cortisol:corticosterone ratio is 5:1 in whales and dolphins that have
been sampled (Thomson & Geraci, 1986; Ortiz & Worthy, 2000) in comparison to
10:1 in humans (Raubenheimer et al., 2006). Cortisol levels have been known to
increase within the first five minutes of exposure to a stressor in humans; in
bottlenose dolphin’s increases have been observed in the first ten minutes (Orlov
et al., 1988). Cortisol levels have been used in the past to gauge how cetaceans
experience, cope, and adapt to stressors in their environments, whether they be
captive or wild (Thompson & Geraci, 1986, St. Aubin et al., 1996, Ortiz &
Worthy, 2010, Spoon & Romano, 2012).
Cortisol levels have been studied in accordance with the stress response
during known stressful activities including capture and handling. Cortisol levels
increased in bottlenose dolphins (Thomson & Geraci, 1986; St. Aubin et al.,
1996) and belugas (St. Aubin & Geraci, 1989, 1992) during capture and handling
processes in most studies. One study conducted on bottlenose dolphins showed
no change in cortisol during capture and handling procedures (Ortiz & Worthy,
2000). Factors such as habituation and desensitization may have contributed to
the static cortisol levels in Ortiz & Worthy, 2000.

20

 

Stressors

Numerous cetaceans, like humans, are self-aware sentient beings (Reiss &
Marino, 2000). Stressors are not interpreted the same between individuals and
species (St. Aubin & Dierauf, 2001). Cetaceans are often living with various
stressors present in their daily lives originating from natural and anthropogenic
sources. Cetaceans’ life history traits include long life spans, late maturity, low
reproductive potential, and feeding near the top of the food chain which makes
them particularly susceptible to anthropogenic stressors (Fair & Becker, 2000). In
the wild, natural stressors like predation, prolonged fasts, extended dives, and
altered weather patterns, coupled with anthropogenic environmental stressors like
increased boat traffic, habitat degradation, pollution, and harassment can induce
stress by increased concentrations of mineral- and glucocorticoids like
aldosterone and cortisol (St. Aubin & Dierauf, 2001). In captivity, most stressors
are of anthropogenic origin because the animals is kept in human care, these
stressors include condensed living spaces, human interaction, chemically treated
water, food quality, man-made social structure, and removal from family bonds.
The stressors faced in captivity likely increase levels of mineral- and
glucocorticoids in individual animals, although for conclusive data more research
needs to be completed.
Like humans, cetaceans individual and group responses to stressors vary.
In some situations experience and acclimation can dull the stress response in some
individuals for possible stressful events. Different individuals may exhibit a
21

 

stress response when new stimuli are placed in an enclosure, but others may not
(St. Aubin & Dierauf, 2001). In some situations where stressors are introduced it
is plausible that heightened response levels in some individuals and/or
populations could predispose them to the chronic effects of prolonged
hypothalamus-pituitary-adrenal axis stimulation including immunosuppression
and reproductive problems (St. Aubin, 2002a).

Acoustic Stressors

Anthropogenic acoustic disturbances are the most studied cause of
cetacean stress (Rolland, et al. 2012). Acoustic disturbances can range from ship
traffic to navy sonar to whale watching boats (Fair & Becker, 2000). Cetaceans
are incredible auditory creatures routinely using acoustic measures to
communicate, navigate, and find prey. Cetaceans are primarily very social
animals with some species such as southern resident killer whales living in
maternal family groups for their entire lives (Ford et al., 2000). This displays the
importance of communication and social interaction within cetaceans.
Odontocetes (toothed whales, dolphins, and porpoises) use echolocation to detect
prey sources and navigate in deep, dark waters, using a complex system of
sending out a pulse of sound from the melon that bounces off of an object and
gets absorbed by the lower jaw allowing the animal to detect in what direction,
what size, and how far away that object is (Berta et al., 2006). It has been
hypothesized that mysticetes (baleen whales) also use auditory cues to detect
density of their small prey sources and for navigation purposes (Berta et al.,
22

 

2006). Increased frequency of anthropogenic auditory prevalence in the marine
environment can disturb every normal behavior of cetaceans including sleeping,
foraging, and mating. Its effects can range from short to long-term displacement
(Fair & Becker, 2000). Exposure to high or cumulative noise levels in cetaceans
can cause elevated stress levels, which have the ability to lead to a compromised
immune system and depressed reproductive functioning (Fair & Becker, 2000).
A recent study conducted by Rosalind Rolland, of the New England
Aquarium proved that heavy shipping noise in the Bay of Fundy causes increased
levels of stress in the endangered population of North Atlantic right whales
(Eubalaena glacialis) (Rolland et al. 2012). This study had obtained fecal
samples from North Atlantic Rights Whales in the Bay of Fundy from 2001-2005
(Rolland et al., 2012). These samples were analyzed in conjunction with noise
recordings taken from the bay during the time of sampling, paying special
attention to the noise recordings from September 2001, the year “9/11” terrorist
events practically halted all commerce (Rolland et al., 2012). The observations
showed that on September 9, 2001, nine ships were observed navigating through
the Bay of Fundy while on September 13, 2001 only three ships were observed
(Rolland et al., 2012). The decrease in ships contributed to a decrease in low
frequency shipping noise, which is around the same frequency North Atlantic
right whales communicate in (Rolland et al., 2012). The results of comparing the
stress hormones in the fecal samples collected in September of 2001 showed a
significant decrease in stress hormones analyzed in the days immediately
following September 11th than before September 11th when shipping was
23

 

unaffected (Rolland et al., 2012). These results were only consistent with
September of 2001, which indicates the North Atlantic Right Whale population
inhabiting the Bay of Fundy have an increased stress response to an
anthropogenic acoustic presence (Rolland et al., 2012). These endangered whales
may be susceptible to immunosuppression and/or repressed reproduction due to
chronic elevated stress levels (Rolland et al., 2012). This study can aide in the
conservation strategies to help increase the population of North Atlantic right
whales by acting as scientific evidence to prove that shipping noise is directly
related to the physical well-being of these highly endangered animals.

Environmental Contaminants

Cetaceans also experience chronic stress effects from exposure to
environmental contaminants (Fair & Becker, 2000). Since many cetaceans have
long life spans and many odontocetes occupy the top of the food chain lipophilic
contaminants have the ability to bio-accumulate in the animals stored lipids
(blubber) (Martineau, 2007). In Southern Resident Killer Whales among other
odontocetes a trend of higher contaminant levels in males and juveniles has been
recorded because females often pass accumulated contaminants to their offspring
through maternal transfer and lactation (Krahn et al, 2007). Lipophilic
contaminants including persistent organochlorine pollutants (POPs), which
consist of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers
(PBDEs), and dichlorodiphenyltrich-lorethane (DDT) among others, are well
known immunosuppressors and can target the adrenal glands and alter stress
24

 

responses (Martineau, 2007). Immunosuppression can cause increased
susceptibility to diseases and pathogens, which could lead to premature mortality
(Martineau, 2007).
Southern resident killer whales (SRKW) reside in the Salish Sea off the
coast of Washington State for around six months every year (Ford et al., 2000).
They often enter the waters of Puget Sound. They have been observed as far south
as Monterey Bay, California and as far north as Southeastern Alaska (Ford et al.,
2000). Because of the animals extended close proximity to civilization they are
constantly exposed to environmental contaminants (Balcomb, 2012). Persistent
organic pollutants (POPs) in combination with other stressors such as an increase
in anthropogenic noise, and a decline in prey have the ability to cause immune
and endocrine system disruption (Krahn et al., 2007).
Detecting immune functions in free-ranging cetaceans is difficult due to
their constant mobility and difficulty in obtaining samples. An approach devised
to analyze immune functions requires comparing a population that is hypothesized
as being contaminated (i.e. inhabiting waters close to shore in urban areas) to
another group of the same species that is less exposed to pollutants (farther off
shore or close to shore in a less unpopulated area) (Martineau, 2007). This
approach helped evaluate the conclusion that beluga whales (Delphinapterus
leucas) living in the highly polluted St. Lawrence Seaway display a more
compromised immune system and were more susceptible to disease and parasites
than belugas inhabiting the waters of coastal Alaska (Martineau, 2007). With
respect to the Southern Resident Killer Whales, a study being conducted by the
25

 

University of Washington’s School of Conservation Biology has reported that
Transient killer whales who feed on marine mammals have the highest levels of
PCB’s in comparison when Northern and Southern Resident Killer Whales which
corroborate past studies conducted on free ranging Northern, Southern residents
and transient killer whales (Ross et al., 2000). At the “Ways of Whales”
workshop on January 28, 2012 at Camp Casey Conference Center (Coupeville,
Whidbey Island) Jessica Lundin’s preliminary research displayed Southern
Resident Killer Whales having substantially higher levels of PCB’s that than of
Northern Resident Killer Whales, who have a larger population and frequently
inhabit less urbanized areas. Ross et al. (2000) found that age, sex, and dietary
preference had direct effects on PCB concentrations in free ranging pacific killer
whales. Lundin went on to show that Southeastern Alaskan resident killer whales
show the smallest levels of accumulated PCB’s because of their more remote
location and low exposure to the toxins. All Southern Killer Whales that were
sampled in a 2007 study have PCB levels that have exceeded the thresholds for
health effects per established in a study done in harbor seals (Krahn et al. 2007).
Increased exposure to toxins and their ability to target adrenal gland function to
alter stress response function, including over and under production of mineraland glucocorticoids (cortisol and aldosterone), can increase impacts on the
animal’s immune system and reproduction when amalgamated (Martineau, 2007).
The ability to link increased levels of toxins to increased levels of stress in
cetaceans can be used to impose improved environmental protection with the

26

 

discharge of the few mentioned toxins still being produced and lead to healthier
chemical regimes.

Food/Prey Scarcity

Food scarcity has long been hypothesized to play a role in stress levels of
cetaceans. In a study conducted by the University of Washington’s School of
Conservation Biology (UWSCB), Samuel Wasser and Katherine Ayres have been
monitoring fecal hormone samples of Southern Resident Killer Whales since 2006
and continue to do so. The southern resident killer whales have undergone
extreme population declines over the past fifteen years, an over 20% decline in
the years between 1995-2005, with little knowledge of why (Krahn et al., 2002).
According to the UWSCB primary hypothesis of this population drop is the
increased stress levels that correlate with a decrease of primary prey supply.
Southern resident killer whales are genetically different from others of
their same species (Morin et al., 2006). One of the reasons these orcas are
categorized differently is because of their primary prey choice, Pacific salmon.
Orcas around the world eat different food sources ranging from other marine
mammals, herring, squid, to even sharks. The Southern Residents obtain 96% of
the nutritional needs from Pacific salmon (Oncorhynchus spp.), 63% are obtained
from exclusively Chinook salmon (Ford et al, 2000). Even in the presence of
other potential food sources, these orcas choose to forage for Chinook salmon
(Oncorhynchus tshawytscha), which are the largest of the salmonoid species in

27

 

the Pacific Northwest and potentially the most threatened (Ford et al, 2000).
Pacific salmon require a clean, disease and parasite-free marine habitat to thrive.
Current salmon runs are declining along the Washington Coast runs are 1.8% of
historic run size; Puget Sound runs are 8%, the Columbia Basin 1.7%, the Oregon
Coast 7%, and British Columbia 36.2% (Lackey, 2000). The combination of
Washington, Oregon, Idaho, and California salmon runs are 5.2% of historic
salmon runs (Lackey, 2000).
In the hundreds of fecal samples collected by University of Washington’s
School of Conservation Biology glucocorticoid and triiodethyonine levels were
measured. Glucocorticoid levels increase in response to acute psychological and
nutritional stress and triiodethyonine, a hormone produced by the thyroid gland is
responsible for the regulation of metabolism, decrease in response to longer term
nutritional stress, but are unaffected by psychological stress lowering metabolism
(Ayres et al., 2012). This study supports the trend of higher glucocorticoid (i.e.
cortisol) levels associated with times of highest nutritional stress (Ayres et al.,
2012). Analysis has rendered the highest glucocorticoid levels in 2007 where
salmon runs were at their recorded lowest, and the lowest levels of
glucocorticoids were recorded in 2009 when salmon runs were more abundant
(Ayers et al., 2012). The analysis has also indicated the lowest stress (lowest
glucocorticoid levels) in southern resident killer whales are recorded in July and
August each year which coincide with peak salmon runs in the Salish Sea (Ayers
et al., 2012). The seasonal trends of glucocorticoid levels reflect stress levels
increasing and decreasing with nutritional stress imposed by primary prey
28

 

abundance (Ayres et al., 2012). This research supports the importance of salmon
protection for the survival of these endangered whales.

Fisheries Practices

Stress in cetaceans is not just limited to acoustic and environmental
origination. Fishery induced stress has been studied in several species, especially
in the waters of the eastern tropical Pacific Ocean (Curry, 1999). In the Eastern
tropical Pacific Ocean tuna purse-seine fisheries are prevalent, using dolphins as
scouts to catch yellowfin tuna (Curry, 1999). Three particular dolphin species are
commonly involved with this practice, pantropical spotted (Stenella attenuate),
spinner (S. longirostris), and common (Delphinus spp.) because of their unknown
involvement with tuna (Hammond, 1983). Three of these dolphin stocks are
currently depleted: the northeastern offshore spotted dolphin, the eastern spinner
dolphins, and the coastal spinner dolphin (Curry, 1999). The methods employed
by purse-seine fisheries have the ability to cause significant stress to the dolphins
involved (Curry, 1999).
Purse-seine tuna fisheries in the Eastern tropical Pacific Ocean often use
the technique of encircling a pod of dolphins that have comingled with a
substantial amount of tuna with a purse-seine net, which can be 1.6 km long and
200 m deep (Curry, 1999). This process can take anywhere from 40 minutes to
over three hours, causing the dolphins caught in the net to huddle at the bottom or
near the surface to seemingly gain the farthest distance from the vessel/s involved

29

 

(Norris et al, 1980). Even though some escapement strategies are employed by the
fishermen to remove the dolphins from the net many get entangled in the net and
drown (Curry, 1999). Although dolphin mortality estimates exceeded 4.5 million
from 1959-1972 (Wade, 2007), estimates throughout the late 1990’s speculate that
over 3,000 dolphins die each year from the effects of tuna purse-seine fisheries in
the Eastern tropical Pacific Ocean (Curry, 1999).
The effects of stress from purse-seine fishing may affect many more
dolphins than it kills. More than half of the dolphins observed when encircled by
purse-seine nets display signs that have generally been accepted as signs of stress
and agitation in cetaceans including headslaps, tailslaps, thrashing, and bunching
(Norris et al, 1980). Other behaviors associated with stress in dolphins that have
been observed are hyperactivity, mostly in spinner dolphins trying to escape from
the net, which can lead to physical exertion, and passive behaviors such as
floating and sinking (Norris et al, 1980). These nets may capture the same pods
of dolphins found in the Eastern tropical Pacific Ocean as frequently as once per
week (Edwards & Perkins, 1998). Using four subsets of current research to
identify responses to stress including laboratory/captive animals, domestic
animals, clinical research, and studies of free ranging animals; the conclusion is
that prolonged exposure to stressors can cause compromised immune and
reproductive function, and is hypothesized to be a cause of the declining numbers
of the northeastern offshore spotted dolphin, eastern spinner dolphin, and the
coastal spinner dolphin stocks (Curry, 1999). These dolphin populations have
shown evidence of acute stress by displaying elevated circulating glucocorticoid
30

 

levels (Curry, 2002). These prolonged, increased glucocorticoid levels can be a
direct reflection of fisheries induced stress in cetaceans. Although other fisheries
such as long line and gillnetting expose cetaceans to stressors, the knowledge
gained from these studies on purse seine netting have lead to the direct
implementation of dolphin escape apparatuses from nets and dolphin by-catch,
but more effective conservation strategies need be employed to protect the three
endangered species chronically harassed by this fishery.

Stress Hormone Sample Collection

The collection method for obtaining stress hormone samples in cetaceans
varies depending upon the captive or wild status of the animal being targeted.
Five employed sample collection methods for stress hormone analysis in
cetaceans are: saliva, blow, fecal, blood, and blubber. All methods could be
employed in each setting but not all are practical. The most common stress
hormone collection method in a captive setting is blood serum samples via
venipuncture. Due to establishing positive reinforcement behavioral protocols
many captive cetaceans exhibit trained husbandry behaviors such as fluke rising
for medical procedures (Schmitt et al. 2010). Previous wild studies often elicited
a stress response in the process by chasing and restraining the animals to obtain a
serum sample (St Aubin et al. 1989). Currently, biopsy sampling of blubber is the
most common in large species of cetaceans, including most mysticetes (Noren &
Mocklin, 2011). Methods such as fecal collection are now being regularly
employed (Hunt et al., 2006). Each method has a different level of invasiveness
31

 

ranging from very invasive to relatively non-invasive depending upon the animal
and setting. The level of invasiveness has the ability to alter the results of the
analysis of stress hormones depending on how the animals reacted to the
collection method. Stress levels and habituation to stressors vary within different
species and individuals of the same species (Fair & Becker, 2000).
The collection of stress hormones in cetaceans varies depending on the
species of cetacean being targeted and if it inhabits a captive or wild setting. Five
sample collection methods for stress hormone analysis that are currently being
utilized in cetaceans are saliva, blow, fecal, blood, and blubber. All methods
could be employed in a captive setting but not all are practical in a wild setting,
such as collection of a blood sample. The most common stress hormone
collection in a captive setting is blood samples because most captive cetaceans are
taught husbandry behaviors such as raising its fluke above water for blood sample
collection, by using positive reinforcement (Schmitt et al., 2010). In the wild
setting biopsy sampling of blubber is still the most common (Noren & Mocklin,
2011) yet more recently researched methods such as fecal collection are now
being regularly employed (Hunt et al., 2006). Each method has a different level
of invasiveness ranging from very invasive (biopsy) to relatively non-invasive
(fecal collection). The level of invasiveness has the ability to alter the results of
the analysis of stress hormones depending on how the animals reacted to the
collection method. Stress levels and habituation to stressors vary within different
species and individuals of the same species (Fair & Becker, 2000).

32

 

Since cetaceans are exposed to a wide variety of stressors by physical,
chemical, and biological factors, these methods of monitoring stress hormones in
cetaceans are an important tool to gauge the physical and emotional health of the
animal. Physical stressors include fishery-interaction, pollution, acoustic
influences, and climate change. Chemical stressors include exposure to
chemicals, harmful algal blooms, metals, pesticides, and oil pollution. Biological
stressors include disease, parasites, decline in prey, and habitat degradation (Fair
& Becker., 2000). A cetacean’s reaction to a stressor can be documented in a
variety of ways including visual surface observation while the animal is in the
vicinity of a stressor or collecting a sample for analysis of stress hormones (Curry,
1999). A thorough conclusion would ideally be based on both a visual
observation of the animal and an analysis of stress hormone production.

Saliva

Saliva can be used as a less invasive source to analyze steroid hormone
levels (Groschl, 2008). It is refuted as a technique that reduces the natural stress
response of the body when collecting plasma and serum. Saliva can be analyzed
to measure stress hormones because it is a carrier of signal molecules, which can
be transported into the salivary glands from blood vessels or produced by the
glands independently (Groschl, 2008). The rate at which these hormones can be
transferred from the blood to saliva is determined by the passage through
lipophilic layers of the capillaries and glandular epithelial cells. Steroids tend to
be transferred through these barriers rather quickly because they are lipophilic
33

 

(Groschl, 2008). Cortisol is a stress hormone produced via the pituitary-adrenal
cortex axis and is responsible for the chronic stress response (Groschl, 2008).
Salivary gluco- and mineralocorticoid samples have been used to measure stress
levels in captive Delphinids (Hogg et al., 2009).
The process of collecting saliva is usually performed with commercial
collection devices that are composed of a nonabsorbent collection pad that must
be kept in the mouth for at least one-minute, and a glass or plastic vesicle used to
house the sample on the pad (Groschl, 2008). Centrifugation is used to remove the
saliva from samples for testing and use. Centrifugations use centrifugal forces to
separate liquid from solid components by rotating at high speeds and require the
recording of force, time, and temperature (Rice University, 2005). Stress
hormones can then be analyzed in a number of ways utilizing immunological and
chromatographic methods (Groschl, 2008). Immunoassays are biochemical tests
that can measure the amount of a substance in a solution. Immunological analysis
involves nonradioactive enzyme-linked immunosorbant assay (ELISA) methods,
which determines if a particular protein is present and renders results with colored
products (Groschl, 2008). Some companies offer FDA-approved assays for
measurement of salivary steroids as an alternative to ELISA (Groschl, 2008).
Chromatographic assays separate a mixture of compounds and chromatographic
methods include combining liquid chromatography with mass spectrometric (LCMS) detection for quantification of salivary steroid hormones, which have been
more reliable than immunological methods (Groschl, 2008). Liquid
chromatography uses a pump to separate compounds and their components, and
34

 

mass spectrometry determines the mass of molecules using a mass spectrometer
(Niessen, 2006). LC-MS is an instrument that includes a high performance liquid
chromatograph connected to a mass spectrometer, which allows for analysis of a
much wider range of compounds (University of Bristol 2005). Testosterone and
progesterone levels were viably measured from a moderate sample size of baleen
whales in this study via the above-mentioned methods (Groschl, 2008).
Cortisol is a key stress hormone that increases the rate of gluconeogenesis
during stress and is frequently measured in salivary analysis. Because cortisol
levels usually rise when an animals is approached with an invasive method such
as venipuncture, saliva samples offer a more reliable standing stress rate by being
less invasive, thus eliciting less of a stress response (Groschl, 2008). A consensus
among the scientific community is that higher levels of salivary cortisol are
collected in people and animals under chronic stress (Groschl, 2008). Some
problems with using saliva as method to measure stress hormones such as
cortisol, arise from a natural enzymatic conversion process. Large portions of
cortisol are converted into cortisone in the salivary glands by 11β-hydroxysteroid
dehydrogenase II, and then to ketoform, which is inactive. (Groschl, 2008). This
process often creates discrepancies in data of cortisol collection including falsely
increased measurements of both glucocorticoids, cortisol and cortisone (Groschl,
2008). Cortisol levels are also influenced by timing of collection because of the
circadian rhythm of natural adrenal secretion (Groschl, 2008). Future research
needs to be completed to test the accuracy of using saliva samples to measure
stress hormones in cetaceans.
35

 

Blow

A relatively new method that has been developed is to use cetacean blow,
the air exhaled out of a cetacean’s blowhole, to measure steroid hormones. This
may also serve as a method to collect mineral- and glucocorticoids such as
cortisol and aldosterone in a less invasive way (Hogg et al., 2005). Currently this
method has been employed to collect and analyze testosterone and progesterone
for reproductive studies in free ranging cetaceans including northern right whales
and humpback whales. Because steroid hormones are defined by the
Encyclopedia Britannica as “any of a group of hormones that belong to the class
of chemical compounds known as steroids; secreted by three steroid glands—the
adrenal cortex (mineral- and glucocorticoids), testes, and ovaries” and can be
grouped by the receptors to which they bind, mineral- and glucocorticoids bind
with androgens (which includes testosterone), estrogens, and progestogens (which
includes progesterone) are all within the five groups of steroid hormones.
Analyses of stress hormones could be completed with similar methods to
testosterone and progesterone analysis (Hogg et al., 2005).
Cetacean blow contains lung mucosa, which is collected by using cotton
gauze, or nylon stocking fastened to a bamboo ring at the end of a pole ranging in
length from 10-15 meters (Hogg et al., 2005). The pole would be extended off the
bow of a small vessel and held above the blowhole of the animal when they
exhaled. Wind, weather, and sea conditions affected the quality of the samples by
interfering with the proximity to the whale and concentration of the sample (Hogg
36

 

et al., 2005). Only a small sample is needed for correct processing usually less
than (50 µL) allowing the validated method of liquid chromatography-mass
spectrometry (LC-MS) that has been used on captive dolphins to be employed
(Hogg et al., 2005). Two forms were evaluated using LC-MS including a gradient
scan for mass-to-change rations and selected ion monitoring (SIM) mode analysis
for testosterone and progesterone (Hogg et al., 2005). This method was the first
documented use of lung mucosa to gather steroid hormones, especially
reproductive hormones.
This collection technique allows for a quick, less invasive method for
gathering steroid hormone samples from wild and captive cetaceans that may
spend little time at the surface (Hogg et al., 2005). The exchange of steroid
hormones in blow samples are hypothesized to retain more data the longer that
animal can stay submerged and the larger the lung volume. Vascularization of
their lungs allows more compounds to diffuse from the blood stream into the lung
mucosa (Dellman & Eurell, 1998). This theory allows blow samples to be viewed
as a mix of organic material instead of just air and water (Hogg et al., 2005). In
theory this process could be used to evaluate other steroid hormones such as
mineral- and glucocorticoids to analyze stress levels in wild and captive
cetaceans.

37

 

Fecal

Fecal samples have been collected and used to determine age and
reproductive rates of free-swimming cetaceans since 2005 (Rolland et al., 2005).
More recently this same fecal collection technique has been applied to measure
fecal glucocorticoids levels to analyze adrenal activity and physiologic stress in
free-swimming cetaceans, including North Atlantic right whales and Southern
resident killer whales (Hunt et al., 2006). This technique has also been employed
on a variety of terrestrial mammals due to the lack of confrontation or interaction
with the animal to obtain the sample. Since glucocorticoids are released by the
adrenal gland in response to a variety of stressors, they end up in the feces by
being released from the adrenal gland into the blood then excreted through the
bile into the gut that renders them as metabolites in feces (Wasser et al., 2000).
Although increased levels of glucocorticoids can be caused by predictable
events such as pregnancy, elevated levels of glucocorticoids generally indicate an
adrenal stressor is present (Balm, 1999). Elevated levels of glucocorticoids have
been reported in a North Atlantic right whale that was fatally entangled in a net
(Hunt et al., 2006). Glucocorticoid levels should be relatively low in juvenile,
pre-adolescent, and non-reproductive animals (Hunt et al., 2006).
Fecal hormone analysis provides some problems including the comingling
of metabolized fecal hormones with many unidentified metabolites that have
unstable antibody affinities (Wasser et al., 2000). Cross-reactively from one

38

 

hormone elevating levels of another may also affect glucocorticoid levels in
cetaceans (Hunt et al., 2006).
Fecal collection can be completed by trailing the animal from as far as a
quarter of a mile or by using a detection K-9 to locate the floating cetacean feces
(Ayres et al., 2012). Detections K-9’s are dogs that are trained to alert their
handler to floating fecal sample locations (Ayres et al., 2012). Samples then are
removed from the water by use of a nylon mesh dip-net attached to a common
boat hook (Hunt et al., 2006) or a specialized apparatus (UWSCB, 2012). As
much of the sample is retrieved as soon as possible and drained of remaining salt
water, then in many cases 50-250g are froze within an hour of collection (-20
degrees Celsius) and stored for later analysis (Hunt et al., 2006). Variation is then
removed from the samples by a process of freeze drying, mixing, pulverizing, and
sifting, this process assists in equalizing hormone content (Wasser et al., 1996).
The samples are then weighed and boiled for 20 minutes in 5 ml 90% ethanol
water or 100% ethanol (Wasser et al., 2000). The sample is then centrifuged for
ten minutes, suspended in 5 ml 90-100% ethanol, vortexed for a minute, and
finally re-centrifuged (Wasser et al., 2000). The sample is then dried, and redissolved in 1.0 ml methanol, and then the supernatant diluted in phosphatebuffered saline (PBS) a technique designed by Wasser et al., in 1994. This
method allows for steroid extraction recoveries (Wasser et al., 1994).
Another assay to analyze glucocorticoid content in fecal samples is by
using a non-boiling, vortexing extraction method (Schwarzenberger et al., 1991).
This method involves the sample being confined in a tube with 2.0 ml 90%
39

 

methanol, vortexed for 30 minutes, and then centrifuged for 20 minutes at 500g
and then the supernatant diluted in PBS. Both extraction methods mentioned
have about a 90-100% hormone accuracy rate and were first tested by recovering
injected glucocorticoid levels in a variety of species (Wasser et al., 2000).
The samples are then cleaned by being passed through a 0.2 micro meter
filter, spin cartridge, and diluted with 5 ml of 80% methanol then analyzed via
High Performance Liquid Chromatography (HPLC) (Wasser et al., 2000). Using
a Radioimmunoassay various cortisol antibodies are then used to examine fecal
GC metabolites including Pantex 031, Incstar CA-1529, CUS R1222, along with a
corticosterone antibody ICN 07-120102 (Wasser et al., 2000). The corticosterone
ICN antibody has been proven to reliably detect adrenal activity in a wild range of
mammalian species, due to its higher cross-reactivity with the dominant fecal GC
metabolites (Wasser et al., 2000). The corticosterone ICN antibody was raised in
rabbits and shows a low cross-reactivity to cortisol along with binding well to
fecal metabolites of cortisol and aldosterone (Hunt et al., 2006).
In North Atlantic right whales, fecal samples reliably measured
immunoreactive fecal glucocorticoids showcasing pregnant/lactating females
along with one animal caught in a net, and a yearling female exhibiting the
highest levels of glucocorticoids, hypothesized from the added stressors of being
pregnant, entangled, and weaning (Hunt et al., 2006). The lowest fecal GC levels
were expressed in immature animals (Hunt et al., 2006). Fecal samples from
North Atlantic right whales also showed an increased concentration of
glucocorticoids with increased ship noise in the Bay of Fundy observed before,
40

 

during, and after the September 11th terrorist events (Roland et al., 2005). Fecal
GC analysis has successfully shown elevated levels at times of hypothesized
physiological or social stress, thus rendering it useful in a vast array of
conservation and management applications, while still remaining the most noninvasive method to conduct stress hormone analysis (Wasser et al., 2000).

Blood

Historically the most commonly practiced method to obtain mineral- and
glucocorticoid levels from animals were from a plasma or serum blood sample
(Sheriff et al., 2011). Stress hormone concentrations can be obtained via
venipuncture, although this method is most commonly practiced in the captive
setting and usually not practical for use on free ranging wild cetaceans. A blood
sample alludes to a clear depiction of the state of the animal at that moment which
is composed of the endogenous cycles, immediate prior experience, and longerterm experiences (Sheriff et al., 2011). Cetaceans like most mammals, have
cortisol as the primary measureable glucocorticoid (Wasser at el., 2000).
In a captive setting blood for stress hormone (ACTH, cortisol,
aldosterone) analysis is usually collected via phlebotomy. Captive cetaceans are
well versed in a variety of husbandry techniques including tail fluke presentation,
which allows relatively easy access to fluke veins (Schmitt et al., 2010). A
sample can then be collected using a three-quarter inch 19-gauge butterflycatheter and 20mL syringe, among other methods (Schmitt et al., 2010). In the

41

 

review of a particular study preformed on beluga whales (Delphinapterus leucas)
compelling results were obtained by incorporating natural diurnal rhythm,
collecting blood three times daily for 5 days at predetermined times which
accounted for natural cycling of hormones; blood collected 20 minutes before and
after a 30 minute wading-contact session for 5 days; and sampled during out of
water examinations where the animals is removed from the water by a stretcher
where blood samples were collected for 20 minutes, 45 minutes, and one hour
after being removed from the water (Schmitt et al., 2010).
Plasma or serum blood samples were collected, plasma is blood collected
via an anti-coagulant and then spun on a centrifuge to remove red blood cells.
Serum is blood obtained without an anti-coagulant and coagulates on its own then
is spun (Sheriff et al., 2011). The main difference between plasma and serum
blood samples is that plasma samples contain fibrinogen and serum samples do
not (Sheriff et al., 2011). Fibrinogen is a protein produced by the liver than acts
as a fibrous coagulant (Medicine Plus, 2012). Plasma samples were placed in
plastic Ethylenediaminetetraacetic acid (EDTA) tubes and serum into serumseparator tubes and then chilled on ice for at least 30 minutes before processing
(Schmitt et al., 2010). The chilling and centrifuge processes minimize steroid
metabolism (Sheriff et al., 2011). The EDTA and serum tubes were centrifuged at
1500 rpm for 10 minutes and the separated plasma and serum were then placed in
plastic vials at -70 degrees Celsius (Schmitt et al., 2010). Storing blood at
temperatures below -20 degrees Celsius in a non-frost-free freezer will maintain
stable glucocorticoid levels (Sheriff et al, 2011). The serum and plasma were
42

 

measured for Adrenocorticotropic hormone (increases production and release of
corticosteroids), cortisol, and aldosterone by automated chemiluminescent
enzyme immunoassays, which are a light-emitting reaction used to monitor an
enzyme label or its products (Kricka, et al., 1987). Each analysis was duplicated
to assess accuracy where cortisol, aldosterone, and ACTH had a less than 5%
variation for intra-assay coefficients (Schmitt et al., 2010). Glucocorticoid
concentrations vary in wildlife from a few nanograms/milliliter to thousands of
nanograms/milliliter so it is important to create a species-specific baseline for the
animals being analyzed (Sheriff et al., 2011). Data then needs to be further
analyzed to create likely baseline stress hormone concentrations for the cetacean
species being analyzed (Schmitt et al., 2010).
The use of stress hormone analysis from blood samples is unique because
unlike other matrices used for stress hormone analysis plasma glucocorticoids
measure total glucocorticoid concentrations (Sheriff et al., 2011). Because free
glucocorticoid concentrations are the critical measurement the equilibrium
dissociation constant of corticosteroid-binding globulin (CBG, the main plasma
protein transport for cortisol) and the maximum corticosteroid binding capacity of
the blood sample is needed (Sheriff et al., 2011). The equilibrium dissociation
constant of CBG is calculated by using saturation binding data and is calculated
for a species as a whole and the maximum corticosteroid binding capacity of the
blood sample is calculated for each individual plasma sample (Sheriff et al.,
2011). The above-mentioned values along with the total glucocorticoid count will
render a free hormone concentration (Sheriff et al., 2011).
43

 

Blood plasma and serum stress hormone analysis is an invasive technique
whether it is employed in captivity or the wild. The animal being sampled is
ultimately being put through a physical stressor for the sample extraction process,
which may lead to altered cortisol and aldosterone levels (Schmitt et al., 2010).
Less invasive techniques such as fecal glucocorticoid monitoring, which render
similarly accurate results, are gaining increased popularity for their ability to be
taken from a distance with less of a physical or emotional stress response from the
animal being tested. This is especially true of cetaceans, which are usually
hyperaware of their situation (Sheriff et al., 2011).

Tissue

Biopsy techniques were developed to obtain skin and blubber samples
from cetaceans in a non-lethal manner (Noren & Mocklin, 2011). Blubber
samples contain steroid hormones, which include mineral- and glucocorticoids
along with other well studied hormones such as progesterone and androgens
(Hogg et al., 2005). To obtain the biopsy sample the use of manual or remote
biopsy methods are chosen based on size and behavior the species being targeted
(Noren & Mocklin, 2011). Smaller cetaceans inhibiting shallow waters can
sometimes be biopsied by both manual and remote methods, while larger
cetaceans are usually biopsied by remote methods (Noren & Mocklin, 2011).
Manual biopsy methods are done by hand and remote biopsy methods by use of
an aluminum pole-mounted biopsy tips or darts being expelled from a crossbow,
compound bow, or gun. Choice of the manual or remote method for obtaining a
44

 

cetacean biopsy sample depend on body size, skin and blubber thickness, and
swimming speed of the targeted animal (Noren & Mocklin, 2011). The size of the
biopsy dart depends upon the depth and structure of the blubber layer being
targeted (Noren & Mocklin, 2011).
A common way to obtain a biopsy on free ranging cetaceans is to use
pneumatic darts fired through a dart gun (Noren & Mocklin, 2011). The use of a
dart gun allows the vessel to be at a greater distance from the animal. Although
accuracy increases as distance decreases, small vessels usually fire the biopsy dart
when the animal is 5-30 meters from the bow for small to mid sized odontocetes
(SWFSC, 2005) and 20-50 meters from the bow for large mysticetes (Noren et al.,
2005). Although techniques and darts can vary a common protocol for gaining
biopsy surveys of free-ranging cetaceans involves blubber samples equating to
less than one gram being collected by using a biopsy dart with a stainless steel tip
between .7cm and 4.0 cm (Fossi et al, 2000). The dart is fitted with an appropriate
sized stop to prevent intrusive penetration and ensured recoil, an average size stop
for targeting killer whales is 2.5 cm according to NOAA’s Southwest Fisheries
Science Center (SWFSC). NOAA’S SWFSC also states that after samples are
collected the darts are thoroughly disinfected between usages. Biopsies are
usually taken between the dorsal fin and the top part of the caudal peduncle,
pending upon aim and species (Noren & Mocklin, 2011). Once the sample is
collected it is immediately put into liquid nitrogen or stored in a cell medium
(Noren & Mocklin, 2011). Glucocorticoids can be assessed by enzyme

45

 

immunoassays, which use enzyme-bound antibodies to detect antigens
(Porstmann et al., 1992).
Because of the invasive nature of employing biopsy-sampling techniques
the behavioral effects of employing these techniques vary among cetaceans, and
many observations may be considered inconclusive because of the large amount
of time spent submerged where observation is usually obstructed (Noren &
Mocklin, 2011). Another invasive aspect of biopsy sampling is the physical
impact on the animal. A biopsy dart it leaves a small wound (Norton et al., 2011),
usually equaling the size of the sample, ~1 gram (SWFSC, 2005). From the
limited amount of data collected via biopsy protocol of free-ranging cetaceans no
adverse heath effects from the wound have been observed and the wound often
heals quickly with relatively little scarring (Noren & Mocklin, 2011). However,
one report of a biopsy dart stopper malfunctioning possibly killed a short-beaked
common dolphin (Bearzi, 2000). Positive physiological response to the biopsy,
which included healing and healed wounds were reported in killer whales
(Barrett-Lennard et al., 1996), indo-pacific humpback dolphins (Jefferson, et al.,
2008), bottlenose dolphins (Weller et al., 1997, Berrow et al., 2002, Parsons et al.,
2003, Gorgone et al., 2008, Bruce-Allen et al., 1985) and southern right whales
(Best et al., 2005). In most species sampled a behavioral response to contact with
the biopsy dart was exhibited ranging from a shaking motion to fleeing the area,
with the stronger responses exhibited by smaller odontocetes (Noren & Mocklin,
2011). Some of the information gained from biopsy sampling of cetaceans
includes genetic information, prey preferences, foraging ecology, contaminant
46

 

loads, and physiological processes such as fertility and stress levels (Noren &
Mocklin, 2011). Because of the massive gain of information that can be used in
conservation of these species and the relatively minor observed disturbance to the
animals being sampled, biopsy sampling is viewed as having more positive than
negative impacts on the species being sampled (Noren & Mocklin, 2011).

Environments


 

Natural and artificial environments differ in many ways for cetaceans. In

the wild many species are able to travel hundreds of miles a day and come in
contact with stressors that would not be present in an artificial environment such
as predators, food scarcity, and pollution. They are able to live in a natural social
structure, rest, hunt, socialize, mate, and travel at will. Artificial environments
often include a forced social structure or no social structure, higher or lower water
temperatures, altered diet, chemically treated water, forced human interaction,
lack of stimuli, and lack of space (Rose, 2011).

Natural (Wild)


 

In the wild Atlantic bottlenose dolphin, beluga whales, and killer whales

occupy different habitats and are often socially bonded with their pods (O’corrycrowe, 2008, Ford, 2008, Wells & Scott, 2008). Killer whales do not have any
known predators, other than humans, but belugas and Atlantic bottlenose dolphins
are subject to predation. Each species maintains a unique diet based on the region
47

 

they occupy. They may be migratory, transient, or resident populations.
Complications with measuring stress hormones in a natural environment include
collection methods and ability to locate and sample the animals without eliciting a
stress response (Noren & Mockland, 2011).


 
 Artificial


 

(Captive)

Artificial environments vary across the world and can range from

multimillion-dollar aquaria to small traveling carnivals with plastic pools.
Cetaceans can be kept outdoors or indoors and in a natural or artificial
environment. The water can be chlorinated, chemically treated, or pumped in
from a natural marine source. Their diet consists of dead fish of varying degrees
of quality depending on the facility they are in, and are often supplement with
excess vitamins and fresh water for hydration. Social structures are constructed
by humans and may consist of the same or different species inhabiting the same
enclosure or solitary confinement. Some are placed in facilities with climates
similar to their natural location others are exposed to climates that differ greatly
from where they would naturally inhabit. Some facilities have regularly
scheduled performances others just have viewing. Predation is usually not an
issue unless aggression within the social structure is exhibited.

48

 

Importance of Cortisol Monitoring

In cetaceans monitoring stress hormones is used to observe the animals
adaptation to environmental changes and physical stimuli (Schmitt et al. 2010). It
is valuable to monitor stress and its effects on an animal’s state of wellbeing
(Schmitt et al. 2010). Free-ranging, wild cetaceans can experience stress in
numerous ways such as noise, predation, fisheries, ecotourism, climate change,
declining food sources, diseases, social issues, pollution, and habitat degradation,
among others (Schmitt et al. 2010). Captive cetaceans can also face a variety of
stressors such as isolation, social instability, depression, lack of stimulation, poor
food quality, special confinement, artificial water, loud, unnatural noises, and
forced human interaction (Rose, 2011). These factors can cause acute
physiological stress responses, which can cause capture myopathy and problems
with thermoregulation. Chronic effects include stress-induced pathologies along
with changes in immune system and reproductive functions (Curry, 1999). The
culmination of acute and chronic stressors can occur and intensify over time
(Curry, 1999). Because of the known stressors to cetaceans, stress level
monitoring in both natural and artificial environments are an important aspect that
can help humans create better environments with lower levels of anthropogenic
stressors for the animals living within.

49

 

Comparing Cortisol Levels in Wild and Captive Animals

Captive studies have developed better methodology for obtaining cortisol
samples. Animals have been taught husbandry commands that are performed on a
daily basis allowing for adaptation for sample collection exposing their superficial
fluke veins, thus negating the stimulus that would elicit a stress response (Schmitt
et al. 2010). A direct comparison between basal cortisol levels of wild and captive
cetaceans cannot be obtained from these methods, a comparison of stress vs.
resting cortisol levels between the two areas and different species can be assessed,
and be a helpful contribution to marine mammal science. Recently, the use of
fecal glucocorticoids has been used for obtaining cortisol levels in wild cetaceans
(Wasser et al. 2000). This methodology is non-invasive and allows for a proper
comparison of basal cortisol levels between wild and captive cetaceans. Fecal
glucocorticoid analysis also produces more reliable basal cortisol estimates than
blood serum concentrations because they reflect the total amount excreted,
whereas blood serum levels have been known to change quickly in reference to a
stressor (Möstl & Palme, 2002). A fecal glucocorticoid sample will reflect
values expressed between 12-24 hours prior to collection depending upon species,
while blood serum samples will reflect in time concentrations, hence the
variability (Möstl & Palme 2002) Based on this thesis research, as detailed below,
I propose that in the future fecal glucocorticoids from both captive and wild
animals will be the most accurate measurement of cortisol levels in wild and
captive cetaceans, and in turn be the best way to assess stress in these animals. To
50

 

date no published study has ever attempted to collect data from fecal matter of
wild and captive cetaceans of the same species for comparison.

Comparisons of Stress Hormones Across Taxa

In a study conducted on West Indian Manatees (Trichechus manatus), a
species of Sirenian, Larkin et al. (2010) concluded when fecal matter is analyzed
and compared in wild and captive West Indian manatees the cortisol levels
depend on what region the animals are being sampled in and the state of captive
care (captive manatees in the USA had higher levels of cortisol than their wild
counterparts and vice versa for manatees tested in Mexico). Confounding factors
that come into play are size, temperature, and number of animals, season, sex,
age, reproductive status, and access to food sources in the wild and captive
environments. In another study conducted by Rangel-Negrin et al. (2009) in the
Yucatan Peninsula this method has been preformed and expressed conclusive
results in spider monkeys (Ateles geoffroyi yucatanensis). Spider monkeys living
in preserved forests exhibit statistically significant lower levels of cortisol than
captive spider monkeys held at zoos and as pets.
In the literature examined a basal cortisol level has been estimated and
accepted for captive cetacean, pinniped, and some terrestrial mammal species
(Thomson & Geraci 1983, Gardiner & Hall 1997, Rangel-Negrin 2009). Wild
estimates are more variable due to gaining access to the sample, as mentioned
above. Confounding factors are very prevalent and can be included or avoided
51

 

depending upon specific study and methodology. Some common confounding
factors include age, sex, size of tank, human presence, seasonality, indoor/outdoor
location, animals housed with, time in captivity, wild or captive born, among
others for captive animals (Spoon & Romano 2012). Wild animals have their
own set of confounding factors including age, sex, reproductive status,
seasonality, anthropogenic impacts (sound, toxins, presence), prey availability,
sample methodology (Ayres et al. 2012). The best way to avoid many
confounding factors is to obtain as much data as possible about the animals being
sampled and their environment. Serum to fecal to salivary cortisol can’t be
directly compared, but trends can be analyzed between them (Wasser, personal
communication). Most published literature to date uses serum cortisol samples.
Like humans, different species will display different basal or resting
cortisol levels to reflect natural circulation rhythm and/or stressors in their lives
(Morisaka et al. 2010). The main animals I have assessed in my literature review
include beluga whales, killer whales, and bottlenose dolphin, each of these species
have different resting circulating levels of cortisol (Schmitt et al. 2010, Suzuki et
al. 1998, St. Aubin et al, 1996). These species of cetacean were chosen because
of the amount of literature published on them both in captive and wild
environments. The literature provides as accurate estimate of baseline circulating
levels of cortisol for captive species of these animals (Schmitt et al. 2010, Suzuki
et al. 1998, St. Aubin et al. 1996), along with attempts to gather wild estimates
(Thomson & Geraci, 1986, Ayres et al. 2012, St. Aubin et al., 1996). The wild
estimates more likely represent a highly to moderately stressed animal than non52

 

stressed animals (St. Aubin et al., 1996). The animals in captivity may represent a
mildly to moderately stressed animal due to constructions of living in an artificial
environment, especially if that animal was wild caught (Rose, 2011).
Most comparisons of cortisol levels in wild and captive cetaceans
conclude that wild animals have higher circulating levels of cortisol, which is in
contrast to many terrestrial species (St. Aubin et al. 1996, Ortiz & Worthy, 2000,
Thomson & Geraci, 1986). This is hypothesized to be true because of the stress
the animals are put through to obtain the samples (Thomson & Geraci, 1986). In
studies conducted on other species with methodology reflecting non-invasive
sample collection, such as fecal, captive animals tend to have higher levels of
resting cortisol than their wild counterparts (Rangel-Negrin et al. 2009). This is
hypothesized to be due to the generic structure and lack of stimuli when living in
a captive environment, among other factors including correlating cortisol levels
with the display of stereotypic behaviors (Liu et al. 2006).
Serum cortisol samples obtained from wild cetaceans cannot be concluded
as normal resting cortisol levels even if the animals are confined in their natural
environment for weeks with nets. It still does not represent normal patterns and
structure, and could, and probably does express a medium to mild stress response
elevating cortisol levels (St. Aubin et al., 2001). As cetacean endocrinology
becomes more recognized as an important tool to analyze cetacean health, more
non-invasive measures are being implemented to gain insight into estimates of
stress levels in free ranging cetaceans by analyzing fecal glucocorticoids. Noninvasive fecal sampling first gained acceptance in the early 2000s
53

 

(Schwarzenberger, 2007, Wasser, 2000) is gaining popularity and providing an
accurate assessment of fecal glucocorticoid levels in free ranging cetaceans
without eliciting a stress response during collection. Once this is the accepted
methodology, and studies end and are published the literature review will greatly
differ. Use of non-invasive sample collection needs to be, and to an extent has
been the excepted method for cetacean endocrinology, especially when referring
to stress hormones. The ability to have little to no influence on the animal’s
behavior when collecting the sample is crucial to obtaining a resting cortisol level.
Although this methodology is rather difficult to conduct in a marine environment,
it is not impossible, in fact it is very plausible and has been deemed effective by
government agencies and cutting edge research institutions such as NOAA and
The University of Washington’s School of Conservation Biology.
In previous literature a common assay and unit for analysis of cortisol
levels would be helpful, radioimmunoassays (RA) are the most widely excepted
forms of analysis and I125 kits seem to be the most widely used (Ayres et al. 2012,
St. Aubin & Geraci, 1989), yet other radioimmuno- kits are still utilized along
with electrochemiluminescence immunoassay (ECL-IA) kits (Noda et al. 2006,
Naka et al. 2007). Differentiations between the two do not seemingly obstruct the
analysis. Published fecal glucocorticoid research on cetaceans is limited, but is
becoming increasingly popular (Rolland et al. 2012, Ayres et al. 2012). Most
cortisol comparisons between wild and captive marine mammals are deemed
inconclusive due to invasive attempts to obtain samples for the wild animals,
although through different methodology (i.e. obtaining samples directly after
54

 

capture, after weeks, and after months) offer a unique insight on the stress
response of wild animals (Thomson & Geraci, 1986). This field of research itself
is rather limited. Some articles that were reviewed include published and
unpublished research on stress hormones in beluga whales (Tryland et al. 2006,
Schmitt et al. 2010, St. Aubin et al. 2001, Spoon & Romano 2012, Lair et al.
1997, St. Aubin & Geraci 1989), killer whales (Ayres et al. 2012, Suzuki et al.
2003, Suzuki et al. 1998, Lyamin et al. 2005), and bottlenose dolphins (Suzuki et
al. 1998, Noda et al. 2006, Naka et al. 2007, Houser et al. 2011, Blasio et al. 2012,
Pedernera-Romano et al. 2006, Thomson and Geraci 1986, Ortiz & Worthy, 2000,
St. Aubin et al. 1996). Each species has unique features and provides confounding
factors for the analysis, but the elimination of all confounding factors would be
impossible. The most reliable studies provided many samples and were preformed
over a long period of time to eliminate many of those factors (Suzuki et al. 1998,
2003) and they allow insights to circadian rhythms and seasonality that were not
taken into account during shorter studies.
Present studies have given the first glimpse of stress hormone levels in
wild and captive cetaceans. It allows a base to draw upon when establishing better
sample collection and analysis. Each study has similar methodology that has been
the standard in marine mammal practice, but isn’t always the most conclusive.
Although, without the previous studies on cetacean endocrinology, cortisol levels
would not be known in many species of wild and captive cetacean (Schmitt et al.
2010). The captive studies have given a baseline of circulating cortisol levels for
captive cetaceans (Pedernra-Romano et al., 2006, St. Aubin & Geraci 1989;1992 ,
55

 

Blasio et al. 2011, Suzuki et al. 1998, Thomson & Geraci 1986). They have
found seasonality differences (Spoon & Romano 2012), diurnal differences
(Morisaka et al. 2010, Schmitt et al. 2010, Suzuki et al. 2003), and reproductive
differences among and within species (St. Aubin et al., 1996). Without these
studies that information would not be available due to constraints in wild animals
without a bias, although with implementation of non-invasive collection methods
all of that will change and wild animals will be able to be conclusively and
continuously assessed (Ayres et al. 2012).
Prolonged exposure to stressors, chronic stress, can cause fatal
complications (Curry, 1999), which are commonly found in animals living in
artificial, captive environments (Marine Mammal Inventory Reports, obtained
from NMFS via FOIA). This study will serve as the first analysis of its kind to
assess the stress levels of wild and captive common bottlenose dolphin, killer
whales, and beluga whales by combining and comparing all published data on
record. The ability the analyze stress in cetaceans can help target what is inducing
the stress response and help alleviate it with a better understanding for these
organisms and their environment, and in time may be applied to conserve
endangered wild cetacean species and their habitat.

Stress Hormone Analysis


 

A variety of immunoassays have been used to determine free cortisol

levels in serum and plasma samples obtained in the studies that were used for this

56

 

systematic review (See Appendix A). The three main categories of assays utilized
in these studies were Radioimmunoassays (RIA) (74% of studies),
Chemiluminscence immunoassays (CLIA) (17% of studies), and Enzyme-limed
immunosorvent (ELISA) which are synonymous with Enzymeimmunoassys
(EIA) ( ~3% of studies) (See Appendices B, C, D). CLIA’s include
chemiluminescentnyzme immunoassays (CLEIA), electrochemiluminescent
immunoassays (ECLIA), and immunochemiluminesence assays (ICLIA). All
three of these types of immunoassays are based on binding competition. Cortisol
from the animal and labeled cortisol compete for binding sites on the anti-cortisol
antibody that has a high affinity and specificity for cortisol. The main difference
between these assays is the detection methods. RIA’s use radioactive detection;
ELISA/EIA’s use photometric detection; and CLIA’s use luminescence detection
(IBL International, Hamburg, Germany). Other cortisol detection methods
utilized in the studies were time-resolved fluoroimmunoassay (TR-FIA) used
~3% and an altered method of the Porter-Sibler chromogens test ( ~3% of
studies). TR-FIA’s are also based on binding competition, but detected via
fluorescence. The Porter-Sibler chromogens test measures the side chain of
cortisol metabolites using spectrophotometry. RIA’s, ELSIA/EIA’s, CLIA’s are
all similar methods for measuring serum cortisol and can produce comparable
results in animals and humans (Singh et al., 1997; Suzuki, personal
communication). TR-FIA’s also produce comparable levels of free cortisol
(Suzuki, personal communication).

57

 

Problems

Captive animals or wild caught animals often died during these studies
due to complications from capture, refusal to eat, infections, or administration of
hormones to mimic the stress response (Thomson & Geraci, 1986, St. Aubin &
Geraci, 1989). In many studies wild animals were chased for hours and held in
nets for several more hours and cortisol levels never exceeded a peak ~3 hours
into the stressful activity even if the perceived stressor continued (Thomson &
Geraci, 1986). The reason for that conclusion is not known but it is postulated that
acute and chronic stress may produce different effects and although finding a
measurable physiological stress response may be productive in certain situations
(Dierauf, 1990), it may prove difficult to research the effects of long-term low
level stress, which could be studied in an artificial captive environment.
Cetacean researchers are beginning to question the value of
chase/capture/release techniques for establishing baseline cortisol levels (St.
Aubin et al., 2013). Despite several decades of research that includes collection of
cortisol levels in captive and wild cetaceans, no researchers to date have
attempted to conduct a systematic review to compare cortisol levels across
environments and species. These elevated cortisol levels often collected as a result
of chase and restraint serve as a display of high-moderate stress in wild cetaceans;
some that have been collected via non-invasive methods, serve as normal
circulating cortisol levels. Although studies in the past, present, and future have
and are being funded to analyze stress hormones in wild cetaceans, few if any, are
58

 

using fecal cortisol levels to establish wild baseline cortisol levels. To date
studies on Southern Resident Killer Whales conducted by the University of
Washington and NOAA are using fecal cortisol levels to assess stress in wild
killer whales (conservationbiology.net). Another project funded by the Office of
Naval Research in conjunction with NOAA and the University of Washington is
collecting stress hormone data from a managed dolphin population to analyze
season variation across multiple matrices, diurnal variation in hormone
production, adrenocortical sensitivity, and thyroid challenges but not collecting
any data from wild cetaceans (Houser et al., 2012). Analyzing individual animals
cortisol levels can lead to many confounding variables such as age, sex, diurnal
rhythm, seasonality, and reproductive status. It is important to reduce
confounding factors when at all possible, by either only comparing mean cortisol
levels or extrapolating data from longer studies where circadian rhythm and
seasonality is taken info effect to produce the most accurate analysis.

59

 

CHAPTER 3
METHODOLOGY

 

Data Collection


 

Systematic reviews are beginning to emerge in ecology-based fields,

including conservation management plans, due to their ability to provide a
quantitative and qualitative assessment of published results (Roberts et al, 2006).
Systematic reviews play an integral role by assembling data sets from different
studies researching a static element and reporting if any trends are apparent. For
this thesis, 27 methodological and reporting aspects have been derived to structure
a successful systematic review. In this review 20 of those steps2 are completed
which follow the format for a functional systematic review of cortisol levels in
three species of wild and captive cetaceans.


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2

 Produce an explicit protocol, explain the background of the review, clearly state a question that the review was the
address, clearly define the question elements, define search terms used to identify sources of evidence, document a detailed
systematic literature search of sources, search for unpublished literature which is held by non-governmental organizations,
governmental departments and/or charities, define inclusion/exclusion criteria for identification of relevant evidence,
document reasons for inclusion/exclusion for each study, undertake an assessment of each studies quality/validity, provide
standardized, documented and repeatable methods for how data was extracted from each of the studies accepted within the
review synthesis, provide a formal assessment and estimation of the possible risk of publication bias of those studies
accepted within the final synthesis of the review, provide a qualitative synthesis of the evidence in the text of the review
and/or tabulated each accepted studies’ findings, undertake a quantitative synthesis of the evidence according to the
methods section, undertake a quantitative analysis of the evidence, investigate any sources of heterogeneity within the
dataset, report key findings, identify an evidence gap/lack of data to properly answer the main question of the review,
identify other evidence gaps or areas lacking knowledge in light of the review’s findings, provide recommendations for
future topics/questions that still require investigation, and advise on the methodology of future experiments.


 

60

 


 

This systematic review consists of data that exists entirely in publications

and unpublished data from published studies. I conducted a text search using the
terms: “cortisol cetacean”, “glucocorticoids cetacean”, “stress cetacean”, “cortisol
Atlantic bottlenose dolphin”, “cortisol bottlenose dolphin”, “cortisol Tursiops
truncatus”, “glucocorticoids Atlantic bottlenose dolphin”, “glucocorticoids
bottlenose dolphin”, “glucocorticoids Tursiops truncatus”, “stress Atlantic
bottlenose dolphin”, “stress bottlenose dolphin”, “stress Tursiops truncatus”,
“cortisol killer whale”, “cortisol orca”, “cortisol Orcinus orca”, “glucocorticoids
killer whale”, “glucocorticoids orca”, “glucocorticoids Orcinus orca”, “stress
killer whale”, “stress orca”, “stress Orcinus orca”, “cortisol beluga whale”,
“cortisol white whale”, “cortisol beluga”, “cortisol Delphinapterus leucas”,
“glucocorticoids beluga whale”, “glucocorticoids white whale”, “glucocorticoids
beluga”, “glucocorticoids Delphinapterus leucas”, “stress beluga whale”, “stress
white whale”, “stress beluga”, “stress Delphinapterus leucas”. When an
applicable article was of interest the data was collected in a separate spreadsheet,
methods were noted, and authors were credited. I choose studies that exhibited
similar methodology and validated assays were used for analysis. I researched
out to all living authors in each study and received some unpublished data from
them. Data that showed individual cortisol levels were analyzed to show mean
cortisol levels to lower the impact of confounding variables. I attempted to obtain
all information of possible confounding variables from all authors that were still
living for possible analysis (See Appendices E, F, G, H).

61

 

I have not personally collect any new data. I am in contact with all known
researchers in the field of cetacean stress hormone endocrinology and am
constantly alerted to new results and publications as this progressed. If a viable
amount of was left before the data had to be analyzed the new data was added into
the systematic review. The data being analyzed are from peer-reviewed journals
such as Marine Mammal Science, Journal of Comparative Psychology, Brain
Behavior and Immunity, Journal of Wildlife Diseases, Aquatic Mammals,
Comparative Biophysiology and Chemistry, Canadian Journal of Fisheries and
Aquatic Science, General and Comparative Endocrinology, and Nature.
Cortisol levels were measured by validated radioimmunoassay (RIA),
chemiluminescent immunoassay (ECLIA, ICLIA, CLEIA), and enzyme
immunoassay methods (EIA/ELISA). Cortisol antibodies used in all three of these
method exhibited comparable hormone-bind in human and animal samples (Singh
et al., 1997). Mean cortisol levels and standard deviations for each group: captive
Tursiops truncatus; wild Tursiops truncatus; captive Orcinus orca; wild Orcinus
orca; captive Delphinapterus leucas; wild Delphinapterus leucas along with
individual whole cortisol numbers from as many of the studies as possible. An
animal/group of animals were considered to be “captive” if they were held in
human care, whether it be in an open or closed containment, for ≥ 1 year. The
unpublished data came from renowned researchers in the field of cetacean
endocrinology that have either chosen to not publish cortisol levels in the studies
they conducted or haven’t published any results yet. Sampling methodology is

62

 

broken down into three categories: wild, non-husbandry, and husbandry (See
Table 1).
Table 1: Sample Methods Abbreviation key. Assistance in understanding the sample methods column in “Basic
Information” tables on Atlantic bottlenose dolphins, killer whales, and beluga whales.

Sample
 Methods
 

Definition
 

Chase,
 capture,
 restraint
 (C/C/R)
 

Usually
 a
 wild
 practice
 of
 chasing
 the
 
animal
 with
 a
 vessel,
 netting
 them,
 hauling
 
onto
 a
 deck
 or
 driving
 into
 shallow
 water
 
to
 obtain
 a
 sample.
 

Involuntary
 non-­‐husbandry
 (NH)
 

A
 captive
 practice
 that
 consists
 of
 driving
 
the
 animal
 to
 shallow
 water,
 draining
 the
 
pool,
 or
 hauling
 the
 animal
 out
 of
 water
 in
 
to
 obtain
 a
 sample.
 

Voluntary
 husbandry
 (H)
 

A
 captive
 practice
 where
 the
 animal
 is
 
taught
 to
 respond
 to
 a
 visual
 command
 by
 
presenting
 a
 body
 part
 (usually
 caudal
 fin)
 
to
 be
 sampled
 then
 is
 rewarded
 for
 the
 
behavior.
 

Cetacean endocrinology is an important but relatively small field that
began in the late 1970’s. Specialization in stress hormone analysis has become
recognized as an accurate way to assess stress levels in cetaceans and is
increasingly seen in current research. Most studies that have been completed
and/or published were from a core group of authors such as: Dr. David St. Aubin,
Dr. Sammuel Wasser, Dr. Miwa Suzuki, Todd Schmitt DVM, Dr. Rudy Ortiz, and
Dr. Tracy Romano. I have used their data with much appreciation and respect.

63

 

Conversion

To more accurately display the data obtained from past studies on cortisol
levels in wild and captive Atlantic bottlenose dolphins, killer whales, and beluga
whales I converted mean cortisol levels to the two most utilized units (one in
Système International (SI) and the other in traditional unites), which were
micrograms/deciliter (µg/dl and ug/dl) and nanomoles/liter (nmol/l). Out of the 25
studies, nine gave mean cortisol levels in nmol/l, ten in µg/dl, five in ng/ml, and
one that measured FGC in ng/g. The only unit that could not be converted to µg/dl
and nmol/l was the FGC study done on wild killer whales because FGC studies
measure dried fecal matter for glucocorticoid metabolites not the parent hormone
of cortisol. The conversions were done in Microsoft Excel for Mac 2011, version
14.3.1, last update-installed 14.3.1 (µg/dl to nmol/l and vice versa) or by an online
medical calculator MedCalc 3000 (http://medcalc3000.com/Basic.htm) (ng/ml to
µg/dl). To convert from nmol/l to µg/dl you need to obtain the molar mass of the
hormone which is 362.46 g/mol to find the conversion factor which is 27.59
according to the Endocrinology Conversion Factors, Michigan State University,
Diagnostic Center for Population & Animal Health
www.dcpah.msu.edu/sections/endocrinology/WEBCD.ENDO.REF.002.pdf).
To convert mean cortisol levels from µg/dl to nmol/l you multiply the
level (µg/dl) by 27.59 and get the level in nmol/l. To convert the mean cortisol
level from nmol/l to µg/dl you divide the level (nmol/l) by 27.59 and get the level
in µg/dl. For example if your mean cortisol level was 0.2 µg/dl and you wanted to
64

 

convert it to nmol/l you would multiply 0.2 x 27.59=5.51 nmol/l. Then to convert
it back you take 5.51/27.59=0.2 µg/dl. I preformed these conversions in Microsoft
excel and with a calculator to double-check for accuracy. To convert the mean
cortisol levels with a unit of ng/ml to µg/dl I used the online medical calculator
with the drop down box and selected ng/ml for the first box and µg/dl for the
second box then typed in the level in the first box and the conversion for µg/dl
appeared in the second box. The math behind this conversion was double
checked by calculator with the knowledge of 1 µg=1000 ng and 1 dl=100 ml.
Once the conversion from ng/ml to µg/dl was complete then the mean cortisol
level was multiplied by 27.59 to obtain the level in nmol/l. These two unit will be
displayed throughout this analysis and in all charts and tables to allow for fluid
viewing, understanding, and access to data.

Statistical Analysis

Statistical analysis software programs were utilized on my personal
MacBook Pro for analysis of the data collected from all studies mentioned below
to assemble accurate mean cortisol levels for each species in a wild and captive
environment along with comparisons between environments, between species,
and in Atlantic bottlenose dolphin an analysis of trends in mean cortisol levels
over time in captivity.


 
 
 
 
 

 

65

 

Microsoft Excel

Microsoft Excel for Mac 2011, version 14.2.5 was utilized for
spreadsheets and descriptive statistics. Descriptive statistics including mean
cortisol levels and standard deviation of the mean3 were preformed by using the
average, standard deviation. All functions were preformed in triple to account for
human error.

Odontocete Physiology


 

Atlantic bottlenose dolphins, killer whales, and beluga whales are part of

the suborder odontoceti, which means toothed whales. One of the most important
features that members of odontoceti possess is a satellite dish shaped skull, which
is thought to give it the ability to echolocate (Berta et al., 2006, p). Echolocation
is a projection of sound that is directed by the melon to locate objects such as prey
and impasses (Berta et al., 2006, p. ). When it reaches an object it then bounces
back and is collected through the lower jaw of the animal to produce a sonar
image of the surroundings and distance to objects. They also possess a thick
spinal cord to help with quick movements to capture prey (Berta et al., 2006).
Odontocetes display other important systems that have evolved to suite an
aquatic lifestyle. These include respiratory, thermal regulation, diving

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3


 Standard
 deviation
 of
 the
 mean
 was
 used
 in
 place
 of
 standard
 deviation
 in
 this
 analysis.
 It
 was
 derived
 from
 the
 
standard
 deviation
 between
 the
 means
 and
 does
 not
 include
 all
 numbers
 obtained
 to
 compose
 the
 original
 mean.
 It
 is
 
used
 to
 show
 variance
 between
 mean
 cortisol
 levels
 obtained
 in
 different
 studies.
 

66

 

adaptations, and advance renal systems. The main adaptation of the respiratory
systems is the regression of the single blowhole to the forward ventral region
(Berta et al., 2006). This allows breathing air at the surface without having to
exert excess energy. Thermoregulation is important because bodies loose heat
faster in water than in air. Blubber and countercurrent heat exchange are the most
important ways that these animals thermoregulate (Berta et al., 2006). Cetaceans
possess veins that run along arteries so blood from cooler areas such as flukes and
fins warm up from traveling close to the warmed arterial blood so minimum heat
is loss. The process can also be reversed by expanding arteries by increasing
blood flow which causes the capillaries and veins along the surface of the skin to
expand which allows some of the arterial blood to return along the peripheral
veins which are close to the surface of the skin and the heat is lost through the
skin (Berta et al., 2006).
Diving adaptations are essential for capturing prey and evading predators.
Key components for diving include adaptations to pressure such as a flexible
ribcage and lack of air sinuses in skull; adaptations to anoxia such as a greater
capacity for oxygen storage and elevated hemoglobin in the blood and myoglobin
in muscle, bradycardia, and peripheral vasoconstriction (Berta, et al., 2006).
Another important adaptation for living in a marine environment is the
ability to osmoregulate. Cetaceans accomplish this by limiting salt intake and
limiting water loss. Limiting salt intake is done by gaining fresh water from prey
tissues along with metabolic water that is released when fats or carbohydrates are
broken down from digestion. Limiting water loss is done by a highly efficient
67

 

reniculate kidney, which produces hyperosmotic urine, and an inability to perspire
(Berta et al., 2006).
The combination of metabolic and anatomic adaptations cetaceans possess
for living in an aquatic salt-water environment and similarities between stress
physiology with terrestrial animals may give cetaceans a unique advantage
(Schmitt et al., 2010). These adaptations allow enhanced escape strategies along
with sensory advantages that come with the imposed stress of breathing air while
residing in a marine environment. The culmination of these marine adaptations
may allow the animal to better adapt or cope with the perception and physiologic
response to a perceived stressor (Fair & Becker, 2000).

Stress in the Wild

Monitoring stress hormones in wild cetaceans allows scientists to gather
reference points from healthy and unhealthy populations. Anthropogenic and
environmental stressors including pollutants, disease, and habitat degradation due
to anthropogenic activities and global climate change are increasingly impacting
cetacean populations (St. Aubin et al., 2001). The frequency of mass mortality
events including strandings has aided in need to search for answers. Current
parameters such as capture and release sampling may not accurately depict
baseline levels of stress hormones due to invasive methodology (St. Aubin et al.,
1996). Baseline stress hormone levels are needed for wild cetaceans so as
anthropogenic and environmental threats continue to increase impacts on stress
68

 

levels can be documented. This is especially important in animals like beluga
whales that inhabit a narrow habitat range that is more drastically impacted by
environmental impacts such as global climate change (Schmitt et al., 2010).
Although adaptations such as strong social networks, that are often observed in
cetaceans, may lessen the impacts of stressors, the increasing appearance of noise,
fisheries, diseases, oil spills, harmful algal blooms, chemical contaminants, and
habitat change will continue to increase the burden (Fair & Becker, 2000).
Reference points of stress hormones are needed to assess the health of the animals
and may assist in the development of better conservation techniques aimed to
lowering anthropogenic and environmental stressors (Tryland et al., 2006).
Some studies such as St. Aubin (1996) claim that wild sample collection
methodology that included surround, capture, and release tactics did not elicit
signs of distress in the animals being sampled. Cortisol levels in these studies
may be subjected to inaccuracy of baseline numbers due to the invasive nature of
sample collection and the onset of the stress response and cortisol increases within
the first ten minutes of exposure to a stressor (Orlov et al., 1991). Even though
cortisol levels are not known to peak until 1-2 hours after confronted by a stressor
(Thomson & Geraci, 1986) and some studies have enacted sample collection in
less than one hour from chase (Ortiz & Worthy, 2010) the impacts that these
chase, capture, release studies may have on wild populations when cortisol levels
do rise is unknown.

69

 

Stress in Captivity

Stress plays a role in the overall health of animals living in both a wild and
captive environment. Cetaceans are often kept in a captive environment due to
their popularity to the general public (Noda et al, 2006). It is important for the
staff at marine mammal venues to monitor the animal’s health, including exposure
to stressors, and continually improves captive conditions to minimize stress (Noda
et al., 2006). This stress management is an essential element of captive care
(Waples & Gales, 2006). Captive animals have increased demands put on them,
because they are compelled to adapt to the new environment (Orlov et al., 1988).
Captive cetaceans face a different set of stressors than their wild counterparts,
which has lead to recognizable health problems (St. Aubin & Dierauf, 2001). A
captive environment usually includes environmental changes such as
transportation and introduction of novel stimuli, which has been reported to
increase cortisol levels in bottlenose dolphins and beluga whales (Spoon &
Romano, 2012, Copland & Needham, 1992, Noda et al., 2006). According to
Waples & Gales (2002) stressors in a captive environmental can include social
factors such as changes in group dynamics, competition over resources, unstable
dominance hierarchies; and physical factors such as changes in food quality,
reduced stamina, confinement, and forced human interaction (Fair & Becker,
2000). Due to the logistical issues with obtaining blood samples from free ranging

70

 

cetaceans, much of what we know about stress hormones in cetaceans come from
captive studies (Medway et al., 1970, Orlov et al., 1988;1991).
It has been proposed that a combination of both behavioral observation
and stress hormone analysis be imposed to more accurately detect stress in captive
cetaceans, although no statistically significant relationships have emerged (Ortiz
& Worthy, 2000; St. Aubin & Dierauf, 2001; St. Aubin et al., 1996; Thomson &
Geraci, 1986; Waples & Gales, 2002; Curry, 1999). There is good reason to
postulate that behavioral observations can be implemented to assess cetacean
well-being (Esch et al., 2009). Behavioral signs of acute stress in dolphins and
belugas have been identified as loss of appetite, social instability, and
vocalizations along with changes in respiration and dive times (Wapes & Gales,
2002, Castellote & Fossa, 2006). In wild cetaceans bursts of energy and lethargy
have been noted in acutely stressed dolphins (Curry, 1999). It is often easier to
detect signs of acute stress in captive animals due to their smaller range and
dependence on caretakers. Problems associated with continuous stress hormone
monitoring of captive cetaceans include the invasive act of blood sample
collection that can lead to infection at collection site and depletion of blood
supply.
Chronic stress can lead to decreased fitness, physiological problems, along
with reproductive and immune suppression (St. Aubin & Dierauf, 2001). Being
able to reconginize the signs and symptoms of chronic stress is of importance to
captive facilities. Behavioral cues of dolphins exhibiting chronic stress are
thought to be associated with changes in behavior such as lethargy and withdrawn
71

 

tendencies, along with a decrease in appetite (Thomson & Geraci, 1986; St. Aubin
& Dierauf, 2001). Internal signs of chronic stress include gastric ulcers,
compromised immune system, and reproductive problems (St. Aubin & Dierauf,
2001; Waples & Gates, 2006). Different species may adapt to stressors in
captivity in different ways. Orlov et al. (1991) proposed that animals that have a
larger range of habitat and diet, such as beluga whales, might have increased
tolerance to stressful conditions when compared to other cetaceans. Behavioral
observation along with adaptation is important to include when evaluating stress
in cetaceans. Combining those parameters with hematology which can show signs
of stress including decreased lymphocytes and eosinophil’s, increases in
neutrophils, and elevated glucocorticoids (Thomson and Geraci, 1986, St. Aubin
& Dierauf, 2001) will allow for an accurate analysis of stress in captive cetaceans.
The importance of monitoring both hematological and behavior parameters to
evaluate stress in both captive and wild cetaceans is of the upmost importance.
Social parameters are being recognized as playing a growing role in
stressors faced by captive cetaceans. The often manipulated and artificial social
structures presented to captive cetaceans have the ability to cause several forms of
social stressors in these highly social animals. Social stressors include threats,
changes in relationship, dominance, and competition. In captive killer whales, a
smaller male, has displayed higher cortisol levels that don’t reflect seasonality
that has been displayed in the other animals in his enclosure (Suzuki et al., 1998;
2003). The inability to escape a dominant individual, which, may not exist in a
wild environment, can lead to chronic stress and even death. Currently, a young
72

 

killer whale captured in the Netherlands was moved to a captive facility in Spain.
Due to the artificial and unstable social hierarchy at this facility the young animal
is exposed to near constant harassment and raking from the other animals (Visser,
personal communication). This type of social problem has not been reported as
observed in wild killer whales (Visser, personal communication). Sweeney 1990,
notes that captive dolphins may experience social stress due to artificial
groupings, social changes, and subordination; if true these behaviors should be
constantly monitored in a captive environment to minimize the possibility of
immunosuppression or even death.
Due to the numerous social problems being reported in captive cetaceans
behavioral monitoring is an important component to evaluate stress in captive
cetaceans but continuous monitoring of stress hormones may play a large role in
increasing quality and quantity of life in these animals. Many cetaceans are
thought to display a defense mechanism, where they masks signs of illness until it
is in relatively advanced stages when symptoms such as loss of appetite and
lethargy may be detected (Waples & Gates, 2006). Because of this
aforementioned defense mechanism it may be important to monitor behavioral
cues in a captive environment to assess stress in animals along with routine
cortisol analysis that could be completed by noninvasive fecal gathering.


 

 

 
73

 


 
 Atlantic bottlenose dolphin (Tursiops truncatus)


 

The Atlantic bottlenose dolphin, otherwise known as coastal bottlenose

dolphin, or Atlantic bottlenose dolphin (Tursiops truncatus) is most likely the
most well-known and most studied cetacean. They are found in warm-temperate
to tropical seas and are widely distributed where surface temperatures range from
10°C-32°C (Wells & Scott, 2008). Their small-medium size and ability to adapt
to human presence has made them a popular subject in wild and captive research
studies. In the wild they live in pods of 2 to 15 individuals, but can congregate
with up to 1000 depending upon location (Wells & Scott, 2008). Females often
stay in loose family groups, nursery groups, or mixed sex juvenile groups and
adult males often wonder on their own or as strongly bonded pairs. They prefer
coastal environments putting them in constant contact with human civilization and
numerous anthropogenic stressors, although an offshore ecotype has recently been
identified. Anthropogenic activities that lead to pollution of their environment
lead to bioaccumulation of toxins in the animals. In some countries such as Japan,
Peru, Sri Lanka, and the Faroe Islands Atlantic bottlenose dolphins are caught for
aquaria and hunted for food (Wells & Scott, 2008).
Atlantic bottlenose dolphins were first publicly displayed at the Brighton
Aquarium in 1883, then at the New York Aquarium in 1914. They are the most
prevalent cetacean in captivity with over 3000 living in aquaria worldwide
(Corkeron, 2008). In the United States about 70% of Atlantic bottlenose dolphins
are used for public display where the rest are used for military or research
74

 

opportunities. They have more success breeding in captivity than the other two
species being studied in this analysis. Their size and intelligence makes them
competent performers in many forms of entertainment such as shows at aquaria,
movies, TV series, and interactive programs such as “swim-with” experiences and
touch pools. Atlantic bottlenose dolphins are also often participants in “Dolphin
Assisted Therapy” (DAT) where they interact with people who have disabilities.
Although no research has been completed that conclusively proves the
effectiveness of such programs. Anthropogenic stressors are prevalent in
captivity due to confinement and dependence (Corkeron, 2008).

Biology


 

Atlantic bottlenose dolphins are in the family Delphinidae, which also

includes killer whales (Orcinus orca). They range in size from 2.5-3.8 meters and
are distributed throughout the world’s oceans (Wells & Scott, 2008). They have a
generalized appearance of a medium sized robust body, falcate dorsal fin, and
gray coloration with a rounded, elongated rostrum. They exhibit a highly social
behavior and have signature whistles and clicks that identify individuals and pods.
They can hunt individually or cooperatively and methods vary depending upon
location and prey source. They are generalists or specialists by region that
commonly prey on squid and fish, demonstrating a preference for sciaenids,
scombrids, and mugilids (Wells & Scott, 2008). They are typically brief divers
usually surfacing twice a minute but can hold their breath around 8 minutes on
average. Juveniles may remain with their mother for up to six years, which
75

 

exhibits a high maternal investment. Their main predators are sharks, killer
whales, and humans. Maximum lifespans are ~60 for females and ~50 for males
(Wells & Scott, 2008).


 
 
 
 
 Status

 

The International Union for Conservation of Nature lists Atlantic

bottlenose dolphins as “least concern” with around 600,000 thought to be living in
the wild.

Wild Studies Examining Cortisol levels

Obtaining cortisol samples from wild Atlantic bottlenose dolphins often
includes a chase, capture, and restraint to collect a blood/serum sample (St.
Aubin, 2001). This methodology makes it challenging for the researcher to
interpret if the act of obtaining a cortisol samples may produce misleading
information due to the invasive act of collection (St. Aubin, 2001). The animals
are often restrained in nets and/or hauled up on boats to obtain a blood/serum
sample, thus stimulating the stress response prior to collection. Many studies use
wild serum cortisol samples to simulate a medium-high stress response rather than
a baseline (St. Aubin et al, 2001). (See Table 2)

 

 

 

 

76

 

Table
 2:
 Atlantic Bottlenose Dolphin Cortisol Studies, Basic information. Species, Author (s) of study mean cortisol levels
were obtained from, whether the study was conducted in a wild or captive environment (black wild/red captive), sample
type, mean cortisol in nmol/L and µg/dl, number of animals sampled in each study, number of samples analyzed to
determine mean cortisol levels in each study, range of individual cortisol levels in nmol/L and µg/dl, sample method, and
assay used for measurement of cortisol levels.

Atlantic
 Bottlenose
 Dolphin
 

Wild:
 
 
Captive
 

Mean
 
F
 

Mean
 
F
 

#
 
Animal
 

#
 
Sample
 

Range
 

Range.
 

Sample
 

Analysis
 

Author
 (s)
 

Sample
 
type
 

nmol/L
 

μg/dl
 

N
 

N
 

nmol/l
 

μg/dl
 

Method
 

Assay
 

Thomson
 &
 Geraci,
 1986
 

serum
 

100
 

3.6
 

38
 

38
 

C/C/R
 

RIA
 

St.
 Aubin
 et
 al.,
 1996
 

serum
 

71.7
 

2.6
 

36
 

36
 


 
33.1-­‐
113.1
 


 
1.2-­‐4.1
 

C/C/R
 

RIA
 

Ortiz
 &
 Worthy,
 2000
 

plasma
 

77.3
 

2.8
 

31
 

31
 

27.6-­‐
154.5
 

1.0-­‐5.6
 

C/C/R
 

RIA
 

Medway
 et
 al.,
 1970
 

plasma
 

66.2
 

2.4
 

8
 

8
 

46.9-­‐
82.77
 

1.7-­‐3.0
 

NH
 

Silber-­‐
Porter
 

Thomson
 &
 Geraci,
 1986
 

serum
 

35
 

1.3
 

2
 

2
 

30-­‐120
 

1.09-­‐
4.35
 

NH
 

RIA
 

Orlov
 et
 al.,
 1988
 

serum
 

90.34
 

3.3
 

40
 

40
 

NH
 

RIA
 

Orlov
 et
 al.,
 1991
 

serum
 

90.3
 

3.3
 

18
 

18
 


 


 

NH
 

RIA
 

Copland
 &
 Needham,
 1992
 

plasma
 

90
 

3.3
 

6
 

6
 


 


 

NH
 

RIA
 

St.
 Aubin
 et
 al.,
 1996
 

serum
 

52.4
 

1.9
 

36
 

36
 


 
13.8-­‐
110.36
 


 
0.5-­‐4.0
 

H
 

RIA
 

Suzuki
 et
 al.,
 1998
 

serum
 

10.5
 

0.38
 

2
 

10
 

5.5-­‐22.1
 

0.2-­‐0.8
 

H
 

RIA
 

Reidarson
 &
 McBain,
 1999
 

serum
 

63.5
 

2.3
 

6
 

6
 

35.87-­‐91
 

1.3-­‐3.3
 

H
 

ICMA
 

Reidarson
 &
 McBain,
 1999
 

serum
 

69
 

2.5
 

2
 

6
 

49.7-­‐
85.5
 

1.8-­‐3.1
 

H
 

ICMA
 

Noda
 et
 al.,
 2006
 

plasma
 

160
 

5.8
 

5
 

5
 

NH
 

ECLIA
 

Ridgeway
 et
 al.,
 2006
 

serum
 

14.3
 

0.52
 

1
 

4
 


 
9.9-­‐18.2
 


 
0.36-­‐
0.66
 

H
 

RIA
 

Pedernera-­‐Romano
 et
 al.,
 
2006
 

serum
 

19.7
 

0.7
 

7
 

7
 

6.59-­‐
64.46
 

0.24-­‐
2.34
 

H
 

RIA
 

Naka
 et
 al.,
 2007
 

plasma
 

10.2
 

0.37
 

5
 

5
 


 


 

H
 

EIA
 

Ridgeway
 et
 al.,
 2009
 

serum
 

44.1
 

1.6
 

1
 

6
 


 


 

H
 

RIA
 

Ortiz
 et
 al.,
 2010
 

plasma
 

u.d.
 

u.d.
 

2
 

2
 


 


 

H
 

RIA
 

Houser
 et
 al.,
 2011
 

serum
 

15.49
 

0.56
 

2
 

9
 

u.d.-­‐39.4
 

u.d.-­‐1.43
 

H
 

RIA
 

77

 

Blasio
 et
 al.,
 2012
 

serum
 

15.5
 

0.56
 

6
 

14
 

11.9-­‐
20.4
 

0.43-­‐
0.74
 

H
 

RIA
 

Blasio
 et
 al.,
 2012
 

serum
 

16.3
 

0.59
 

4
 

6
 

13.2-­‐
20.4
 

0.48-­‐
0.74
 

H
 

RIA
 

Suzuki
 &
 Komaba,,
 2012
 
(UP)
 

serum
 

35.87
 

1.3
 

3
 

77
 

8.27-­‐
110.36
 

0.3-­‐4.0
 

H
 

TR-­‐FIA
 

Ortiz & Worthy, 2000

31 wild Tursiops truncatus (17 M, 14 F) were caught off the coast of
Beaufort, North Carolina and monitored for plasma adrenal steroids (cortisol,
aldosterone, adrenocorticotropin) and arginine vasopressin (AVP). AVP is
neurohypophysial hormone that is derived from a preprohormone that is
synthesized in the hypothalamus. Its primary function in mammals is to retain
water and constrict blood vessels. It plays an active role in homeostasis because
of its regulation of water, glucose, and salts in the blood. It was analyzed in this
study to see if correlations existed between AVP and any of the adrenal steroids.
Samples were taken within 40 minutes from the fluke vein and were analyzed via
a commercially available RIA kit, DPC, L.A, CA. Cortisol had significant
positive correlations with corticosterone; and showed a correlation that was not
statistically significant with aldosterone. A significant correlation between
cortisol and AVP was not observed.
Cortisol levels ranged form 1.0-5.6 µg/dl, and showed no differences in
samples taken within or above 20 and 36 minutes. This may be due to the
78

 

observation of cortisol levels peaking between 1-3 hours in cetaceans (St. Aubin
et al., 2001). The majority of the animals in this study were said to have not
experienced a physiological stress response within the 40 minutes of capture and
sampling. It is important to keep in mind that each animal differs in their stress
response so it may be illogical to conclude that all free-ranging bottlenose
dolphins do not experience an acute stress response within 40 minutes of
sampling and cortisol levels are not biased when invasive sampling methods are
present. Time of year was not indicated so seasonality may also play a part in the
cortisol levels, typically spring/summer months contribute to lower cortisol levels
than fall/winter months (Orlov et al., 1988).

Wild/Captive Studies Examining Cortisol Levels
A compilation of studies that researched cortisol levels in both wild and
captive populations of Atlantic bottlenose dolphins.

St. Aubin et al., 1996

Adrenal hormone analysis was completed on 36 wild and 36 “semidomesticated” captive Atlantic bottlenose dolphins. The captive animals were
part of a Naval Program from two states and included 18 males and 18 females
between the ages of 4-33. The percentage of wild caught/captive born animals
was not clear. Blood samples were collected via voluntary behavior from their
tail flukes irregularly between February 1988 and March 1992. Assaying was
79

 

performed using a commercially available RIA kit, NCS Diagnostics, Willowdale,
Ontario. Serum cortisol levels ranged from 0.5-4.0 mg/dl. These levels were
deemed close to a baseline cortisol sample for captive bottlenose dolphins due to
the process of voluntary collection and amount of time kept in captivity.
For comparison, 36 (18M;18F) wild caught Atlantic bottlenose dolphins
were captured, sampled, then released in Sarasota Bay, FL as part of the Sarasota
Dolphin Research Program between June 1988 and July 1990. 14 of the samples
were taken in winter months; 22 in summer months to account for seasonal
variability. The capture process was conducted by encircling the animals with
nets then bringing them onboard a vessel for examination and sample collection.
Times between the capture process and blood collection ranged between 23-260
minutes, with a mean of 80 minutes. Assaying was performed using a
commercially available RIA kit, NCS Diagnostics, Willowdale, Ontario. Serum
cortisol levels ranged from 1.2-4.1 mg/dl and were slightly elevated in females.
These cortisol levels were similar to ranges observed in other wild bottlenose
dolphins captured from sampling (Thomson & Geraci, 1986).
Age or season did not seem to have an effect on cortisol levels. Adrenal
hormones showed the greatest variance among wild and “semi-domesticated”
captive animals, with the “semi-domesticated” animals displaying lower levels of
cortisol, although distress was not behaviorally observed. The increase of adrenal
hormones in the wild dolphins suggests a mild stress response. Wild animals
sampled within an hour of capture showed similar levels of cortisol as the captive
animals. The similar levels of cortisol between both groups of dolphins may be
80

 

have occurred because cortisol levels may not have reached their peak thought to
occur between 1-2 hours or because the “semi-domesticated” animals were also
exhibiting a mild stress response.

Thomson & Geraci, 1986

Ten captive bottlenose dolphins between the ages of 4-15 were sampled in
this study to test for stress associated with capture and handling. In June 1983 the
first 3 captive bottlenose dolphins kept in a outdoor sea pen, and all females
around 4 years old. Each animal was captured and sampled two different ways,
samples were collected from the flukes or dorsal fin during both capture methods.
Calm-capture, where the animal was corralled in its sea pen and hoisted onto a
stretcher within 10 minutes and then moved to a carrier and kept wet for up to 6
hours; and chase-capture where the animal was chased for 3 hours prior to being
handled the same as mentioned above. Each of these techniques occurred on
alternating days. Blood samples were taken before the animal was removed from
the water (10 minutes after capture), after it was in a carrier (15 minutes after
removal from the water), along with at 30 minutes, 1, 3, and 6 hours after being
kept in the carrier, and once before the animal was released back into the water.
Cortisol levels for each experiment were analyzed by RIA, New England Nuclear,
Boston, MA. Serum cortisol levels were 30 and 40 nmol/L in the first samples
collected after calm-capture, but were elevated to 80-120 nmol/L after the first
hour they were removed from water where the stayed for the remainder of the 6
81

 

hours. This suggests that even calm-capture evokes the stress response in
bottlenose dolphins. After chase-capture initial cortisol levels were 60 and 80
nmol/L and evened out in the range of 60-100 nmol/L for the next six hours.
4 days after the conclusion of the capture study 1 of the dolphins refused
to eat for 3 days. The other two animals were used in another study to test the
effects of synthetic ACTH on adrenal hormone production. For this study the
animals were captured within 20 minutes and given 50 IU of Cortrosyn with
blood samples collected at 1, 1.5, 3, 3.5, 4 and 5 h after administration of the
drug. Cortisol levels were 60-100 nmol/L after Cortrosyn injection, which is
simulated ACTH although it is unclear whether cortisol levels rose because of
arousal or Cortrosyn injection. One of the animals in this study died within 48
hours of injection and the necropsy showed congested adrenal glands.
In October of that same year seven other captive dolphins between the
ages of 8 and 15 were used to compare the effects of synthetic ACTH to the first
three animals. Using a similar protocol except for elongated capture and restraint
times (~4 hours total) 3 of the animals were given 50 IU of Cortrosyn, the other 4
were given the same amount of ACTHar. The animals showed similar patterns of
mildly elevated cortisol levels after injections but it is also unknown whether
capture and handling or injection played a role in cortisol concentrations. A
dolphin in this study died within 5 days of injection with ACTHar and showed
signs of adrenal congestion.

82

 

Spanning the entire time of the experiments blood samples from 38 wild
bottlenose dolphins were also collected. The animals were chased then held in a
net for up to 5 hours while samples could be collected before the were lifted onto
a platform for blood collection. Mean serum cortisol level of the wild dolphins
was 100 nmol/L, which was similar to captive dolphins after chase-capture and
synthetic ACTH injection, which suggests a mild stress response due to sample
collection methods. Although additional synesthetic ACTH injection after capture
and handling did not rise cortisol above peak levels displayed after capture and
handling, due to the deaths of 2/9 dolphins given the synthetic hormone it may be
dangerous to elicit a stress response by stimulating the adrenal cortex in already
stressed bottlenose dolphins.

Captive Studies Examining Cortisol Levels

Captive Atlantic bottlenose dolphin serum cortisol levels have been
estimated to vary between 0.6-3.6 µg/dl among different ages and sexes
(Thompson & Geraci, 1986; Orlov et al., 1988; St. Aubin et al., 1996; Suzuki et
al., 1998; Ortiz & Worthy, 2000). When the animals are adjusted to a captive
environment and are taught common husbandry practices, such as displaying the
fluke vein, blood/serum collection can be completed within minutes, and the
results most likely produce the most accurate baseline for cortisol levels (St.
Aubin et al., 2013) within this species. Captive studies have show that age and
season does not play a role in cortisol production in semi-domesticated Atlantic
bottlenose dolphins (St. Aubin et al. 1996). Research has shown that Atlantic
83

 

bottlenose dolphin (Tursiops truncatus) typically exhibit lower cortisol levels than
Indo-Pacific bottlenose dolphins (Tursiops aduncas) (Suzuki, personal
communication). (See Table 2)

Orlov et al., 1988
In a study conducted by Orlov et. al., in 1988 40 wild bottlenose dolphins
were sampled during “normative” conditions. A standard RIA kit was used for
analysis. A serum cortisol range of 4-9 nM were displayed after capture when
exposed to experimental three and six hour stressors to discover how well they
adapt to stressors. The animals were given a tranquilizer prior to capture to
decrease the acute stress response. This may skew control cortisol levels by
affecting normal free ranging cortisol levels. The results concluded that due to
shifts of adaptive hormones that are active in the acute and chronic stress response
(cortisol, insulin, triiodothyroine, and thyroxin) the hormonal changes of the
stress response in these animals reacted in a similar way as terrestrial mammals.
Animals were captured and moved to a marine base in the former USSR.
The experimental stress occurred through a process of lifting the animals out of a
pen and placing them in a seawater-filed bath for 3 and 6-hour periods. During
the three-hour experimental stress, blood was taken from the “tail vein” at 15, 30,
45, and 60 minute intervals; during the six hour experimental stressor samples
were obtained by the same methodology at 1, 3, and 6 hour intervals. During the
study on days 1,3-4 razing the animals out of the pen and into the bath exhibited
higher cortisol levels than the actual immobilization in the bath did. Increases in
84

 

cortisol occurred within one hour of raising the animals into the bath, then lead to
relatively stable levels during immobilization (3 and 6 hours) until transport back
to the pens. These stable levels of mean cortisol may reflect a “recovery period”.
Mean cortisol levels peaked at 24 hours, then decreased from day 3-4 to day 7,
although the levels never reached the established “norm” during those periods.
Possible issues with data include the knowledge of bottlenose dolphins exhibiting
seasonality in cortisol levels displaying high levels in fall/winter than
spring/summer months. This may be due to an evolutionary process, the increase
of unfavorable environmental factors occurring in fall/winter months. An
interesting note is that the author describes one of the animals sampled during the
experimental stress as “aggressive” which is rare, and two as docile.

Orlov et al., 1991

Eighteen captivity adapted bottlenose dolphins that were kept at the
Institute of Evolutionary Morphology and Animal Ecology, USSR, were sampled
for this study. Blood was taken from the fluke vein and analyzed by a RIA kit
produced by Sorin, France. Seasonal variation trends were recorded similar to
Orlov 1988, displaying higher serum cortisol levels in winter/spring and lower
levels in summer/fall. Ion concentrations differed more dramatically in the
bottlenose dolphins than in beluga whales that were also being sampled. Detailed
methodology was absent on the bottlenose dolphin sampling times and other
parameters because this study was mostly focused on beluga whales and
85

 

translated. It is probable that methodology was similar to the protocol in Orlov et
al., 1988. Mean cortisol levels for the animals were identical to the results for
Orlov et al., 1988 but were similar to other studies on wild bottlenose dolphins,
not captivity adapted animals.

Blasio et al., 2012


 

Ten (6 F, 4 M, 3 born in captivity, 7 wild caught) captive Atlantic

bottlenose dolphins were sampled for serum cortisol levels in open (n=6) and
closed (n=4) facilities. 20 samples (14, open; 6 closed) were taken from the
caudal vein during routine voluntary husbandry practices. Assessment was
preformed using a solid-phase RIA kit, Cort CT2 Bio International, France.
Cortisol levels ranged from 0.43-0.74 µg/dl in open faculties and 0.48-0.74 µg/dl
in closed facilities, these levels fall in the range of cortisol levels from other
captive studies, which is the relatively large range of 0.4-3.6 µg/dl (Ortiz &
Worthy, 2000, St. Aubin et al., 1996, Suzuki et al., 1998). Confounding factors
include health, age, gender, and season. No statistically significant differences
were found in in cortisol levels between animals living in open and closed captive
environments. Although behavior differences such as more time spent resting in
closed captive environments were observed.

86

 

Copland & Needham, 1992

Six captive dolphins (4M;2F) were moved by air from one facility to
another in Australia. One dolphin was 6 months another was 4 years, the
remaining 4 were ≥10 years. The animals were last fed over 24 hours before
transport. At 5 AM on the date of transport the adult animals were given 3 mg/kg
of diazepam to reduce travel anxiety. Serum samples were taken ~4 hours after
the diazepam was administered, ~1.5 hours after capture, and ~6.5 hours before
the second samples. Plasma cortisol was measured by Ameriex Cortisol RIA kit,
code IM. 2021, Amersham Pty. Ltd., North Ryde, New South Wales 2113,
Australia. Serum cortisol rose during transport and other classic stress indicators
of the leukogram such as lymphopaenia (low lymphocytes) and eosinopaenia (low
eosinophil granulocyte) observed. The results show that even when given an antianxiety drug these dolphins still exhibited a stress response during transport.

Houser et al., 2011

Four (3M;1F) adult Atlantic bottlenose dolphins (2 control; 2 experimental)
were housed individually in above groups pools for a 10 day period and sampled
for cortisol and aldosterone. The control animals were housed in ambient water
temperature, where the experimental animals were exposed to decreased water
temperatures in the range of 4.2-16.6°C. Blood serum samples were collected
every 2-3 days between 0745-1000 hours by voluntary husbandry behavior, tail
fluke presentation. Samples were analyzed via RIA kit TKCO1, Siemens
87

 

Healthcare Diagnostics, Deerfield, Illinois 60015-0778, USA. Cortisol levels
were elevated in the experimental animals that were exposed to declining
temperatures, although cortisol levels were not as high as observed in capture and
handling studies. These results may demonstrate that bottlenose dolphins exhibit
a more intense stress response to capture and handing than to lowering water
temperatures. It is interesting to note that one control animal had cortisol levels
below detection in some samples, and the other control animal displayed a higher
cortisol level on day 4 in the pool than both of the experimental animals. This
demonstrates the variability in recognition of a stressor between different animals
of the same species. The study was preformed to see how thermal stress affected
cortisol and aldosterone production.

Naka et al., 2007

Blood samples were obtained voluntarily from the fluke vein of 5 captive
bottlenose dolphins (age 4-22) kept at Kamogawa Sea World, Chiba, Japan. The
animals were healthy and on no medications at the time of sampling. Samples
were collected when the animals were in a natural “floating” position, and then 10
minutes after the pool was drained in a “landed” position. Plasma cortisol was
measured using a Cortisol EIA kit, Cayman Chemicals, Ann Arbor, USA. Mean
cortisol concentrations rose from 3.7 ng/ml in the floating position to 10.6 ng/ml
in the landed position, although the increase was not statistically significant.
Heart rate was recorded in three of the animals in floating and landed position and
displayed a higher but not statistically significant value in landed animals.
88

 

Plasma cortisol levels of the captive animals in this study fall within the range of
known levels assessed in wild members of the same species (St. Aubin et al.,
1996). Mean cortisol levels expressed in wild Atlantic bottlenose dolphins could
be exhibiting a low-moderate stress response due to the chase and capture
methodology for sample collection (Ortiz & Worthy, 2000).

Noda et al., 2006

Ten captive female bottlenose dolphins weighing between 200-250 kg
were utilized in this study. All were wild caught in waters surrounding Japan and
kept at a captive facility for at least five years. Before being transported to
another facility 6 hours away, 6 of the animals treated with a total of 40 mg/kg of
bovine lactoferrin/day for seven days. Lactoferrin a glycoprotein secreted in
bodily fluids. It is known for its ability to contribute positively to the immune
system and protect animals from pathogens by increasing the activity of
neutrophils. The five control animals were not treated. All animals were deemed
healthy and sexually mature. Blood serum samples were taken before transport
after the pool was drained and the animals were loaded into transport units. After
6 hours of transport another sample was taken upon arrival at the receiving
facility. Samples were taken from superficial vessel on the ventral side of the
fluke. Analyses of cortisol levels were determined by electrochemiluminescence
immunoassay using rabbit antibody.

89

 

Before transport mean serum cortisol levels were higher than referenced
cortisol values, but showed no significant variation between groups. After
transport the treated group showed decreased levels of mean serum cortisol while
the untreated groups levels remained the static. The animals in this study
displayed higher levels of cortisol than members of the same species sampled in a
captive environment (St. Aubin et al., 2001) before transport. This could be due
to the amount of time taken to drain the pools (~3 hours) before the first blood
sample was taken. These same animals exhibited lower levels of mean serum
cortisol before the experiment, making it difficult to separate the stress of draining
the pool vs. the stress of the actual transport. After transport the group treated
with lacoferrin displayed a reduced levels of mean serum cortisol, which may
indicate that bovine lactoferrin, may reduce cortisol levels in bottlenose dolphins
during transport.

Ortiz et al., 2010
Two captive male bottlenose dolphins we sampled voluntarily from their
caudal flukes for blood serum. Two samples were obtained over 2 days while the
animals were eating regularly (5 kg/d). The next samples were taken during a
fasting period of 38 hours at 14, 24, and 38 hours. After the 38 hour fast dolphins
were fed their normal diet for 24 hours where samples were collected at 5, 12, and
24 hours. Cortisol was measured by a commercial radioimmunoassay, DPC, LA,
CA, USA. Plasma cortisol and aldosterone levels were undetectable or below
sensitivity for the assay used in this experiment. This would make the levels
lower than commonly seen in other captive and wild members of the same species
90

 

(Thomson & Geraci 1986; St. Aubin et al., 1996; Ortiz and Worthy 2000, Noda et
al. 2006, Naka et al., 2007, Suzuki et al., 1998).
The seemingly lack of circulating cortisol in these two animals could be
due to reduced activity and age or adrenal atrophy, which occurs when the adrenal
hormones are chronically used to the point of atrophy. Fasting has been known to
cause an increase of circulating cortisol in seals (Ortiz et al., 2001;2003), although
documentation has never been published on cetaceans. The results of this study
may be used to indicate the possibility of adrenal atrophy in captive bottlenose
dolphins exposed to chronic stressors; display that the small levels of
glucocorticoids present in many samples from bottlenose dolphins may indicate
that a low stress response is occurring due to detection; or arise from a technical
mistake during the sample collection or analysis protocol.

Reidarson & McBain, 1999

Two male captive dolphins, ages 10 and 13, were given a single oral dose
of dexamethasone at .11 mg/kg. Dexamethasone is a corticosteroid that is
clinically used when adrenal hormone production is low. It is used to treat
inflammation and allergies in humans (http://www.nlm.nih.gov). Blood samples
were taken voluntarily from the fluke vein immediately post-dose, then 1, 1.5, 2,
7, and 17 days thereafter. Analysis was conducted by immunochemiluminescence
assay, Chiron Diagnostics, East Walpole, Massachusetts 02032, USA. Within 24
91

 

hours they animals expressed lympohopenia and neutrophilia, usually
characteristic of a stress leukogram. Cortisol levels decreased from between 2-3
µg/dl at administration to undetectable levels after 24 hours of administration and
returned to detectable levels within 48 hours. The base cortisol levels collected at
administration of the medication reflect levels collected from a control group of
six bottlenose dolphins sampled similarly without the administration of a
medication (1.3-3.3 mg/dl). Cortisol levels may have decreased with
administration of dexamethasone due to its ability to suppress the release of
ACTH. It is important to note that dexamethasone is a known anti-inflammatory
agent and immunosuppressant.

Ridgeway et al., 2006

Five 120-h (5 day) dolphin vigilance session were conducted between
July and February. Blood samples were taken immediately prior to and after 120h vigilance sessions from voluntary fluke presentation, a common husbandry
behavior. Analysis conducted with a commercially available RIA kit, Diagnostic
Products Corporation, L.A., CA) validated by Romano et al. (Romano et al.,
2004). Cortisol levels were within normal ranges (6.6-3.6 µg1!! ) (St. Aubin et al.,
1996) and no changes were observed before and after the experimental sessions.
These results may display that cortisol along with other hormones associated with
the stress response such as epinephrine and norepinephrine do not fluctuate during
sleep deprivation, which was supposed to be simulated by continuous vigilance
92

 

over 5 day periods. The detection rate for these two animals was 87-99%. These
tests were conducted between July and February, a seasonal variation in cortisol
levels have been shown in bottlenose dolphins displaying lower cortisol levels in
summer/fall months and higher levels in spring and summer months (Orlov et al.,
1988). Since cortisol levels did not rise pre and post study it could be
hypothesized that uni-hemispherical sleep patterns occur in bottlenose dolphins,
which allows auditory vigilance without showing adverse reactions of sleep
deprivation. It should also be noted in this study that the fish “rewards” for
correctly pressing the paddle to identify the 1.5-s sound were part of the daily
diet, which may have been motivation for the animals to stay vigilant during
nocturnal hours where respiration rates and reaction rates were reportedly slower.

Ridgeway et al., 2009

Two captive bottlenose dolphins (1M;1F) were tested for auditory
vigilance in three 72 and four 120 h experiments. In the experiments the animals
were trained to press a paddle when a 1.5 s goal tone at 70kHz was played
throughout at varying times when a 0.5 s tone was more constant. Upon pressing
the paddle for the correct tone the animal received a fish “reward” which was
actually part of the standard 24-hour diet. Serum cortisol levels were collected
from the 26-year-old female dolphin immediately prior to and post one 120h
vigilance session. Blood/serum samples were taken from the fluke vein during
voluntary presentation and analysis was conducted with a commercially available
93

 

RIA kit, Diagnostic Products Corporation, L.A., CA) validated by Romano et al.
(Romano et al., 2004).
Cortisol levels were normal at 1.2 µg/d! !! pre and 2.0 µg/d! !! post 120h
session (St. Aubin et al., 1996). The results corroborate a similar study conducted
by the same primary author conducted in 2006. Displaying results that captive
bottlenose dolphins may not show classic signs of sleep deprivation, including
increased cortisol levels, after 120h near constant vigilance sessions due to unihemispherical sleep. As noted in the pervious study seasonality, as well as the
fact that the food “reward” that was part of the normal diet of the animal may
have played a part in the vigilance due to hunger. In non-experimental conditions
captive dolphins are rarely fed a portion of their diet in the nocturnal hours, due to
displaying characteristics of a diurnal species.

Suzuki et al., 1998

Two bottlenose dolphins (1M;1F) that were wild caught but held in
captivity for at least 13 years were sampled twice a month from September to
December via voluntary tail fluke presentation. Samples were collected in the AM
hours. The total range of mean serum cortisol levels between both animals was
1.6-6.5 ng/ml with the female showing slightly higher levels. These two animals
displayed high levels of cortisol upon arrival at the facility, but has since
decreased and stabilized. During this study an RIA system for serum cortisol
measurement specifically in bottlenose dolphins and killer whales was developed.
94

 

Two antibodies were tested: FKA404 reacted only with a cortisol fraction;
FKA402 completely cross-reacted with 21-DOC which is a precursor to cortisol.
This study not only observes cortisol levels of two captive bottlenose dolphins,
but also warns in cross reactivity of certain antiserums when analyzing cortisol
levels in cetaceans. The mean cortisol levels for 2 animals in this study were
lower than in previous studies (Medway et al., 1970;St. Aubin et at., 1996) which
could be due to cross reactivity issues in the other assays or due to acclimation
and/or individuality.

Medway et al., 1970

Eight bottlenose dolphins that were housed at the Montréal Aquarium
were sampled in this study. Blood samples were collected via venipuncture from
the dorsal or ventral flukes. After initial blood samples were taken 2 of the
animals were injected with 10 mg of dexamethasone and sampled again in 8
hours. Dexamethasone is known to reduce cortisol levels in humans within 24
hours. Analysis was preformed by a modification of the Silber-Porter method
(Peterson et al., 1957). The range of plasma cortisol levels from the initial
samples were 1.7-3.0 µg/100 ml, 8 hours later the two animals injected with
dexamethasone had a plasma cortisol level <1 µg/100 ml. It is important to note
that control samples were not taken after 8 hours in the 6 other animals that were
not injected with dexamethasone so a comparison in not applicable. It is thought
that cortisol levels peak within 1-2 hours of confrontation with a stressor

95

 

(Thomson & Geraci, 1986), so it is plausible to hypothesize that either all the
animals sampled at the start of this study were experiencing a mild stress
response, dexamethasone may have depressed plasma cortisol levels, or normal
circulating levels of plasma cortisol in these 8 animals are <1 µg/100 ml.

Pedernera-Romano et al., 2006

Four bottlenose dolphins (2F;2M) housed in two different aquaria in
Mexico City were trained to give saliva samples before each first meal. Both
aquaria had similar space and water conditions, along with entertainment shows
and therapy sessions. The dolphins were all wild caught and between 8-20 years
old and have spent at least 2.5 years in captivity. Salivary cortisol measurement is
applicable because of its less invasive nature and ability to be used in long-term
cortisol studies without the possibility of bacterial infection from a puncture mark
created during venipuncture. Cortisol is found in saliva because of its known
solubility of in lipids of cell membranes. The saliva samples were collected via
trained voluntary behavior between 9-930h.
A control group of 7 dolphins from a different aquarium in Puerto
Vallarta, Mexico were used to obtain time-matched samples of saliva and
blood/serum cortisol levels. In these animals 4 were in captivity for 5 years
between the ages of 9-11 and trained to voluntarily allow serum and saliva
samples. The remaining 3 dolphins in the control group have been in captivity for
96

 

1 years between the ages of 4-5 had to be restrained for sampling. Samples from
these animals were taken between 1000-1100 hours with saliva obtained 3-4 after
serum collection. Cortisol from both types of measurements was measured in RIA
kits (Cort CT2, CIS Bio International, France).
The time-matched study displayed that saliva cortisol values represented
about 27% of blood values with a correlation value of .73 between time-matched
values. That percentage is similar to humans and some other primate species.
Serum cortisol ranged from 6.59-64.46 nmol ! !! while saliva cortisol levels
ranged from 1.43-15.72 nmol ! !! . Saliva cortisol levels were elevated in the
animals that had been kept in captivity for a year when compared to the animals
that had been in captivity for five years. The samples obtained from the four
animals for sole saliva analysis displayed the individuality of stress reactions
between members of the same species in similar conditions with some animals
displaying detectable levels of cortisol 3/31 days and others showing detectable
cortisol levels only 11/31 days. The adopting of less invasive techniques such as
saliva sampling in captive cetaceans may allow health assessments relating to
stress to occur more often without the adverse effects of possible infection and
loss of blood which can occur from constant blood hormone monitoring
techniques.

97

 

Suzuki & Komaba, 2012

I received unpublished data from Miwa Suzuki and her colleague
Masayuki
 Komaba
 who collected the serum cortisol samples in three Atlantic
bottlenose dolphins kept at Kujukushima
 Aquarium
 -­‐
 Umikirara
 (Nagasaki,
 Japan).
The animals were sampled between May 26, 2009 and September 13, 2011 during
all seasons. Samples were obtained from the animals during husbandry
procedures from the fluke vein, from the animals when lifted from the enclosure
in a stretcher, and when some of the animals were on antibiotics. Antibiotics did
not significantly affect the cortisol levels (p>0.05) in the animals so they were
included in the mean cortisol levels for this analysis. Animals that had samples
taken after being lifted from the pool did display a significant increase (p=0.05) in
cortisol levels and were excluded from the mean cortisol levels for this analysis.
77 samples were collected. The samples were analyzed by TR-FIA, DELFIA
system, verified for cortisol in bottlenose dolphins by Suzuki et al (1998),
PerkinElmer, Walthom, Massachutes.

Killer Whale (Orcinus orca)


 

The Killer Whale or orca is easily recognized and widely distributed. It is

second only to humans as the most widely distributed mammal on Earth (Ford,
98

 

2008). It is found in all oceans and most seas, from the Arctic to the Antarctic,
but prefers coastal temperate waters. Three subspecies and ten ecotypes have
been identified. The subspecies have differing mitochondrial DNA that suggest
some groups have not interbred for centuries (Morin et al., 2006). The three
subspecies include fish eating residents, mammal eating transients and fish eating
offshores. Depending upon the subspecies pods can vary from tight knit family
groups that never leave their mothers in certain resident populations, to smaller
hunting groups in transient populations, to large groups of up to 100 in the seldom
studied offshore populations (Ford, 2008). Their wide coastal distribution makes
them a prime target for research both in the wild and captivity, and also puts them
in close proximity to many anthropogenic stressors. They are still caught for
aquaria in countries such as Russia and Japan (Ford, 2008). They are not usually
hunted for consumption due to the high accumulation of toxins found in their
blubber.
The first killer whale to be put on display was captured in 1964 with the
purpose of being used as a model for a sculpture at the Vancouver Aquarium.
The Seattle aquarium quickly followed by capturing the first killer whale for
exhibition purposes in 1965. These animals were caught from local resident
populations and between 1965-1973. 45 members of the Southern Resident orca
community were captured and another 13 died in the process. This was about half
of the population at that time. Only one animal out of the 45 captured is still alive
and on display at the Miami Seaquarium. In 1976 wild killer whale captured were
outlawed in Washington State due to the negative impacts on the local population.
99

 

Orca captures ceased in the US in 1976 as well since no other state had a local
population. According to a constantly updated web resource called “orcahome.de”
that tracks captures, births and deaths of killer whales in captivity worldwide
maintained by a collaboration of killer whale enthusiasts, based on publications of
all types. 131 orcas have been caught for aquaria worldwide, 12 are alive at the
time of publication. Currently 46 orcas are in captivity worldwide
(http://www.orcahome.de/orcastat.htm). The mortality rate of infants, defined as
≤6 months, in captivity is 50.2%, the wild figure is unknown due to difficulty of
observation (Rose, 2011). In captivity orcas can be grouped in artificial pods or
kept alone. Anthropogenic stressors are prevalent in captivity due to confinement
and dependence (Rose, 2011).

Biology


 

Killer whales range in size from 6-9 meters and are the largest member of

the Delphinidae (Ford, 2008). They have striking black and white coloration with
an eye patch that varies in size depending upon ecotype. They are sexually
dimorphic with males being substantially larger and possessing a much larger
appendages including dorsal and pectoral fins. They can be individually
identified by their unique saddle patch and dorsal fins (Ford, 2008). They are
thought to be the most highly socialized animal other than humans and pods have
developed their own distinct dialect (Ford et al., 2000). They usually hunt
cooperatively and have developed specialization for different ecotypes and
regions. They are typically brief divers usually surfacing every 10-20 seconds
100

 

but can hold their breath for over 15 minutes if necessary. Juveniles will remain
with their mothers until the next calf is born, usually around 6 years, and may stay
for their entire lives. Killer whales have no natural predators other than man.
Maximum lifespans in the wild around ~90 for females and ~60 for males,
average lifespans are in the 50’s for females and 30’s for males (Ford et al.,
2000). In captivity the average lifespan is around 25 years for both males and
females (Rose, 2011).

Status


 

The International Union for Conservation of Nature lists killer whales as

“Data Deficient” with an estimated 50,000 thought to be living in the wild.
Certain subspecies such as the Southern and Northern Resident communities are
listed as endangered in the United States and Canada.


 
 
 
 Wild

Studies Examining Cortisol Levels

Collecting cortisol samples from wild orcas have recently evolved. Due to
their large size and inhabitation of harsh climates chase, capture, and release is
not a logical strategy to obtain a blood/serum sample. In the past, tissues samples
were obtained by use of a biopsy gun, which shoots a pneumatic dark at the
animal during a focal follow (Noren & Mocklin, 2011). This method is relatively
invasive and has the potential to cause an infection at the sample site and cause
the animals to avoid research boats in the future. Currently in Washington state
101

 

the University of Washington’s School of Conservation Biology along with
NOAA are collecting fecal samples from killer whales to analyze cortisol
concentrations. These samples can be collected from up to ¾ of a mile away and
are relatively non-invasive. Fecal cortisol takes around 12-24 hours to metabolize
so the sample collected reflects the conditions of the previous day (Ayres,
personal communication). In a recent study fecal cortisol samples were used to
determine that Southern Resident Killer Whales exhibit higher levels of cortisol
when their primary prey source is low, and vise versa (Ayres et al., 2012). The
same study also demonstrated that SRKW cortisol levels were highest when a
smaller number of recreational, commercial, and research vessels were around,
suggesting that prey availability plays a larger role in stress levels than human
vessel presence (Ayres et al., 2012). This methodology allows access to baseline
cortisol levels of wild killer whales, something that has yet to be established in
Atlantic bottlenose dolphins and beluga whales.

Ayres et al., 2012


 

The endangered Southern Resident Killer Whales are composed of three

pods (J, K, and L) and often inhibit the inland waters of the Salish Sea from MayOctober where they forage for their primary food source Chinook salmon
(Oncorhynchus tshawytscha). Declines in Chinook salmon runs in the western US
may create stress for these animals; this is deemed the inadequate prey
hypothesis. While inhabiting the Salish Sea commercial, recreation, and research
102

 

vessels often observe the animals, sometimes in large quantities that may also act
as a stressor; this is called the vessel impact hypothesis.

While in the inland

waters SRKWs often feed on Frasier River Chinook salmon run which are usually
at their peak in August-September, the same months vessel numbers peak. The
inverse is often true as well, with the Chinook run lower in spring/early summer,
along with fewer vessels.
This study obtained 154 fecal samples from the 88 members of the three
pods between 2007-2009. Most samples were obtained from J pod (n=113). Fecal
samples allow noninvasive sample collection by trailing the animals from up to ¾
mile away and scooping floating fecal matter for stress hormone analysis. RIA kit
!"#

I corticosterone, #07-120103; MP Biomedicals, Costa Mesa, CA was used

for fecal hormone metabolite analysis. Results of analysis concluded that
glucocorticoid levels were lowest when Fraser River Chinook salmon were
peaking, although vessel abundance was also peaking at that time, and vice versa.
The range of fecal glucocorticoids in these animals was 500-3,500 ng/g. The
comparison between fecal GCs of male SRKWs (n=34) for a stranded male killer
whale that later had to be euthanized varied greatly (1000 ng/g for SRKW males;
28,000 for stranded male). The results of this study demonstrate that prey
abundance plays a larger role in elevating glucocorticoids than vessel presences.
It should be noted that during years of low prey abundance vessel impacts might
evoke more of a physiological reaction in the stress response.

103

 

Captive Studies Examining Cortisol Levels

Orca serum cortisol levels have been estimated to be around 0.4 µg/dl in a
stable captive environment (Suzuki et al., 1998). Captive orcas are often taught
husbandry practices that allow routine medical procedures to take place without
exciting the stress response. These practices include presentation of the fluke for
routine blood sampling. These blood serum samples most likely represent a
baseline cortisol level for captive orcas. It is unknown if a cortisol baseline for
captive orcas could be applied to wild orcas because of differences in
environment and lifestyle. A study has shown that factors such as time of day and
season can play a role in cortisol production of captive orcas. Killer whales have
exhibited decreasing cortisol until 18:00 hours then fluctuations until increasing
the following morning (Suzuki et al., 1998). Seasonality played a factor in the
males and female orca being studied. The pregnant female showed cyclic
changes in cortisol concentrations at 4-month intervals (Suzuki et al., 1998) while
the males exhibited lower cortisol concentrations in the summer than in the
winter. Elevated mean cortisol levels in captive killer whales have been
documented in the autumn, over spring (Suzuki et al., 2003). Another study
showed that cortisol levels in two females killer whales during pregnancy and 3-5
weeks post partum found no significant increases in cortisol levels (Lyamin et al.,
2005), although the time of sample collection during the pregnancy was not listed
so it could be within the similar 4-month intervals that Suzuki et al. revealed. (See
Table 3)
104

 

Table
 3: Killer Whale Cortisol Studies, Basic information. Species, Author (s) of study mean cortisol levels were obtained
from, whether the study was conducted in a wild or captive environment (black wild/red captive), sample type, mean
cortisol in nmol/L and µg/dl, number of animals sampled in each study, number of samples analyzed to determine mean
cortisol levels in each study, range of individual cortisol levels in nmol/L and µg/dl, sample method, and assay used for
measurement of cortisol levels.

Killer
 Whale
 

wild:
 
captive
 

mean
 
F
 

mean
 
F
 

#
 
Animal
 

#
 
Sample
 

Range
 

Range
 

Sampling
 

Analysi
s
 

Author
 

Sample
 
type
 

nmol/
L
 

μg/dl
 

N
 

n
 

nmol/L
 

μg/dl
 

Method
 

Assay
 

Suzuki
 et
 al.,
 
1998
 

serum
 

5.52
 

0.2
 

3
 

18
 

n.d.-­‐11
 

n.d.-­‐
0.4
 

husbandr
y
 

RIA
 

Suzuki
 et
 al.,
 
2003*
 

serum
 

8.28
 

0.3
 

7
 

319
 

n.d.-­‐
52.42
 

n.d.-­‐
1.9
 

husbandr
y
 

RIA
 

Lyamin
 et
 al.,
 
2005
 

plasma
 

7.17
 

0.26
 

3
 

3
 

2.8-­‐22
 

0.1-­‐
0.8
 

husbandr
y
 

RIA
 

Lyamin
 et
 al.,
 
2005
 

plasma
 

9.66
 

0.35
 

3
 

3
 

8.3-­‐13.8
 

0.3-­‐
0.5
 

husbandr
y
 

RIA
 

*Supplemental
 data
 from
 study
 utilized.
 
 

Lyamin et al., 2005


 

Three female killer whales were sampled by voluntary fluke presentation

when pregnant and then again 3-5 weeks post partum. The samples were taken to
attempt to discover what was responsible for the decrease in sleep behavior after
the calves were born. Although no statically significant increases were found
between cortisol levels when the animals were pregnant vs. 3-5 weeks after birth
of their calf, there was greater variation in the range of cortisol in the pregnant
animals (0.9-7.4 ng m! !! ) than 3-5 post partum (2.8-4.1 ng m! !! ). This study
depicts that killer whale mothers and neonates displaying very little resting
behavior during the first 3 weeks of giving birth and birth, and that resting
behavior slowing increases from 4-8 weeks while returning to pre-calf resting
patterns at around 8 weeks throughout adulthood.
105

 

Suzuki et al., 1998

Three killer whales (2M;1F) that were wild caught but held in captivity for
at least 10 years were sampled twice a month from September to December via
voluntary tail fluke presentation. Samples were collected in the AM (900-1000h)
and PM (1600-1700h) hours to account for the circadian rhythm of cortisol
production often observed in terrestrial diurnal mammals. The total range of
mean serum cortisol levels between all animals in the AM was 1.4-4.0 ng/ml and
0.2-2.8 nm/ml in the PM. Although the mean cortisol levels in all of the animals
tended to decrease in the PM only the female’s cortisol levels were significantly
different between the AM and PM. The difference in cortisol levels between the
female and two males could differ due to social rank. Naturally killer whales are
matriarchal and in captivity females often harass males. One male that showed
the lowest diurnal mean cortisol variation has been observed getting harassed by
the female in this study. Those observations may depict that social rank plays a
part in cortisol production.
During this study an RIA system for serum cortisol measurement
specifically in bottlenose dolphins and killer whales was developed. Two
antibodies were tested: FKA404 reacted only with a cortisol fraction; FKA402
completely cross-reacted with 21-DOC which is a precursor to cortisol. This study
not only observes cortisol levels of three captive killer whales, but also warns in
cross reactivity of certain antiserums when analyzing cortisol levels in cetaceans.
Seasonality could have also played a part in the mean cortisol levels of the
106

 

animals being studied other Delphinids have shown seasonal patterns in cortisol
production.

Suzuki et al., 2003

Three wild caught captive killer whales (2M; 1F), being kept at
Kamogawa Sea World, Japan were utilized in the study to determine diurnal and
seasonal patters in killer whales. For the diurnal change study 2, 11-year-old
captive killer whales (1M; 1F) wild caught in 1988 were sampled via
venipuncture through voluntary tail fluke presentation at 0900, 1200, 1500, 1800,
2100, 2400, 0300, and 0600 h. Serum cortisol levels were measured by RIA
developed specifically for bottlenose dolphins and killer whales by the main
author of this study in a previous study. Minimum levels of cortisol were
observed at 2400; maximum levels at 0600, but a pattern of fluctuations
developed between 1800 and following morning. The female tended to display
higher cortisol levels and greater fluctuations throughout the day than the male in
this study, sometimes peaking at 2100.
The annual change study obtained samples from 3 killer whales (2M; 1F)
wild caught but maintained at the same facility for at least 10 years and sampled
in the same methodology twice a day between 0900-1000 and 1600-1700. All the
animals were adults, but the female in the annual change study was pregnant.
Serum cortisol levels were analyzed by the same RIA mentioned above. Males
displayed seasonal variation with higher cortisol levels recorded in fall and winter
107

 

months, similar findings have been reported in bottlenose dolphins (Turisops
truncatus) in winter months (Orlov et al., 1988). The pregnant female exhibited
cyclic cortisol concentrations every four months, but this may be due to her
pregnancy during the time of the study. Cortisol concentrations were significantly
lower in the female and one of the males in the AM than the PM. The other male
sampled showed a trend of this diurnal patter, but results were not significant for
him.

This could be due to his low social rank, which may burden him with a

more constant stressor. It is important to note that Progesterone showed a negative
correlation with cortisol, increasing when cortisol decreases. Stress hormones
like cortisol are known reproductive hormone suppressants.


 
 Beluga


 

Whale (Delphinapterus leucas)

Beluga whales are also known as white whales. They inhabit the Arctic

and subarctic. They are medium sized and live in fluid pods of 2 -10 individuals,
although they can form herds composed of 1000s (O’corry-Crowe, 2008).
Females are known to form large nursery groups while adult males may be seen
singly or form a separate pod of 6-20 individuals. Due to the harsh climate
belugas take part in a predictable yearly migration usually returning to their natal
sites in the spring (O’corry-Crowe, 2008). They tend to inhabit coastal areas,
which made them a target for research and commercial harvesting. A distinctive
population of belugas inhabiting the St. Lawrence Seaway in Canada has
unusually high cancer rates due to smelting operations in the twentieth century.
At the time of death some of the animals were so polluted with heavy metals and
108

 

organohalogens they had to be labeled as hazardous waste. Increasing
anthropogenic threats causing habitat degradation such as oil and gas
development in the Arctic and global warming continue to add obstacles to beluga
survival (O’corry-Crowe, 2008).
Beluga whales were one of the first cetaceans to be held in captivity (cetabase.com). In 1861 a beluga from the St. Lawrence Seaway was put on display at
Barnum’s Museum in New York. Currently about 200 belugas are captive
throughout North America, Europe, and Japan. Although some facilities have had
success with breeding captive belugas the gene pool in low and new wild caught
specimens are still regularly used to stock aquaria. Some aquaria offer “swimwith” programs with belugas. Most belugas in captivity are used for public
display but a small fraction is used in military exercises. Anthropogenic stressors
are prevalent in captivity due to confinement and dependence.

Biology


 

Belugas are in the family Monodontidae. They range in size from 3.5-5.5

meters and are easily identifiable by their white coloration. The exhibit many
differences from Atlantic bottlenose dolphins and killer whales including lacking
a dorsal fin, un-fused neck vertebrae, rounded melon, thick blubber, yearly
molting, and small appendages (O’corry-Crowe, 2008). These adaptations allow
for better maneuvering and thermoregulation in the Arctic ice packs. They are
highly social within their groups and can make up to 50 different vocalizations

109

 

(O’corry-Crowe, 2008). They have developed signature calls for individuals and
in one case have even mimicked human speech. They hunt in groups and
individually to feed on fish, crustaceans, sandworms, and cephalopods. They have
the ability to dive up to 1000 meters and remain submerged for 25 minutes when
hunting or navigating dense ice packs. Juveniles stay with their mother for
around 3-4 years until another calf is born (O’corry-Crowe, 2008). Their main
predators are killer whales, polar bears, and humans. Males and Females live on
average 35-50 years in the wild, but maximum lifespan is around 80 years old
(O’corry-Crowe, 2008).


 
 
 Status

The International Union for Conservation of Nature lists beluga whales as
“near threatened” with an estimated 150,000 alive in the wild. Certain
populations including the Cook Inlet belugas near Anchorage, Alaska along with
the Hudson and Ungava Bay pods in Canada are listed as endangered. Two other
populations in Canada, the Cumberland Bay and St. Lawrence River estuary
belugas are listed as threatened.

 

 
 
 
 
 Wild

Studies Examining Cortisol Levels

Obtaining cortisol samples from wild beluga whales has numerous
obstacles resulting from location and habitat preferences, but their habituation to
110

 

specific estuaries in summer make them a target for researchers (St. Aubin &
Geraci, 1989; 1992). Previous studies conducted on wild beluga whales have
obtained blood/serum samples from chase, capture, and restraint (St. Aubin &
Geraci, 1989; St. Aubin et al., 2001). Since many cetaceans show an increase of
cortisol during onset of the acute stress response with cortisol levels peaking
within 1-2 hours, the invasive methodology preformed to gather blood/serum
samples may not depict an accurate cortisol baseline (St. Aubin et al., 2001).
Chase, capture, and transport of the belugas all caused elevations in serum cortisol
levels (St. Aubin & Geraci, 1989). Further studies determined that serum cortisol
did not show age or sex related differences in wild beluga whales that were
sampled during tagging or capture, although an increase in cortisol did occur in
both sexes and all ages of animals tested (St. Aubin et al., 2001). (See Table 4)

Table 4: Beluga Whale Cortisol Studies, Basic information. Species, Author (s) of study mean cortisol levels were obtained
from, whether the study was conducted in a wild or captive environment (black wild/red captive), sample type, mean
cortisol in nmol/L and µg/dl, number of animals sampled in each study, number of samples analyzed to determine mean
cortisol levels in each study, range of individual cortisol levels in nmol/L and µg/dl, sample method, and assay used for
measurement of cortisol levels.

Mean
 
F
 

#
 
Anima
l
 

#
 
Sampl
e
 

Range
 

Range
 

Sampl
e
 

Analys
is
 

Wild:
 
Beluga
 

Captive
 

Mean
 
F
 

Author
 

Sample
 

nmol/
L
 

μg/dl
 

N
 

N
 

nmol/L
 

μg/dl
 

Metho
d
 

Assay
 

St.
 Aubin
 &
 Geraci,
 
1989
 

Plasma
 

90
 

3.3
 

42
 

41
 

18-­‐196
 

0.7-­‐7.1
 

C/C/R
 

RIA
 

St.
 Aubin
 &
 Geraci,
 
1992
 

plasma
 

110
 

4
 

10
 

158
 

80-­‐204
 

2.9-­‐7.4
 

C/C/R
 

RIA
 

St.
 Aubin
 et
 al.,
 
2001
 

plasma
 

89.1
 

3.2
 

183
 

115
 

15.5-­‐
204.4
 

0.6-­‐7.4
 

C/C/R
 

RIA
 

Tryland
 et
 al.,
 2006
 

serum
 

125.1
 

4.5
 

21
 

21
 

53-­‐219
 

1.9-­‐7.9
 

C/C/R
 

CLEIA
 

Orlov
 et
 al.,
 1991
 

serum
 

82.6
 

2.9
 

10
 

16
 

NH
 

RIA
 

Schmitt
 et
 al.,
 2010
 

serum/
 
plasma
 

49.7
 

1.8
 

3
 

104
 

H
 

CLEIA
 

serum
 

27.6
 

~1
 

7
 

27
 

H
 

CLEIA
 


 
18.2-­‐
115
 


 
0.66-­‐
4.17
 

Spoon
 &
 Romano,
 

 


 

111

 

2012
 

St. Aubin & Geraci, 1989


 

Forty-two sub-adult beluga whales were captured in the Seal and Churchill

River estuaries in western Hudson Bay during July 1985 and 1970. Large adults
and females with calves were purposely avoided to reduce confounding factors
such as age and reproductive hormones. Blood/serum samples were taken during
restraint within one hour. Analysis was conducted using a commercial !!"# RIA
kit, New England Nuclear, Boston, MA . Cortisol levels ranged from 18-196
nmol !!! . All but six of the animals were released immediately after the
samples were collected. It has been documented that cortisol levels often peak
within 1-2 hours of capture and restraint (Thomson & Geraci, 1986). That
knowledge may contribute to the mean cortisol level calculated from the animals
sampled within an hour of capture representing a low-medium stress response and
not an accurate assessment of a basal or baseline level. The exact time that
cortisol levels begin to rise during the cetacean acute stress response is unknown,
but it is plausible to hypothesize that changes in cortisol levels being occur
immediately after presented with a stressor.
Six belugas that were captured for serum samples were taken into
captivity for 10 weeks. Blood samples were taken 30 minutes to one hour after
capture, when they arrived at the holding facility after 2-3 hours of transport, and
irregularly during the next 70 days. Cortisol levels were highest after the 2-3 hour
112

 

transport to the holding facility, which could be viewed as a medium-high stress
response, where most showed a declining trend during the first five days in a
captive environment. Spikes were documented in most animals between capture,
transfer (2-3 hours), and at irregular intervals during the first five days in the
holding facility. One animal’s cortisol levels peaked prior to its first day in
captivity and slighting rose for the preceding five days, perhaps indicating that
different individuals may adapt to stress in different ways.
An interesting note in this study is that out of 21 samples taken within an
hour of capture the mean cortisol level is only slightly higher than the mean
cortisol level determined from 77 samples of the six animals taken captive in the
recovery phase, days 7-70 (both numbers between 80-120 nmol !!! ). This could
be due to the mean cortisol levels at both capture and after 70 days in captivity
indicating a baseline for the animals in both environments or indicate that mean
cortisol levels collected during acute stress within one hour of capture are similar
due to acute stress still being exhibited in a captive environment up to 70 days
later.

St. Aubin & Geraci, 1992


 

Ten juvenile beluga whales (8M; 2F) were captured in the Churchill River

in Manitoba, Canada in July 1988 (4) and July 1989 (6). Blood samples were
collected from the caudal peduncle 30-40 minutes after capture (10-20 minute
approach followed by up to 20 minutes to obtain sample). Samples were analyzed

113

 

by commercially available RIA kits, NCS Diagnostics, Willowdale, Ontario. The
mean cortisol level for the 10 animals ranged from 80-205 nmol/L.
The animals were then transported to holding facilities and held for up to
five days and then released. Cortisol levels remained steady or increased from
capture throughout the first 24 hours at the hold facility. Samples were collected
at the holding facility at low and high tides. During low tides the pools were
drained to 0.8 meters, which caused an increase in cortisol levels by at least 10
nmol/L in over 77% of the animals. The inverse occurred with 70% of the
animals exhibiting a cortisol level of at least 10 nmol/L 5-6 hours after being kept
in shallow water, about 30 minutes before the pool was refilled to 1.65 m.
These results illustrate that cortisol levels were altered in these animals by
perceiving a stressful event (ie pool draining) and on some occasions spiked to
mean levels at capture during these events. It is important to note that 2 animals
were given an injection of TSH (10 IU of bovine TSH) after initial blood
sampling at capture and two more after being taken into the holding facility.
Although adrenal hormones like cortisol play a role in thyroid hormone
production, the reverse is not thought to occur. Application of TSH could play a
role in the analysis of mean cortisol levels during the time these animals were
analyzed in captivity.

114

 

St. Aubin et al., 2001


 

Over fifteen years 183 beluga whales were sampled in the Canadian

Arctic. The animals were sampled during attempts to apply tracking instruments
(55), obtained during capture for aquaria, research, or carcass from Inuit hunters.
The animals were mostly sampled during the summer, all other variables such as
age, season, sex, stock, and year were randomly selected. 151 animals were live
captured. The approach lasted from 5-15 minutes. 32 of the samples were from
animals shot for food by Inuit hunters where chase time was similar. Samples
were analyzed by commercially available RIA kits, NCS Diagnostics,
Willowdale, Ontario. Cortisol showed no age or sex related differences. Cortisol
samples were analyzed for 115/183 animals displayed a range of 15.5-204.4
nmol/L.

Tryland et al., 2006


 

Between 1996 and 2001, 221 blood/serum samples were collected from 21

wild beluga whales in three areas off Svalbard, Norway. The whales were were
live captured and restrained while blood was collected. Amount of time from
start of chase to sample collection was not noted in article, but personal
communication with Dr. Tryland has rendered the time of 10-20 minutes.
Samples were analyzed via a commercially available RIA kit, Immunite One
115

 

system, DPC, L.A., CA. Cortisol concentrations were comparable but higher than
previously reported for beluga whales sampled in the Canadian Arctic waters,
although other blood chemistry levels were similar. Cortisol concentrations
ranged from 53-219 nmol/L. These samples may display elevated cortisol levels
in comparison to samples collected during studies on the Canadian belugas due to
the relatively extended time from chase, capture, and sample collection (St. Aubin
et al., 2001). Cortisol levels are thought to peak in cetaceans about 1-2 hours after
exposure to a stressor (Thomson & Geraci, 1986).

Captive Studies Examining Cortisol Levels

Captive studies conducted on stress hormones of beluga whales are more
prevalent than wild studies. The captive beluga whale serum cortisol levels have
been estimated to vary between 0.7-3.2 µg/dl among different ages and sexes (St.
Aubin et al., 2001). Blood/serum is collected via a husbandry practice in a
captive environment (Schmitt et al., 2010, Spoon & Romano, 2012, St. Aubin et
al., 2012). The act of displaying the fluke for unhindered access to the fluke vein
can alleviate stress during collection. Captive beluga whale cortisol levels have
shown a circadian rhythm similar to those in terrestrial mammals with higher
concentrations in the morning than in the evenings (Schmitt et al., 2010). Cortisol
levels have differed in belugas that had been translocated to a new captive
environment with a peak during arrival at the new enclosure and decent to
baseline levels once acclimated (Spoon & Romano, 2012). (See Table 4)

116

 

Schmitt et al., 2010


 

Three long-term captive beluga whales estimated to be around the same

age (19) at time of study, residing in an outdoor exhibit were sampled to
determine baseline, diurnal, and stress induced changes of stress hormones in the
autumn (September and October). The animals were all wild caught from the
Hudson Bay, non-reproductively active, and deemed healthy at time of study.
Blood serum samples were obtained from the ventral fluke veins by signaling the
animals to display their fluke via common husbandry behavior. Over 6-8 weeks
Blood was collected under these conditions three times daily to reflect a baseline
reading. Blood was also collected 20 minutes before and 20 minutes after a
wading-contact session, where 6-8 public participants entered the pool and were
tactility active with the animals, and during an out of water examination. For an
out of water examination to occur the animal must be separated from its group
and directed into a medical pool which is quickly drained so the animal can be
placed on a stretcher, lifted up, and placed on the pool deck (Stage 1). During the
time on the pool deck medical procedures including an endoscopy are preformed
lasting 45 minutes to 1 hour (Stage 2). Blood samples were taken 20 minutes
after initial separation for the exam, following the 45-minute to 1-hour exam,
along with 1, 12, 16, and 24 hours post exam. Samples were measured by
automated chemiluminescent enzyme immunoassay, IMMULITE Coat-A Count,
DPC, L.A., CA.

117

 

Baseline mean serum cortisol levels ranged from .66-4.17 µg/dL with a
mean level of 1.8 µg/dL. Baseline endogenous cortisol concentrations from these
animals were higher in the morning than afternoon and evening, displaying a
cyclic trend that has also been documented in captive killer whales (Suzuki et al.,
1998). Mean cortisol levels along with others stress hormones including ACTH
and aldosterone were all significantly elevated during the out of water exam.
Mean cortisol concentrations rose 4X baseline levels (3.8 µg/dL) in stage one of
the out of water examination peaking at 7.9 µg/dL in stage two, then returned to
3.8 µg/dL one hour following the exam, and then returned to the baseline level of
1.8 µg/dL, 12 hours post out of water exam. The changes in mean cortisol levels
during pre-post and during the out of water examination provides a timeline of an
acute stress response on belugas. Past studies on beluga whales have indicated
that seasonality effects cortisol levels, with samples taken in spring more elevated
than samples taken in fall (Orlov et al., 1991). No statistically significant results
were found in samples taken pre and post wading contact session, but time of day
was not indicated so it may play a part in the analysis.

Spoon & Romano, 20124


 

This study observed changes in cortisol levels in beluga whales associated

with a predicted stressor, which was the introduction of seven beluga whales to
their enclosure. 3 resident beluga whales at the Mystic Aquarium (Mystic, CT,

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4


 The
 mean
 cortisol
 levels
 obtained
 from
 seven
 animals
 in
 Spoon
 and
 Romano
 (2012)
 was
 estimated
 based
 on
 individual
 
cortisol
 levels
 posted
 in
 a
 graph
 in
 the
 publication.
 It
 is
 possible
 that
 the
 mean
 cortisol
 level
 may
 be
 slightly
 skewed.
 

118

 

USA) were introduced to 4 belugas being transported from the John G. Shedd
Aquarium in Chicago. Baseline samples were obtained from the animals being
transported along with the three belugas that reside at the Mystic Aquarium.
“Arrival” samples were taken from the transported belugas immediately after
transport and before introduced in the new environment; and from the resident
belugas within 5 days of the transported animals arrival. “Acclimation” samples
were taken from the transported belugas 5-6 months after they arrived and from
the resident belugas 4, and 8 weeks after the arrival of the transported animals.
All blood serum samples were taken via venipuncture from voluntary fluke
presentation except the samples obtained when the transported animals were still
restrained for transport. Cortisol levels were analyzed using chemiluminescent
enzyme immunoassay, Immulite, Siemens, L.A., CA, USA.
The three resident belugas displayed elevated levels of E and NE but did
not display any statistically significant differences in mean serum cortisol
concentrations between baseline, arrival, and acclimation periods. All 4
transported belugas showed statistically significant differences in cortisol levels
from baseline, arrival, and acclimation, rising from baseline to peak during the
arrival phase and decreasing during the acclimation phase. This observation may
indicate that transport is an acute stressor to beluga whales. All whales displayed
an increase in E and NE, while just the transported whales had increased cortisol
levels, indicating that the process of relocating and introducing these animals
causes an environmental change that may be perceived by these animals as a

119

 

stressor. It also may indicate that the transport was a greater stressor than the
environmental changes that the resident belugas were exposed to.

Orlov et al., 1991
Ten beluga whales that were captured off of Russia were studied during
adaptation to a captive lifestyle in this study. Blood samples were taken 1, 3, 8,
and 11 days after the animals were captured and transported to a Research
Institute in Vladivostok, Russia. After the initial study on adaptation the animals
were held for a year to study seasonality of hormones along with other blood
constituents. The animals were kept in 6 feet of water to allow for blood sample
collection from the fluke vein. Serum cortisol was analyzed by a RIA assay
produced by IBOKh, Belorussian SSR or Sorin, France. Seasonality was found in
the animals, displaying lower levels of cortisol in spring/fall and higher levels in
winter/spring. This may be due to the harsh climatic impacts they face living in
the arctic. Mean cortisol levels for the were similar to other studies conducted on
wild captured animals and only held for a short amount of time, not an entire year.
Acute stress was noted by increase in cortisol levels within days 1-4 with a
rapid adaptation process that stabilized levels on days 8-11. Adaptability differed
among animals in this study, with cortisol levels varying among individuals.
Stressor experiments were preformed on 3 of the animals after the initial testing
phase and the results concluded that the animal going through the greatest stressor
exhibits the highest cortisol levels. Two of the animals were subjected to
immobilization on a porolon bed; the larger of the two animals displayed the
120

 

highest levels of cortisol. This is thought to be due to the development of internal
organ compression syndrome what was expressed in a more drastic manner in the
animal weighing the most. The third animal was immobilized by being put in
tank and displayed lower cortisol levels than the two animals that were presented
with more severe immobilization experiments. A reference was not made
between the cortisol levels of the animal kept in the tank to the animals kept in sea
pens that were not exposed to any additional stressor after capture.

121

 

CHAPTER 4
RESULTS

Wild vs. Captive Mean Cortisol Levels Within Species

Mean cortisol levels5 within each species living in both a wild and captive
environment are impacted by sample collection methodology6 (C/C/R, H, NH).
Producing descriptive statistics on mean cortisol level and standard deviation of
the means by combining all findings of cortisol levels in available published data
allows for the most accurate interpretation of cortisol levels found in wild and
captive members of these three species of cetacean.

Atlantic Bottlenose Dolphin

Mean cortisol levels differ greatly between studies completed on wild and
captive Atlantic bottlenose dolphins. Sampling methodology may play a large part

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5

 All cortisol samples that were analyzed in this analysis come from studies that employed sample collection via
venipuncture unless otherwise noted in the text. The samples were either blood serum or plasma and cortisol levels are
similar and comparable within each medium. The decision to use either serum or plasma in each analysis is based on the
original author and their preferred assay used for cortisol detection. For more detailed information on each of these topics
within each species please refer to the various tables supplied throughout this analysis.


 

6


 Husbandry (H) and non-husbandry (NH) practices will be referenced frequently in this analysis. For the purpose of this
analysis husbandry practices, sampling, or methodology refers to instances where the animal is trained to present its tail
fluke for voluntary sample collection. Non-husbandry practices, sampling, or methodology refers to instances when the
animal is sampled using forceful techniques such as lifting or removing from the tank and/or being forced into shallow
water for involuntary sample collection. All wild sample collection techniques employ chase, capture, restraint (C/C/R)
techniques.


 

122

 

in the results of these types of studies. Results from all studies on mean cortisol
levels in wild
 and captive studies Atlantic bottlenose dolphin display higher mean
cortisol levels in captive animals sampled using non-husbandry practices (range:
35-160 nmol/L), followed closely by wild studies that employ chase, capture,
restraint sampling techniques (range: 71.7-100 nmol/L). The lowest mean cortisol
levels were found in captive animals that were sampled using husbandry practices
(range: 10.5-69 nmol/L).
All wild studies that assessed mean cortisol levels in wild Atlantic
bottlenose dolphins display higher mean cortisol levels than any captive study that
employed husbandry sampling methodology. All the wild studies display mean
cortisol levels exceeding 70 nmol/L (range 71.7-100 nmol/L) while over half the
studies (7/12) on the captive animals sampled via husbandry practices display
mean cortisol levels that were under 20 nmol/L (range 10.5-69 nmol/L).
Mean cortisol levels of wild and captive Atlantic bottlenose dolphins that
were sampled utilizing non-husbandry methods are similar. Only one captive
study exceeds all of the wild studies and only 1/3 of the captive studies display
mean cortisol levels lower than the levels obtained in all of the wild studies. The
range of mean cortisol among wild studies is 71.7-100 nmol/L and 35-90.3 nmo/L
in captive animals sampled via non-husbandry methodology.
Mean cortisol levels derived from 3 studies completed on wild Atlantic
bottlenose dolphins are similar to mean cortisol levels from six studies on captive
Atlantic bottlenose dolphins that utilize non-husbandry sampling, but are on

123

 

average much higher than the cortisol levels obtained from ten studies that
employ husbandry sampling (Figure 1).

Mean
 CorMsol
 Levels
 in
 Tursiops
 Truncatus
 
180
 

Mean
 CorMsol
 (nmol/L)
 

160
 
140
 
120
 

Wild
 

100
 
80
 

NH
 

60
 

H
 

40
 
0
 

1986
 
1996
 
2000
 
1970
 
1986
 
1988
 
1991
 
1992
 
2006
 
1996
 
1998
 
1998
 
1999
 
1999
 
2006
 
2006
 
2007
 
2009
 
2011
 
2012
 
2012
 
2012
 

20
 


 

Figure 1: Mean cortisol levels from three studies on wild animals sampled (N=105, n=105)7 and 16
captive studies including 10 captive studies that employ H sampling8 (N=93, n=408) and six captive
studies that employ NH sampling (N=79, n=79).


 

Mean cortisol levels were significantly higher in wild Atlantic bottlenose

dolphins than in captive ones. The mean cortisol levels are more greatly
differentiated when comparing the results of wild animals to the ten studies on
animals that were sampled utilizing captive husbandry sampling. When
comparing mean cortisol levels from three wild studies to six captive studies that
employed non-husbandry sampling techniques the mean cortisol levels were very
similar. Great differences existed in mean cortisol levels of captive animals that
were sampled via husbandry and non-husbandry practices.9 Cortisol samples

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
7


 N=number
 of
 animals
 sampled,
 n=number
 of
 samples
 analyzed
 

 Ortiz et al. (2010) which, produced undetectable cortisol levels were included.
 
9

 Ortiz
 &
 Worthy
 (2010)
 was
 not
 included
 in
 the
 comparisons
 because
 cortisol
 levels
 were
 undetectable.
 The
 study
 sites
 
8

adrenal
 atrophy
 as
 a
 possible
 explanation.
 The
 RIA
 used
 for
 analysis
 in
 their
 study
 is
 a
 common
 commercially
 available
 kit
 
(Immulite,
 DPC)
 used
 in
 5
 other
 studies
 on
 cortisol
 levels
 in
 Atlantic
 bottlenose
 dolphins
 for
 this
 analysis
 and
 has
 an
 

124

 

collected by practicing non-husbandry sampling
 methodology are much higher on
average than samples collected by husbandry practices.
Based on studies, average mean cortisol levels10 are higher in Atlantic
bottlenose dolphins that have been sampled utilizing both non-husbandry
methodology in captivity and chase, capture, restraint methodology in the wild.
Large differences are present when comparing mean cortisol samples from wild
and non-husbandry collection methods to mean cortisol levels collected via
husbandry practices (Figure 2). The mean cortisol level derived from three
studies on wild Atlantic bottlenose dolphins is 83.0±15.011 nmol/L (3.0±0.54
µg/dl). The mean cortisol level obtained from 10 studies on captive animals that
voluntarily allowed sample collection to occur though trained a husbandry
practice is 30.6±20.6 nmol/L (1.1±0.7 µg/dl). The mean cortisol levels from six
studies on captive animals that employed involuntary non-husbandry sampling
methodology is 88.3±41.2 nmol/L (3.2±1.5 µg/dl).

 


 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
analytical
 sensitivity
 of
 5.5
 nmol/L
 (0.2
 μg/dl).
 
 Contamination
 of
 samples
 during
 collection
 or
 analysis
 could
 also
 explain
 
the
 undetectable
 cortisol
 levels
 (Siemens
 Healthcare,
 personal
 communication).
 
10

 Unconventional descriptive statistics were used in this analysis due to the heterogeneous nature of the data of this
project. Since individual cortisol values for each animal from each study were not available when compiling mean cortisol
levels an average mean cortisol levels is displayed. This value is a “mean of means” or synthesized means.
 
11


 Unconventional descriptive statistics were used in this analysis due to the heterogeneous nature of the data. Since

individual cortisol values for each animal from each study were not available a standard deviation of the means was used.
The standard deviation of the means is a standard deviation of mean cortisol levels. It is important to note in this study that
the standard deviations given within each group are not normal standard deviations and are only being used to depict
variance among the mean cortisol levels being examined.

125

 

Mean
 CorMsol
 (nmol/L)
 

Average
 Mean
 CorMsol
 Levels
 in
 Wild
 and
 
CapMve
 Tursiops
 truncatus
 
100
 
90
 
80
 
70
 
60
 
50
 
40
 
30
 
20
 
10
 
0
 

83
 

88.3
 

Wild
 
30.6
 

Wild
 

NH
 

NH
 
H
 

H
 


 

Figure 2: Mean cortisol levels in Atlantic bottlenose dolphins from three wild studies (N=105, n=105),
ten captive H studies (N=93, n=408), and six captive NH studies (N=79, n=79).

Cortisol Collection Time in Wild Atlantic Bottlenose Dolphins


 

Conflicting estimations of when cortisol levels begin to increase in

response to an acute stress response have been published (Ortiz & Worthy, 2000,
St. Aubin et al., 1996, Ortiz et al., 1991, Thomson & Geraci, 1986). Two studies
claim that cortisol levels do not rise in significant levels until an hour after chase
ensues. To evaluate these claims I compared specific results of those two studies
with all data from captive NH and H studies. These two studies conducted on wild
Atlantic bottlenose dolphins contest the claims that sample collection time of <1
hour does not reflect elevated cortisol levels (Ortiz & Worthy, 2000, Aubin et al.,
1996). Ortiz and Worthy (2000) sampled wild Atlantic bottlenose dolphins for
cortisol levels within or below 36 minutes of chase and greater than 36 minutes
126

 

after chase ensues. The mean cortisol levels between those two groups did not
differ by much. In St. Aubin et al., 1996, wild Atlantic bottlenose dolphins were
sampled within an hour of chase ensuing and displayed similar results to both
groups sampled in Ortiz and Worthy (2000). Both of these studies concluded that
cortisol levels are not greatly impacted and little acute stress is shown when
animals are sampled within an hour of the chase (St. Aubin et al., 1996, Ortiz and
Worthy, 2000). When these results are compared to captive studies on Atlantic
bottlenose dolphins they reflect similar values to cortisol samples that were
obtained from six studies using non-husbandry sampling and display greatly
elevated values when compared to cortisol samples of animals from ten studies
that utilized captive husbandry collection methods (Figure 3).

Mean
 CorMsol
 (nmol/L)
 

Mean
 CorMsol
 Levels
 in
 Tursiops
 truncatus
 based
 
on
 Sampling
 Methodology
 and
 Time
 
100
 
90
 
80
 
70
 
60
 
50
 
40
 
30
 
20
 
10
 
0
 

80
 

88.6
 
74.5
 

71.7
 

74.5
 

31
 

<36
 min
 
wild
 

>36
 min
 
wild
 

<60
 min
 
wild
 

>60
 min
 
wild
 total
 

NH
 

H
 


 

Figure 3: The columns referencing 36 minutes is data from Ortiz & Worthy (2000), the columns
referencing 60 minutes is data from St. Aubin et al. (1996), the NH columns is based on six studies, and
the H column represents ten studies.

127

 

Killer Whale12

Mean fecal glucocorticoid (FGC) levels derived from one study (Ayers et
al., 2012) that collected 154 samples from Southern Resident Killer Whales
displayed a mean FGC level of 1008.27 ng/g. Mean cortisol levels collected from
nine captive killer whales sampled via voluntary husbandry practices from three
studies is 7.66±1.75 nmol/L (0.28±0.06 µg/dl). FGC’s are measurements of fecal
metabolites rather than measurements of the parent hormone cortisol, thus, direct
comparisons cannot be made between mean levels of FGC’s and mean cortisol
levels, which is the reason these levels are not depicted in this study. It is possible
for trends in FGC’s to be compared to trends in cortisol levels to depict stress.
Mean cortisol levels derived from four studies that employed husbandry
sampling depict relatively similar results (Figure 4). In three of the four studies in
this analysis, one or all of the animals being sampled were pregnant although that
variable does not seem to effect the small variation in mean cortisol levels
analyzed.


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12


 Analysis
 was
 only
 performed
 on
 captive
 killer
 whale
 mean
 cortisol
 levels
 because
 the
 wild
 study
 measures
 FGC
 
metabolites
 not
 cortisol
 so
 the
 numbers
 cannot
 be
 directly
 compared.
 

128

 

Mean
 CorMsol
 Levels
 in
 CapMve
 Orcinus
 orca
 

Mean
 CorMsol
 (nmol/L)
 

12
 
9.66
 

10
 

8.28
 
7.17
 

8
 
6
 

5.52
 

Husbandry
 

4
 
2
 
0
 
1998
 

2003
 

2005
 

2005
 


 

Figure 4: All three cortisol levels of captive killer whales were including (N=10, n=325), Lyamin et al.
(2005) conducted two separate analyses.

Beluga Whale

Mean cortisol levels are slightly higher in beluga whales that were
sampled in the wild (range: 90-125.1 nmol/L) when compared to captive belugas
that were sampled utilizing non-husbandry methodology (range: 82.6-117.5
nmol/L). Mean cortisol levels from captive belugas as a whole (husbandry and
non-husbandry sampling) was nearly half that, and mean cortisol levels obtained
from animals sampled via husbandry techniques were the lowest (range: 27.6-49.7
nmol/L).
Mean cortisol levels from studies on wild and captive beluga whales that
were sampled utilizing non-husbandry methodology were strikingly similar

129

 

(range: 82.6-125.1 nmol/L). Captive animals sampled using husbandry
methodology were much lower (range 27.6-49.8).
Mean cortisol levels in wild beluga whales and captive NH were
consistently higher than cortisol samples from captive H animals (Figure 5). Mean
cortisol levels derived from four studies on wild beluga whales is 103.55±17.3
nmol/L (3.75±0.63µg/dl). Mean cortisol levels from two captive studies that
employ non-husbandry sampling is 100.1±24.713 (3.6±0.9). Mean cortisol levels
derived from two studies on captive beluga whales sampled through husbandry
methodology is 38.65±15.63 nmol/L (1.4±0.57 µg/dl).

Mean
 CorMsol
 Levels
 in
 Wild
 and
 CapMve
 
Delphinapterus
 leucas
 

Mean
 CorMsol
 (nmol/L)
 

140
 

125.1
 

117.5
 

110
 

120
 
100
  90
 

89.1
 

82.6
 

Wild
 

80
 

NH
 

49.7
 

60
 
40
 

27.6
 

H
 

20
 
0
 
1989
  1992
  2001
  2006
  1991
  2010
  2010
  2012
 


 

Figure 5: Mean cortisol levels from 4 studies on wild beluga whales (N=256, n=335), from two captive
H studies (N=10, n=131), two captive NH studies14 (N=13, n=33)


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
13


 One study, Spoon & Romano, 2010, the mean cortisol levels was estimated by averaging cortisol levels from a figure in
the publication.
 
14


 Another aspect of Schmitt et al.’s (2010) study was included in this graph, to display an attempt to provoke stress in a
captive beluga by taken the sample during an out of water examination.

 

130

 

Mean cortisol levels are significantly higher in the wild beluga whales that
were sampled when compared to the captive studies that employ husbandry
sample collection methods. The mean cortisol levels are extremely similar when
comparing the studies on wild belugas to the studies on captive belugas that were
sampled via non-husbandry practices. Mean cortisol levels were also much
higher in captive animals that were sampled via non-husbandry methodology
when compared to animals sampled utilizing husbandry methodology (Figure 6).

Average
 Mean
 CorMsol
 Levels
 (nmol/L)
 

Average
 Mean
 CorMsol
 Levels
 in
 Wild
 and
 
CapMve
 Delphinapterus
 leucas
 
120
 
100
 
80
 
60
 
40
 
20
 
0
 

103.55
 

100.1
 

38.7
 

Wild
 

NH
 

H*
 

*one mean cortisol level was estimated by evaluating figure in
publication

 


 
Figure 6: Mean cortisol levels from four studies on wild and three studies on captive beluga whales
were included (N=276, n=482).

Wild vs. Captive Mean Cortisol Levels Among Species

In all three species, mean cortisol levels obtained from wild studies are


 

higher than cortisol levels obtained in captive studies as whole (Figure 715). Wild

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
15


 Killer whales are data deficient in wild studies
 

131

 

sampled beluga whales display the highest mean cortisol levels among wild
studies (range: 90-125 nmol/L) and captive beluga whales display the highest
mean cortisol levels among the captives species studied (range: 27.6-82.6
nmol/L). Captive killer whales sampled for cortisol concentrations displayed the
lowest mean cortisol levels of all wild and captive species sampled (5.5-9.7
nmol/L).

Mean
 CorMsol
 (nmol/L)
 

Average
 Mean
 CorMsol
 Levels
 in
 3
 Species
 of
 
Wild
 and
 CapMve
 Odontocete
 
120
 

103.55
 

100
 

100.1
 
83
 

88.3
 

80
 
60
 

38.65
 

40
 

30.6
 
7.66
 

20
 
0
 
wild
 Dl
 

NH
 Dl
 

H
 Dl
 

wild
 Tt
 

NH
 Tt
 

H
 Tt
 

H
 Oo
 

Species/Sampling
 

Figure 7: All 30 studies were included (N=563, n=1399)


 

Among captive beluga whales, Atlantic bottlenose dolphins, and killer

whales sampled in previous studies that included both husbandry and nonhusbandry sampling methodology beluga whales displayed the highest mean
cortisol levels (range: 27.6-82.6 nmol/L), followed closely by Atlantic bottlenose
dolphins (range: 10.5-160 nmol/L). Mean cortisol levels from captive killer
whales were much lower than both other species, but this may be due to all
studies that collected cortisol levels in captive killer whales only practiced
husbandry sampling methodology (range: 5.5-9.7 nmol/L).
132

 

Wild and Captive Atlantic Bottlenose Dolphin and Beluga Whale

Mean cortisol levels derived from four studies conducted on 187 wild
beluga whales is 103.55±17.3 nmol/L (3.75±0.63µg/dl). The mean cortisol level
derived from three studies on 105 wild Atlantic bottlenose dolphins is 83±15.0
nmol/L (3.0±0.54 µg/dl). Results from comparing mean cortisol levels from
studies on wild beluga whales to studies on wild Atlantic bottlenose dolphins are
similar. Similar mean cortisol levels were also observed when comparing all
captive studies on each species, including those utilizing husbandry practices, and
those utilizing non-husbandry practices.

Captive Killer Whale and Atlantic Bottlenose Dolphin


 

Mean cortisol levels in three studies on 10 captive killer whales that were

sampled utilizing husbandry methodology were compared to ten studies on 93
captive Atlantic bottlenose dolphins where husbandry practices were also utilized
for sample collection. The results displayed higher cortisol levels in captive
Atlantic bottlenose dolphins than in captive killer whales when husbandry
sampling was employed.

Captive Beluga Whale and Killer Whale

 

Three studies that assessed mean cortisol level in 10 captive killer whales
via voluntary husbandry sampling methodology were compared to two studies
that assessed mean cortisol levels in 10 captive beluga whales that were sampled
133

 

utilizing voluntary husbandry practices. Mean cortisol levels were significantly
higher in the captive belugas.

Trends in mean Cortisol Levels Throughout Time in Captivity

Assessing trends in mean cortisol levels throughout time in captive
Atlantic bottlenose dolphins may help determine if evolved captive care
contributes to decreased exposure to stress in these animals. Studies have
analyzed cortisol levels in captive Atlantic bottlenose dolphins since 1970 and are
ongoing. Captive care has evolved immensely in the past 42 years due to a better
understanding of cetacean needs. Analyzing mean cortisol levels throughout time
in captivity will display if the captive animals are currently experiencing
statistically significant decreases in stress and may assist in developing more
effective animal husbandry and care.

Atlantic Bottlenose Dolphins

Many of the early studies on cortisol levels of this species utilized nonhusbandry sampling techniques. All published studies prior to 1996 utilized NH
sampling, whereas only one study after 1996 employed NH sampling. Visually, a
slight declining trend may look apparent, but due to the possible impacts of
sampling methodology a definitive trend cannot be conclusively assessed due to
less stress in the environment over time (Figure 8).

134

 

180
 
Mean
 CorMsol
 (nmol/L)
 

Mean
 CorMsol
 Levels
 in
 CapMve
 Tursiops
 truncaus
 from
 
1970-­‐2012
 
160
 
140
 
120
 
100
 
80
 
60
 
40
 
20
 
0
 
1960
 

1970
 

1980
 

1990
 

2000
 

2010
 

2020
 

Non-­‐Husbandry:
 Husbandry
 


 
Figure 8: Mean cortisol levels in captive Atlantic bottlenose dolphins from 1970-2012. All 17 studies on
captive animals were included (N=172, n=487). Some studies conducted multiple analyses or supplied
supplemental data16 that account for the extra points.

In this comparison between mean cortisol levels obtained from studies
conducted from 1996-2012 a trend line was undetectable. Studies that employed
non-husbandry sampling methodology were excluded from the trend map (Figure
9).


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
16


 Blasio et al., 2012, Reirdson & McBain et al., 1999, Suzuki et al., 1998
 

135

 

Trends
 in
 Mean
 CorMsol
 Levels
 in
 CapMve
 
Tursiops
 truncatus
 
Mean
 CorMsol
 (nmol/L)
 

80
 
70
 
60
 
50
 
40
 
30
 
20
 
10
 
0
 
1994
  1996
  1998
  2000
  2002
  2004
  2006
  2008
  2010
  2012
  2014
 

Figure 9: Mean cortisol levels collected in 10 studies on captive Atlantic bottlenose dolphins that
utilized H sampling17 (N=91, n=406). Some studies conducted multiple analyses or supplied
supplemental data that accounts for the extra points18. The two figures in 1999 seem like outliers in the
data set19.


 


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
17


 Ortiz et al. (2010) was not included because the cortisol levels were undetectable
 

 Blasio et al., 2012, Reirdson & McBain et al., 1999, Suzuki et al., 1998
 
19

 The
 authors,
 Reirdson
 &
 McBain
 were
 contacted
 via
 email
 and
 assured
 me
 they
 followed
 the
 published
 procedures
 but
 
18

those
 two
 points
 seem
 anomalous
 enough
 to
 take
 into
 consideration
 when
 assessing
 this
 figure.
 

136

 

CHAPTER 5
DISCUSSION

Analysis

This systematic review displays that sample methodology is a consistent
confounding variable in every research questions that was asked. Because of the
unanticipated impact that sampling methodology had on results it would be
inappropriate to portray any definitive results. Nevertheless, some interesting
trends were observed within each research question. The lack of definitive
answers underscores the need to design and implement a protocol that would
decrease the impact of sampling methodology when collecting samples for
cortisol analysis.
Despite the problems with sampling methodology, I can make some
preliminary conclusions about my four hypotheses based on this systematic
review. First, comparison of mean cortisol levels between wild and captive
members of the same species provided insight on whether sampling methodology
affects the results and demonstrated the importance of implementing new
methodology for future studies. Second, comparison of mean cortisol levels from
studies on wild Atlantic bottlenose dolphins that were sampled in under one hour
to captive studies, assisted in displaying that due to invasive sampling
methodology the acute stress response may be activated upon chase initiation and
137

 

display elevated levels of cortisol when sampled in <60 minutes. Third,
comparison of mean cortisol levels between species assessed if a species may
respond to a similar stressor (capture or captivity) in varying degrees. Fourth,
analyzing the trends in mean cortisol levels of captive Atlantic bottlenose
dolphins assisted in determining if the animals stress levels have declined in a
captive environment as captive care has improved due to heightened knowledge
of cetacean needs.

Wild vs. Captive Mean Cortisol Levels Within Species

My first research hypothesis was that captive members of the same species
would exhibit higher levels of cortisol than their wild counterparts due to the
constant presence of anthropogenic stressors. The results actually displayed
elevated cortisol in wild species. These results are significant because invasive
sampling methodology conducted on the wild cetaceans utilized for this study
may be the cause of raised cortisol levels. The invasive nature employed to obtain
the sample for cortisol analysis may have increased cortisol before and during the
collection process. These results suggest the important of implementing
noninvasive sampling methodology to accurately assess resting cortisol levels in
wild cetaceans during future studies. Because sampling methodology is such a
pronounced confounding variable it would be inaccurate to conclude that captive
cetaceans have a lower resting cortisol level than wild cetaceans or to assume that
captive cetaceans are “less stressed” than their wild counterparts.

138

 

Atlantic Bottlenose Dolphin

The mean cortisol levels were similar in wild studies conducted on
Atlantic bottlenose dolphin and in samples collected in captivity via nonhusbandry methods. The similar mean cortisol levels throughout these studies
may indicate that the stress evoked from wild sample collection methods
involving chase, capture, restraint may be similar to the stress that arises from
involuntary captive sampling methodology, which includes moving the animals to
shallow water (Medway et al., 1970), out of water examinations (Thomson &
Geraci, 1986, Orlov et al., 1988;1991 ), and out of water examinations before or
during transport (Copland & Needham, 1999, Noda et al., 2006) even if the
animals have been exposed to this activity in the past. The mean cortisol levels
expressed during chase, capture, restraint of wild Atlantic bottlenose dolphin and
involuntary, non-husbandry practices in captive Atlantic bottlenose dolphins may
be exhibiting a mild-moderate stress response depending upon the individual
animal (St. Aubin et al., 1990).
Mean cortisol levels obtained from the studies conducted on wild Atlantic
bottlenose dolphins were on average higher than mean cortisol levels obtained
from the studies conducted on captive Atlantic bottlenose dolphins using both
husbandry and non-husbandry sampling methods. These results display the
activation of the stress response during chase, capture, and restraint and the
difficulty associated with trying to obtain baseline cortisol levels in wild Atlantic
139

 

bottlenose dolphins. An even greater difference in mean cortisol levels was found
between the mean cortisol levels collected in the studies on wild animals and the
mean cortisol levels collected from the animals in captive studies that only
employed husbandry sampling. This number may be the greatest display of
variation between mean cortisol levels that are collected when the HPA axis is
stimulated and when it may not be in Atlantic bottlenose dolphins. This aspect of
the study is focusing mainly on comparing mean cortisol levels derived from an
acute stressor, so that is not to say that the animals in captivity are not
experiencing a chronic stressor in some way. It is just depicting the difference in
mean cortisol levels in these animals when different sample collection methods
are employed.
The average of mean cortisol obtained from studies on captive Atlantic
bottlenose dolphins sampled by husbandry practices was significantly lower than
samples obtained from non-husbandry methodology. These results may display
that animals of the same species that are often involuntarily exposed to invasive
medical procedures may not get conditioned to those actions and still exhibit a
stress response when compared to members of the same species who voluntary
participate in sample collection. Thomson & Geraci (1986) noted that captive
Atlantic bottlenose dolphins that were often exposed to out of water examinations
still exhibited higher cortisol levels which are associated with a stress response
during these examinations.

140

 

Cortisol Collection Time in Wild Atlantic Bottlenose Dolphins

My second research hypothesis stated that based upon findings in previous
studies cortisol levels would be lower in cetaceans that were sampled within an
hour of chase initiation. The two studies, Ortiz & Worthy (2000) and St. Aubin et
al. (1996), that collected cortisol samples in wild Atlantic bottlenose dolphin
populations infer that because the samples were collected in under one hour from
chase initiation, the acute stress response was not activated to the point where
cortisol levels would noticeably increase. Because of that inference the studies
hypothesized that their samples may reflect near baseline cortisol levels in wild
Atlantic bottlenose dolphins. Ortiz and Worthy (2000) find no significant
differences in cortisol levels collected <36 minutes from chase initiation and
cortisol levels collected in samples >36-60 minutes from chase initiation (Figure
10).

Figure 10: From Ortiz & Worthy (2000), mean cortisol levels in wild Atlantic bottlenose dolphins
sampled within 36 or after 36 minutes from chase initiation.


 

141

 

St. Aubin et al. (1996) stated that when wild Atlantic bottlenose dolphins were
sampled within one hour of encirclement their cortisol levels were not statistically
significantly different to levels collected from “semi-domesticated” dolphins that
were sampled using husbandry practices, even though the only variable that
displayed statistical significance in variation of cortisol levels was status (wild or
semi-domesticated) (Figure 11).

Figure 11: From St. Aubin et al. (1996), mean cortisol levels in wild Atlantic bottlenose dolphins
sampled within an hour of capture.


 

Although both of these studies collected their cortisol samples within on
hour of chase initiation, the average mean cortisol levels were much higher than
they were in captive studies were husbandry methodology was employed. The
mean cortisol levels from these two wild studies were very similar to mean
cortisol levels obtained from captive animals sampled via non-husbandry
practices. These results suggest that wild chase, capture, restraint sample
collection methodology that occurs in under one hour and involuntary captive
sampling methodology elicit similar stress responses in Atlantic bottlenose
dolphins and the wild collection methods employed even when sample are
collected in under one hour do not reflect baseline cortisol levels in wild Atlantic
bottlenose dolphins.

142

 

Their hypothesis, which states that if sample collection occurs within an
hour after chase initiation the acute stress response is not active enough to display
significant increase in cortisol levels, is not supported by the data collected for
this analysis. When compared to all data available on mean cortisol levels of wild
and captive Atlantic bottlenose dolphins, the compiled data is in fact leaning
towards supporting the alternate hypothesis: that the acute stress response is
activated to a point where cortisol levels rise within the first 36 minutes of chase
initiation until sample collection. The preliminary results from analysis of the
data echoes the need for non-invasive wild sample collection methods if a cortisol
baseline in wild cetaceans is ever to be established.

Killer Whale

Data on mean serum/plasma cortisol levels in wild killer whales was
unavailable and to the best of my knowledge non-existent. One study on fecal
glucocorticoids assessed in free ranging killer whales (Ayers et al., 2012) is
referenced but cannot be compared to captive killer whale mean cortisol levels
due to differences in the hormones assessed. Fecal glucocorticoids analyze the
binding rate of cortisol metabolites and serum/plasma measures the binding rate
of the parent hormone, cortisol. Trends can be analyzed between the two
methodologies but for this analysis that protocol is not applicable. Killer whales
were still included in this analysis because of the data present in captive animals
that can be used to analyze comparisons between other species of odontocetes
kept in captivity.
143

 

Beluga Whale

The similar mean cortisol levels between studies of wild beluga whales
and the studies of captive beluga whales using non-husbandry practices to collect
samples suggests that wild sample collection methods involving chase, capture,
and restraint evoke HPA stimulation and cortisol production similar to the stress
that arises from involuntary captive sampling methodology, which includes
moving the animals to shallow water (Orlov et al., 1991) and out of water
examinations (Schmitt et al., 2010). The small variation between samples
obtained in captivity via husbandry and non-husbandry sampling methodology
could be due to the small sample sizes or a difficulty for these animals to adapt to
a captive environment. If a difficulty of adapting to an artificial environment
contributes to the higher levels of cortisol in husbandry sampled captive beluga
whales is in complete dissonance with Orlov et al.’s (1991) prediction that
because beluga whales are exposed to a greater numbers of climatic and
nutritional stressors they would display greater adaptation to stressful situations
than other cetaceans. In contrast, beluga whales display the highest mean cortisol
levels recorded in this review when sampled via husbandry protocol in captivity.
Averages of mean cortisol levels were much higher in the wild studies
when compared to captive studies that employed husbandry sampling. These
results display the difficulties in attempting to obtain baseline data in wild and
captive beluga whale populations due to invasive sampling methodology.
144

 

Baseline cortisol levels in wild beluga whales will not be established through
currently employed invasive serum/plasma collection methods. This information
also aides in challenging the few studies (St. Aubin et al., 1996, Ortiz & Worthy,
2010) that suggest that baseline cortisol samples are able to be obtained from wild
cetaceans if sampling is completed in under one hour, before cortisol levels are
thought to peak in cetaceans exposed to acute stressors (Thomson & Geraci, 1986,
St. Aubin et al., 1996)

Wild vs. Captive Mean Cortisol Levels Among Species

My third research hypothesis predicted that mean cortisol levels would
vary among the different species being studied. A prediction that larger cetaceans
that are seemingly less likely to adapt well to captivity would display lower levels
of mean cortisol was not observed in this review. No significant difference was
found between mean cortisol levels of any of the three species living in captivity
and wild when compared to each other20. My study suggests these three species of
odontocetes may have similar levels of circulating cortisol and adapt in a similar
manner to sample collection in the wild and captivity. Some studies have
suggested a negative correlation between size of animal and cortisol production,
but that was not apparent in this analysis.
Utilizing descriptive statistics such as mean cortisol levels and standard
deviation of the means to detect differences and similarities of mean cortisol

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
20


 Killer
 whales
 were
 not
 included
 because
 of
 lack
 of
 data.
 

145

 

levels between species of odonotocete may assist in assessing adaptation to
stressors in wild and captive members of these species. It is important in both
wild and captive environments to be aware of how sensitive to stressors cetaceans
are and by comparing mean cortisol levels between three species of odontocetes it
may be possible to assess if one species is more reactive to stressors than others,
or if one species may naturally produce higher of lower levels of cortisol than
another.
Mean cortisol levels obtained from wild studies are higher than cortisol
levels obtained in captive studies as a whole, in both beluga whales and Atlantic
bottlenose dolphins. (Killer whales are data deficient in wild studies). Wild
sampled beluga whales display the highest mean cortisol levels among wild
studies (range: 90-125 nmol/L) and captive beluga whales display the highest
mean cortisol levels among the captives species studied (range: 27.6-82.6
nmol/L). Captive killer whales sampled for cortisol concentrations displayed the
lowest mean cortisol levels of all wild and captive species sampled (5.5-9.7
nmol/L).

Wild and Captive Atlantic Bottlenose Dolphin and Beluga Whale

Although mean cortisol levels obtained from studies on wild beluga
whales were on average greater than levels in wild Atlantic bottlenose dolphins, a
146

 

difference is not detectable. These results suggest that chase, capture, restraint
methodology employed for blood serum/plasma collection activates the HPA axis
and increases cortisol levels in both beluga whales and Atlantic bottlenose
dolphins, two species of cetacean that are often researched in the wild and
captured for use in captive displays. Even though beluga whales seem to display
higher cortisol levels in almost all wild and captive studies a larger variation in
average mean cortisol levels is found in wild beluga whales and wild Atlantic
bottlenose dolphins. When captive members of these species are sampled the
variation decreases. The similarities in mean cortisol levels between captive
belugas that were sampled via husbandry practices and captive Atlantic bottlenose
dolphins that were sampled via husbandry practices, and captive belugas that
sampled by non-husbandry practices and captive Atlantic bottlenose dolphins that
were sampled by non-husbandry practices help display that both of these species
of captive cetacean produce similar levels of cortisol when kept in a captive
environment. Variation in cortisol production due to sampling methodology
reflects similar trends in changes among these species.

Captive Killer Whale and Atlantic Bottlenose Dolphin

Captive killer whales display significantly lower mean cortisol levels
when compared to captive Atlantic bottlenose dolphins that were sampled through
voluntary husbandry practices. One of the original hypotheses stated that larger
147

 

odontocetes may not adapt to captivity as well as smaller members of that family,
but mean cortisol levels obtained from all the animals included in captive
husbandry sampling were 7.66 nmol/L in killer whales and 30.6 nmol/L in
Atlantic bottlenose dolphins. This was the only aspect of species to species
comparisons that yielded significant results. The original hypothesis was still
rejected even though these results were yielded because the smaller species,
Atlantic bottlenose dolphin displayed higher mean cortisol levels than the larger,
killer whale species. It is important to note that both killer whales and Atlantic
bottlenose dolphins are in the same family, Delphinidae, and these results may
assist in conveying that cetacean species within the same family may either
produce different amounts of cortisol, react to stressful situations in different
manners, or display different abilities of adaptation to captivity. It is also
important to note that in 2/3 studies (Lyamin et al., 2005, Suzuki et al., 2003) 4/6
animals were pregnant which could influence mean cortisol levels.

Captive Beluga Whale and Killer Whale

Average mean cortisol levels were greater in captive beluga whales when
compared to captive killer whales when animals were sampled using husbandry
methodology, although the numbers were not significantly different. These results
may assist in corroborating Orlov et al.’s (1991) hypothesis which states that
cetaceans that are exposed to frequent climatic and dietary stressors may possess
an ability to adapt to other stressors, such as introduction to a captive
148

 

environment, better than other species. Killer whales and beluga whales are in
different families of odontocetes, killer whales a member of Delphinidae and
beluga whales a member of Monodontidae. Both species are larger than Atlantic
bottlenose dolphin and can inhabit polar seas, whereas Atlantic bottlenose
dolphins reside is tropic and subtropical climates (Perrin et al., 2002). The data
displays that the mean cortisol levels from nine killer whales were the lowest
(7.66 nmol/L) and mean cortisol levels from the ten beluga whales were the
highest (38.65 nmol/L). It is also important to note that in 2/3 studies on killer
whales (Lyamin et al., 2005; Suzuki et al., 1998) 4/6 animals were pregnant
which could influence mean cortisol levels.

Captive Killer Whales, Beluga Whales, and Atlantic Bottlenose
Dolphins

Captive killer whales tend to display lower mean cortisol levels when
sample collection involved voluntary husbandry practices when compared to
captive Atlantic bottlenose dolphins and captive beluga whales. A negative
correlation between body size and cortisol production has been proposed (Suzuki,
personal communication), but not conclusively confirmed. This correlation was
not observed in this review. If greater body mass does correlate with lower
cortisol levels, lower cortisol levels would be expected to be found in captive
beluga whales and higher cortisol levels in captive Atlantic bottlenose dolphins.
The opposite was observed in this review, although small sample sizes could have

149

 

skewed results. More research should be completed to analyze this possibility of a
correlation between cortisol production and body mass in cetaceans.
Orlov et al. (1991) proposed that animals that are exposed to frequent
climatic and dietary extremes might possess heightened adaptation ability to a
captive environment. This hypothesis may explain why killer whales show lower
levels of mean cortisol when sampled through husbandry methodology in a
captive environment, although one would assume that beluga whale would also
possess this trend, which they do not. Beluga whales displayed the highest levels
of mean cortisol when husbandry sampling methodology was employed (38.65
nmol/L), followed by Atlantic bottlenose dolphins (30.55 nmol/L), and killer
whales (7.66 nmol/L). Wild beluga whales also displayed the largest mean
cortisol levels (103.6 nmol/L) followed by Atlantic bottlenose dolphins (83
nmol/L). Studies on cortisol levels in wild killer whales are data deficient.
Studies that minimize confounding variables, especially stimulation of the HPA
axis during sample collection, must be implemented to more accurate assess
cortisol along with other stress hormone levels in wild and captive cetaceans
before accurate conclusions can be drawn.

Trends in Mean Cortisol Levels Throughout Time in Captivity

My fourth research hypothesis stated that a declining trend in cortisol
levels should be observed in captive Atlantic bottlenose dolphins through time.
All studies that obtained mean cortisol levels in captive Atlantic bottlenose
150

 

dolphins that were conducted before 1996 employed non-husbandry sampling
methodology. Most of these studies yield mean cortisol levels that are higher than
studies post-1996 that employed husbandry-sampling techniques. The range of
mean cortisol levels between 1970 and 1992 is 35-90.3 nmol/L. The range of
mean cortisol levels between 1996-2012 is 0-160 nmol/L. A positive or negative
trend in studies conducted on captive Atlantic bottlenose dolphins that analyzed
mean cortisol levels between 1996 and 2012 could not be detected. All studies
included in the analysis employed husbandry sampling methodology and
displayed a range of 15.5-69 nmol/L.
The lack of significant differences found in mean cortisol levels of captive
Atlantic bottlenose dolphins over a study time of 1970-2012 may help combat the
claim that captive marine mammal facilities have minimized stressors throughout
years by learning more about captive care and animal enrichment over time. All
of the studies up until 1996 employed non-husbandry sampling, which most likely
contributes to the elevated cortisol levels in those studies. When the studies that
utilized non-husbandry methodology were eliminated from the figure, a definitive
decreasing trend is not observed. Two points from the same study in 1999
(Reirdson & McBain) may be viewed as outliers, even though the author’s have
been contacted to verify sampling protocol. Their results were extremely high
when compared to all other studies on captive Atlatnic bottlenose dolphins
sampled via husbandry practices. The levels from their study actually visually
appear more similar to cortisol levels that were obtained via non-husbandry
sampling in other studies utilized in this review. If those two points are eliminated
151

 

from the figure the analysis looks even less persuasive. These results, or lack
thereof, should be used to display the need for non-invasive stress hormone
sampling methodology such as FGC analysis in captive situations used to develop
a system to minimize stress for captive cetaceans kept in a restricted and
dependent artificial situation.

Findings in Comparison with Other Studies

Thompson & Geraci (1986) found that cortisol concentration in dolphins
increased within 10 minutes of capture and peaked at about 90 minutes. Orlov et
al. (1988; 1991) noted that an increase of cortisol in bottlenose dolphins occurred
within 5 minutes of a stressor and a process of partial stabilization then recurrent
intensification occurred 6-12 hours later, with a peak around 30-45 minutes. St.
Aubin & Geraci (1989) noted that cortisol levels may increase, but not until after
30 minutes in their study on beluga whales. St. Aubin et al. (1996) and
Thompson and Geraci (1986) both noted that cortisol levels don’t elevate
significantly until 1-2 hours after exposure to a stressor. Differences in mean
cortisol levels in bottlenose dolphins typically range from 70-150 nmol/L in wild
animals sampled by capture/forced restraint, which are thought to be experiencing
a mild stress response (Thomson & Geraci, 1986; St. Aubin et al., 1989), 210-490
nmol/L in stranded animals, thought to be experience a high stress response, and
50 nmol/L in captive animals that are accustomed to handling, thought to be close

152

 

to baseline levels (Houser et al., 2011). Thompson & Geraci (1986) proposed that
30-40 nmol/L are baseline cortisol concentrations in bottlenose dolphins and those
numbers reflect baseline cortisol levels in dogs and rabbits (Thompson & Geraci,
1986).

Complications in using Cortisol as a Primary Indicator of Stress

Confounding variables and natural variation that are thought to effect
cortisol levels in cetaceans are age, sex, reproductive status, wild or captive status,
season, and time of day. The sample collection method and analysis procedure
can also cause variability in results. Although there are arguments for and against
the influence of these variables I have tried to minimize them in this study by
using only mean cortisol levels and studies that have used similar methodology
and analysis. Studies and aspects of studies that were attempting to artificially
activate acute stress in cetaceans were excluded from this analysis.
Orlov et al. (1991) found that bottlenose dolphins exhibited higher levels
of cortisol in the winter and spring than in summer and fall. The reason for
variability in cortisol levels within seasons may come from evolutionary
adaptations for feeding, with high levels of glucocorticoids having the ability to
inhibit feeding in animals anticipating energy declines (Houser et al., 2011).
Beluga whales that often face harsh climatic conditions in the Arctic have
displayed a seasonal trend as well. Orlov et al. (1991) discovered higher levels of
153

 

cortisol in 10 beluga whales captured off Russia in the spring and winter months.
Suzuki et al. (2003) found similar seasonality trends in captive male killer whales
exhibiting higher cortisol levels in winter than summer months. Concurrently, St.
Aubin et al. (1996) found that season did not have an effect on cortisol levels in
18 wild and 18 semi-domesticated bottlenose dolphins.
Age is thought to play a part in cortisol production in cetaceans. Suzuki
(unpublished data) has observed trends of lower cortisol levels in older animals.
In a study conducted by Ortiz et al. (2010) two captive male bottlenose dolphins
ages17 and 23, displayed undetectable levels of cortisol in each of their 16
samples. The hypothesis for these results is adrenal atrophy though it is unlikely
that age played a large role because the mean lifespan of bottlenose dolphins is
around 45 years (Perrin et al., 2002). St. Aubin et al. (2001) found no age related
differences in cortisol levels in 115 wild caught belugas.
Cortisol production in cetaceans has been found exhibit diurnal circadian
trends similar to humans. Studies have shown evidence of higher cortisol levels in
the morning with lowest levels in the evening in killer whales (Suzuki et al., 1998;
2003). Beluga whales were found to display consistently higher cortisol levels in
morning hours than evening hours (St. Aubin et al., 2001;Schmitt et al., 2010).
The lowest levels of cortisol how been displayed between noon and midnight in
beluga whales (St. Aubin & Ridgeway, unpublished data) and killer whales
(Suzuki et al., 1998).

154

 

Although cortisol levels are thought to differ by sex no statistically
significant results have been produced. St. Aubin et al. (1996) found no
difference in cortisol levels obtained from 36 samples of 18 wild and 18 semidomesticated dolphins. It may be plausible to attribute differences in cortisol
levels between males and females to social ranking. Killer whales for example,
exhibit a matriarchal society, Suzuki et al. (1998;2003) found that of the three
animals sampled (2M:1F) the smaller, less dominant male showed less variability
in cortisol production throughout the diurnal and seasonal study. More research
will have to be done to establish conclusive results.
Wild or captive status can play a large part in fluctuating cortisol levels in
cetaceans. As noted in the methodology section wild animals are often sampled
via chase, capture, and restrain methods that is likely to elicit a stress response
(Tryland et al, 2006, St. Aubin et al., 1996). Ortiz & Worthy (2000) found no
difference in mean cortisol concentration of wild bottlenose dolphins sampled
below 20 minutes to 40 minutes of capture. This could be due to the fact that
cortisol levels are thought to peak in cetaceans between 1-2 hours of introduction
to a stressor (Thomson & Geraci, 1986). St. Aubin et al. (1996) found no
differences in cortisol levels relating to age or sex in his study conducted on 18
wild and 18 semi-domesticated dolphins, but did find variance between the wild
and captive animals. The wild animals displayed higher cortisol levels, which
may be due to the methodology used to obtain the sample.
Thompson & Geraci (1986) found that even captive animals that were
constantly exposed to handling displayed higher levels of cortisol when being
155

 

handled. Noda et al. (2006) and Reirdson & McBain (1999) found that
transporting captive bottlenose dolphins causes elevated cortisol levels. Naka et
al. (2007) found that bottlenose dolphins increased their cortisol level when their
pools were being drained before sample collection. Spoon and Romano (2012)
found that in four beluga whales that were transported from one facility to another
displayed elevated levels of glucocorticoids that were statistically significant from
baseline samples taken before transport; arrival samples taken upon arrival to the
new facility; and acclimation samples taken 5-6 months after arrival at the new
facility. Orlov et al. (1991) did find that 10 wild caught beluga whales that
showed elevated cortisol levels in the first four days of being captive, stabilized
on the 11th day. In the same study he found that wild caught bottlenose dolphins
that were held in captivity for 7 days showed a peak in cortisol levels at 24 hours
that began to decrease from day 3-4, never reached the established “normal”
levels for the animals being held.
Due to the low values found in cetaceans along with the narrow range and
small fluctuations among stressors the ability for cortisol to be used as an absolute
value of stress in cetaceans may be unreliable (St. Aubin & Dierauf, 2001).
Studies on dolphins and beluga whales have displayed that the bound fraction of
cortisol that is normally measured in standard immunoassays is ≤50% of the total
hormone (St. Aubin & Geraci, unpublished data). The small amounts of bound
hormone may mean that greater quantities of free cortisol are circulating through
the body and able to cause greater effects on the animal (St. Aubin & Dierauf,
2001). The measurement of cortisol levels along with other stress indicative
156

 

blood parameters such as aldosterone levels, and lymphocyte and eosinophil
counts, and behavioral observations may be the best method to clearly evaluate
stress in cetaceans (St. Aubin & Dierauf, 2001).
Although cortisol is still used to gauge the effects of acute stressors on
cetaceans it may be a more reliable gauge of chronic stress. A hypothesis exists
that questions the ability of the cetacean adrenal gland to only store and produce
small amounts of cortisol within a limited timeframe (Schmitt et al., 2010). If that
hypothesis is correct it may not be possible to use cortisol levels to reliably
measure acute stress. Fecal glucocorticoid measurements may be the most
accurate way to monitor chronic stress in cetaceans due to the noninvasive
sampling methodology and less variable fluctuations associated with them
(Washburn, 2004). The importance of shifting to noninvasive sampling methods
for stress hormone analysis in cetaceans should not be overlooked. Stress can be
induced by human presence and increase with invasive methodology (Ortiz &
Worthy, 2000; Tryland et al., 2006; St. Aubin et al., 1989). Since cortisol levels in
cetaceans may begin to rise within 10 minutes of perceived stressor (Orlov et al.,
1988; Thompson & Geraci 1986), results for baseline cortisol levels may become
skewed. Baseline cortisol levels are important to be able to analyze
environmental and anthropogenic impacts on the stress of wild and captive
cetaceans. The shift towards FGC measurement and standard immunoassay
analysis would allow for the creation of a collaborative database where
comparisons could accurately be made and a better understanding of how

157

 

numerous stressors impact the health and prosperity of numerous cetaceans
species throughout the onset of global climate change.

Complications in Methodology

When analyzing data on stress hormones in cetaceans it is important to
note many confounding variables and try to reduce them as much as possible. The
individual stress reaction of each animal will vary (Orlov et al., 1988; Esch et al.,
2009; Houser et al., 2011), seasonal hormone fluctuations have been noted in
several studies (Orlov et al, 1991; 1988; Suzuki et al., 1998; 2003), and the
methodology of sample collection has the ability to alter results (Tryland et al.,
2006; Schmitt et al., 2010; St. Aubin & Geraci, 1989). An ongoing disagreement
about when cortisol levels begin to increase in response to an acute stress
response in cetaceans is prevalent in the literature. Cortisol levels in cetaceans are
thought to increase from within 5 minutes of a perceived stressor according to
some researchers (Orlov at al., 1988), not until 35 minutes after a stressor is
encountered (Thomson & Geraci, 1986, St. Aubin & Geraci, 1989), up to an hour
after chase initiation (St. Aubin et al. 1996). This implication has the ability to
affect all serum and plasma cortisol samples obtained from wild cetaceans
whenever beginning of chase to sampling time is greater than five minutes. All
wild studies utilized in this systematic review list chase to sample collection time
as >10 minutes (St Aubin & Geraci 1989;1992: St. Aubin et al., 2001; Tryland et

158

 

al., 2006; Ortiz & Worthy, 2000; St. Aubin et al., 1996; Thomson & Geraci,
1986). It has been noted that mean cortisol concentrations in wild captured
beluga whales correspond to mean cortisol concentrations of captive beluga
whales placed in a stretcher for blood sampling, an act that is categorized as a
“moderate” stressor (Schmitt et al., 2010). Thomson & Geraci (1986) found that
mean serum cortisol levels in wild dolphins were similar to levels obtained in
captive dolphins following a simulated calm- and chase capture.
It is important to monitor stress hormones of cetaceans in the wild. The
knowledge gained from such studies could lead to better management practices
and conservation efforts (Ortiz & Worthy, 2000). Although, it is thought that
some of the current methodology employed could skew results due to activation
of the HPA axis prior to and during sample collection, noninvasive stress
hormone methodology exists in the form of fecal glucocorticoid monitoring.
Although several populations of wild dolphins are continuously monitored via
chase, capture, release methodology, including the Sarasota resident population
monitored by the Sarasota Dolphin Research Project out of MOTE Marine
Laboratories, studies have shown that even captive dolphins that are consistently
exposed to the medical practice of being taken out of their tank for evaluation still
display signs of HPA axis activation in the form of elevated cortisol (Thomson &
Geraci, 1986). If the HPA axis is producing glucocorticoids in response chase,
capture, release methodology it may be possible that baseline cortisol levels are
not plausibly obtained from this practice (Mancia et al., 2008). Several studies
advise that the sample collection methodology may cause an increase in cortisol
159

 

levels prior to sample collection (Tryland et al., 2009; Orlov et al., 1988; St.
Aubin & Geraci, 1989; Schmitt et al., 2010).
Some studies indicate that if sample collection occurs within 40 minutes
of chase the sample does not indicate a physiological stress response (Ortiz &
Worthy, 2000; St. Aubin et al., 1996). In my analysis of 12 captive studies on
cortisol levels using similar plasma/serum collection obtained during routine
husbandry behavior and analyzed by comparable immunoassays on Atlantic
bottlenose dolphin (Tursiops truncatus), mean cortisol levels are significantly
higher in the two wild studies. The mean cortisol level and standard deviation in
83 individual animals obtained from 186 samples was 75.5±21.54 nmol/L
(5.9±6.5 µg/dl) (St. Abuin et al., 1996; Suzuki et al., 1998; Reidarson & McBain,
1999; Reidgeway et al., 2006; Pedernera-Romano et al., 2012; Naka et al., 2007,
Ridgeway et al., 2009; Houser et al., 2011, Balsio et al., 2012; Suzuki & Komaba,
2012), while the mean plasma/serum cortisol levels obtained from a total of 67
wild bottlenose dolphins from Ortiz & Worthy (2000) and St. Aubin et al. (1996)
was 74.5±4.0 (2.7±0.1 µg/dl). These wild sampling methods may have the ability
to physiologically impact the animals utilized for these studies (Mancia et al.,
2008). We are unsure of the degree to which these animals could be impacted in
their daily routines post-sampling. In captive studies where animals were exposed
to an acute stressor cortisol levels have retuned to baseline levels within 24 hours;
in studies where wild animals were brought into a captive environment it has
taken up to 11 days (Orlov et al., 1991). Field studies conducted on wild
cetaceans most likely do not reflect resting cortisol levels (Schmitt et al., 2010),
160

 

which makes comparisons of these results difficult. Although sampling stress
hormone sampling of wild cetaceans is important to monitor, especially in
threatened or endangered populations, it is important to continue to attempt to
determine the effects of capture on the wild animals after sampling occurs (Ortiz
& Worthy, 2000).
Captive animals are exposed to numerous stressors that differ from wild
animals but have the same ability to lower immune system function (Noda et al.,
2006). Stress levels in captive animals can originate during the chase and capture
process, which has been shown by wild chase, capture, and release studies (St.
Aubin & Geraci, 1989;1992, St. Aubin et al., 2001). It may continue through the
transportation process, where cortisol levels have been shown to elevate (Noda et
al, 2006; Copland & Needham, 1992), and into the introduction to new
environmental and social parameters, where belugas that were transported from
one facility to another showed increased cortisol levels until six months after
introduction to the new facility (Spoon & Romano, 2012). The decreased immune
function resulting from increased cortisol levels has the ability to lower several
types of white blood cells and increase the risk of infection (Noda et al., 2006;
Thomson & Geraci, 1986; St. Aubin & Dierauf, 2001). Stress hormone
monitoring is important because elevations in cortisol levels are a precursor to
decreased levels of lymphocytes and esponophils which can lead to infection (St.
Aubin & Dierauf, 2001), which is one of the primary causes of death in captive
dolphins kept in aquariums (Medway, 1980).

161

 

In captivity sampling methodology differs greatly from wild sample
collection. Often animals are trained to perform a husbandry technique such as
displaying their fluke for blood sample collection. Some animals that are newly
captive, young, or disobedient may have to be lifted from their enclosure for
blood sampling to occur. The act of lifting the animal from their tank for sample
collection often excites the HPA axis and increases glucocorticoid production
(Schmitt et al., 2010; Thomson & Geraci, 1986). The increased cortisol levels
associated with HPA axis activation when animals are not conditioned to
husbandry sampling practices may represent a mild to moderate stress response
(Schmitt et al., 2010). The data that most likely represents true baseline cortisol
levels in captive animals is sampled from long-term captive residents that have
been trained to present the sample collection site during husbandry practices with
seasonal and diurnal trends noted (Suzuki et al., 2003).
In captivity serum constituents including cortisol levels may be influenced
by several environmental and social factors such as diet, forced human
interaction, social conflict, human dictated movements, or illness (Waples &
Gales, 2002).

Adaptation is an important factor to evaluate in captive cetaceans

(Orlov et al., 1988). If the animals cannot adapt to restricted living space, changes
in character of nutrition, along with constant human interaction and dependence
they may be subject to chronic stress (Orlov et al., 1988; Blasio et al., 2012).
Captive studies also are limited by their low sample sizes especially for captive
killer whales and beluga whales (Schmitt et al., 2012). Even in Atlantic
bottlenose dolphins, which are relatively prevalent in captive facilities with
162

 

numbers estimated as over 3,000 worldwide, it is often difficult to coordinate
studies between different facilities (Schmitt et al., 2010). It is known that stress in
animals causes behavioral changes, which could lead to mental and physical
health issues (Waples & Gales, 2002). To implement a continuous stress
hormone monitoring program in captive environments where animals are
susceptible to infection and disease, it is crucial to have alternative, non invasive
procedures to establish baselines and detect fluctuations in cortisol levels which
could be used to monitor immune system health (Pedernera-Romano et al., 2006).
Fecal glucocorticoid monitoring has the ability to be implemented in both wild
and captive environments so baseline cortisol levels could be logged in a database
and made available to all scientists and animal caretakers.

Complications in Immunoassay Analysis


 

Although all of the RIA’s, CLIA’s, EIA’s, and TR-FIA’s aside from the

Porter-Sibler chromogens tests have be confirmed to display comparable levels of
cortisol in cetaceans, it is important to be aware of the differences in detection of
the machine, antibody used, and possible contamination during processing. RIA’s
have been the most commonly used assay to measure free cortisol levels in
cetaceans a shift to the other assays is taking place because of the risk and hassle
of working with and disposing radioactive isotopes. CLIA’s and EIA’s are being
employed more frequently than RIA’s currently due to their high sensitivity and
the ease of operation. Liquid chromatography-tandem mass spectrometry (LC163

 

MS/MS) is starting to be used in cortisol analysis because of its highly sensitive
and specific tests developed for cortisol measurement, but the high cost and laborintensive process is not widely utilized at the moment (Babic et al, 2011).
When choosing and comparing the appropriate assay to measure cortisol
levels it is important to perform parallelism tests between standard and serum
samples along with tests on intra- and inter-assay coefficients of variation and
spike recovery tests. It is also important to note the assay sensitivity to cortisol.
Many studies lack this important detail in their publications, which can be an
important factor when some samples are listed as an amount of cortisol that is not
detectable. You will also have to determine if the cortisol levels in the matrices
you are examining are comparable. Serum and plasma samples are directly
comparable to each other, saliva cortisol levels are 27% of serum/plasma levels in
bottlenose dolphins (Pedernera-Romano, 2006), which is similar to the
saliva:serum/plasma cortisol ratio in humans. Doing matched tests on the same
animals allows comparisons to be made.
Fecal cortisol levels are not directly comparable to serum/plasma and
saliva samples because cortisol metabolites are metabolized in the liver before
they can be measured in the samples (Wasser et al., 2000). The most accurate way
to compare fecal cortisol levels to cortisol levels collected in other matrices is by
comparing trends (Wasser, personal communication). Fecal glucocorticoid
metabolites (FGM) may be a more accurate way to assess effects of stress that are
accumulated over time and can be analyzed by current commercially available
RIA kits. The unique ability to assess the effects of stress over time is unable to
164

 

be assessed in single blood samples that along with being invasive also are not
able to be used in a constant manner to assess the cumulative effects of stress in
animals (Wasser et al., 2000). When measuring FGM one must take into account
the lag time for the species being sampled. In cetaceans the lag time is ~24 hours
(Ayres, personal communication). The nature of collecting FGM in both wild and
captive environments would be less likely to elicit a stress response than chase
capture and even husbandry methods. In the wild animals can be trailed by up to
¾ of a mile and samples can be collected from the surface (Ayres et al., 2012) or
in tropical and subtropical climates inversion collection can occur below the
waters surface (Parsons et al., 2003) A transition to non-invasive FGM method for
analyzing cortisol levels in cetaceans living both in captivity and the wild may
provide the best baseline for normal resting cortisol levels in these animals living
in both a wild and captive environment. A long-term daily monitoring program of
captive cetaceans could be implemented due to non-invasive methodology
(collecting feces from the bottom of the tank or from the filter) and could lead to
better care and health monitoring.

165

 

CHAPTER 6
CONCLUSION

 

Conclusions Drawn from Results

Although it wasn’t an anticipated conclusion of this analysis, sampling
methodology played a large role in elevation of mean cortisol level in wild and
captive Atlantic bottlenose dolphins, killer whales, and beluga whales. Mean
cortisol levels obtained from blood samples during wild studies that employed a
chase, capture, restraint protocol were strikingly similar to mean cortisol levels
obtained from involuntary non-husbandry blood sample collection in captive
animals. Significant differences were found between mean cortisol levels
obtained via husbandry and non-husbandry practices for captive Atlantic
bottlenose dolphins and beluga whales. Studies conducted on captive killer
whales only employed husbandry practices to obtain blood samples for cortisol
analysis so no comparison could be made between husbandry and non-husbandry
practices. Mean cortisol levels in the captive killer whales seem to be
significantly lower than mean cortisol levels in captive beluga whales and
Atlantic bottlenose dolphins that were sampled using similar methodology. These
results display the importance of taking sample collection methodology into
account when interpreting cortisol levels of wild and captive cetaceans when
sampled during chase, capture, restraint and involuntary non-husbandry practices,
such as raising the animal out of the water, placing the animal on a stretcher, or
moving the animal to shallow water to obtain a sample.

166

 

The implications of sampling methodology on cortisol levels in wild
sampled and non-husbandry sampled cetaceans may more clearly reflect a mildmoderate stress response than a baseline reading even if samples are collected
within an hour of the perceived stressors appearance. Ortiz & Worthy (2000) and
St. Aubin et al. (1996) concluded that if sample collection were secured within 30
minutes of the chase in wild Atlantic bottlenose dolphins would not display
elevated levels of cortisol indicating HPA axis activity and activation of the acute
stress response. If HPA activation was not displayed in wild studies where chasesample collection occurred within 30 minutes, it is thought the cortisol levels
obtained from those studies could reflect baseline cortisol levels of wild animals.
It is believed that the most accurate baseline cortisol levels in Atlantic bottlenose
dolphins have been obtained during voluntary husbandry sample collection in a
captive environment (Suzuki et al., 1998;2003). The results of this analysis found
that mean cortisol levels obtained from samples in Ortiz and Worthy (2000) and
St. Aubin et al. (1996) that were collected in within 60 minutes of chase were
significantly higher when compared to mean cortisol levels from studies that
employed husbandry sampling on captive Atlantic bottlenose dolphins. In fact,
mean cortisol levels were extremely similar when the levels obtained from Ortiz
and Worthy (2000) an St. Aubin et al. (1996) were compared to mean cortisol
levels obtained from Atlantic bottlenose dolphins that were sampled following
involuntary non-husbandry practices, such as removing the animal from its
enclosure or cornering the animal in shallow water.
The results of this analysis suggest that baseline cortisol levels in
cetaceans may not reflect accurate levels when obtained by current standard
sampling methodology, such as chase, capture, restraint methods in wild animals
(Lair et al., 2006, St. Aubin et al., 1996, Thomson & Geraci, 1986). It is also
167

 

unknown if cortisol samples obtained from voluntary husbandry sampling
methodology in captive cetaceans accurately reflect baseline cortisol levels
because many environmental and social factors may conflict or alter results when
naturally free-ranging animals are confined (Orlov et al., 1988). The acute stress
response in cetaceans has been documented in captive Atlantic bottlenose
dolphins and beluga whales in various studies (Orlov et al., 1991, Schmitt et al.,
2010, Thompson & Geraci, 1986), but less in known about the chronic stress
response, which has the greater likelihood of causing physical and mental
impairments (St. Aubin & Dierauf, 2001).

Recommendations for Further Research

When the Marine Mammal Protection act was amended in 1997 a priority
was given to establish methodology that would be able to evaluate stress in
dolphins (Esch et al., 2009) via The International Dolphin Conservation Program
Act (IDCP Act, U.S. Pubic Law 105-42). The priority status of stress evaluation
in cetaceans is due to the increasing amount of anthropogenic and environmental
stressors introduced into the marine environment. Increased cetacean mortality
recently observed coupled with mass stranding events causes concern over the
general health of cetaceans, and the impact that other factors such as pollution and
disease have on overall stress levels (Esch et al., 2009). Wild collection of
animals for captivity has caused entire populations of some cetacean species to
become endangered. Keeping cetaceans in captivity may also impact the stress
response due to their social nature, intelligence, diverse diets, and large natural
168

 

ranges (Southern Resident Killer Whale Recovery Plan, 2006). In many animal
species exposure to chronic stress can lead to reproductive problems,
immunosuppression and even death (St. Aubin & Dierauf, 2001). In the past,
stress hormone analysis has been used to evaluate changes in individual or
specific populations of animals to different stimuli (Schmitt et al., 2010) To
accurately measure stress in cetaceans non-invasive sample collections methods
may need to be implemented (Wasser et al., 2000). Scientists and researchers will
only be able to analysis the impacts of environmental and anthropogenic stressors
once resting values for healthy animals are established in both wild and captive
environments (Esch et al., 2009).
Studies documenting the stress response in cetaceans have been conducted
by measuring the presence of cortisol in response to stressors and synthetic
stimulants (Thomson and Geraci, 1986, Orlov et al., 1988;1991, St. Aubin &
Geraci 1989;1992, St. Aubin et al., 1996;2001;2013, Suzuki et al., 1998;2003,
Ortiz and Worthy, 2000, Spoon & Romano, 2010). Stress can be measured in
cetaceans by monitoring a variety of hormones and blood parameters other than
cortisol. Thyroid hormones (T3 and T4) can be measured to record nutritional
stress (Ayres et al., 2012, St. Aubin & Geraci, 1989). St. Aubin & Geraci (1989)
discovered that T3 levels were suppressed for a 10-week captive period and that
stimulation of ACTH further decreased the levels in beluga whales. Noda et al.
(2006) found that when bottlenose dolphins were exposed to an elongated
preparatory process (>3 hours) before being transported that the animals exhibited
a stress response that was more clearly indicated on the leucogram than
169

 

monitoring of cortisol levels. A typical stress leucogram in cetaceans displays a
decrease in lymphocytes and eosinophil’s. Non-invasive ways to measure stress in
cetaceans may be completed by behavioral analysis in both captivity and the wild,
although no past studies have been able to correlate cortisol with behavioral state
(St. Aubin and Dierauf, 2001). Inconsistencies in what is deemed a “stressed”
behavior state may impact proper analysis and cause ambiguity. Castellote and
Fossa (2006) discovered that acoustically monitoring captive belugas could be an
effective method of monitoring stress and adaptation in captive animals.
Stress hormone analysis, hematology, and behavioral observations have
the greatest ability to assess stress in cetaceans when used in tandem. The
amalgamation of interdisciplinary practices to create a more accurate core of
cetacean stress levels is extremely important. These methods can create a better
ability to assess environmental and anthropogenic impacts on wild and captive
cetaceans (Spoon and Romano, 2012) and in turn lead to conservation and captive
care techniques. Combining different methodologies of stress monitoring in
cetaceans and establishing a standard non-invasive method of sample collection
and cortisol analysis such as FGM, along with an accepted standard for analysis,
may allow for studies to be more accurate compared and the most accurate results
yielded. The construction of a FGC database on wild and captive cetacean
species would allow for accurate comparisons between species inhabiting
different environments and become the first tool to compile true baseline levels of
cortisol in these animals.

170

 

Appendices
Appendix A: Immunoassay Information Table. Will assist in understanding “Assay Information” tables utilized to detect
cortisol levels in Atlantic bottlenose dolphins, killer whales, and beluga whales.

Immunoassay
 

Abbreviation
 

Detection
 Method
 

Radioimmunoassay
 

RIA
 

Radio-­‐active
 

Enzyme
 immunoassay
 

EIA
 

Photometric
 

Enzyme-­‐linked
 immune
 
sorbent
 assay
 

ELISA
 

Photometric
 

Chemiluminscence
 
immunoassay
 

CLIA
 

Luminescence
 

Electrochemiluminescent
 
immunoassay
 

ECLIA
 

Luminescence
 

Immunochemiluminescence
 
assay
 

ICMA
 

Luminescence
 

Chemiluminescentenzyme
 
immunoassay
 

CLEIA
 

Luminescence
 

Time
 resolved
 flurometric
 
immunoassay
 

TR-­‐FIA
 


 
Flurometric
 

Appendix B: Atlantic Bottlenose Dolphin Cortisol Studies, Assay information. Author (s) of original study on cortisol
level, sample type, assay method, assay producer, location of where the assay was produced, analytical assay sensitivity in
µg/dl and nmol/L.

Author
 
Thomson
 &
 
Geraci,
 1986
 
St.
 Aubin
 et
 al.,
 
1996
 
Ortiz
 &
 Worthy,
 
2000
 
Medway
 et
 al.,
 
1970
 
Thomson
 &
 
Geraci,
 1986
 
Orlov
 et
 al.,
 
1988
 
Orlov
 et
 al.,
 
1991
 

Sampl
e
 type
 

Assay
 
Metho
d
 

Assay
 
System/Kit
 

serum
 

RIA
 

I
 ^125
 

Producer
 
New
 England
 
Nuclear
 

serum
 
plasm
a
 

RIA
 

Immulite
 

Intermedico*
 

Location
 
Boston,
 
Mass
 
Willowdale
,
 Ontario
 

Immulite
 
Chromogen
s
 Test
 

DPC*
 

plasma
 

RIA
 
Silber-­‐
Porter
 

serum
 

RIA
 

1
 ^125
 


 
New
 England
 
Nuclear
 

serum
 

RIA
 

Kort-­‐I-­‐H^3
 


 

serum
 

RIA
 

Kort,
 I
 ^125
 

IBOKH
 

Sensitivi
ty
 μg/dl
 

Sensitivit
y
 nmol/L
 

0.36
 

10
 

0.2
 

5.5
 

L.A.,
 Cali
 

0.2
 

5.5
 


 
Boston,
 
Mass
 
USSR
 or
 
France
 
Sorin,
 
France
 


 


 

0.36
 

10
 


 


 

0.36
 

10
 

171

 

Copland
 &
 
Needham,
 1992
 
St.
 Aubin
 et
 al.,
 
1996
 
Suzuki
 et
 al.,
 
1998
 

plasma
 

RIA
 

Amerlex
 

serum
 

RIA
 

serum
 

RIA
 

Immulite
 
FKA404
 
antibody
 

Reidarson
 &
 
McBain,
 1999
 

serum
 

ICLIA
 


 

Chiron
 
Diagnostics
 

Reidarson
 &
 
McBain,
 1999
 

serum
 

ICLIA
 

Noda
 et
 al.,
 2006
 
Ridgeway
 et
 al.,
 
2006
 
Pedernera-­‐
Romano
 et
 al.,
 
2006
 

plasma
 

ECLIA
 


 
rabbit
 
antibody
 

Chiron
 
Diagnostics
 
Roche
 
Diagnostics
 

New
 South
 
Wales,
 AUS
 
Willowdale,
 
Ontario
 
Tokyo,
 
Japan
 
East
 
Wapole,
 
Mass
 
East
 
Wapole,
 
Mass
 
Indianapoli
s,
 Indiana
 

serum
 

RIA
 

Immulite
 

DPC*
 

serum
 

RIA
 

I
 ^125,Cort
 
CT2
 

Naka
 et
 al.,
 2007
 
Ridgeway
 et
 al.,
 
2009
 

plasma
 

EIA
 

serum
 

Ortiz
 et
 al.,
 2010
 
Houser
 et
 al.,
 
2011
 
Blasio
 et
 al.,
 
2012
 
Blasio
 et
 al.,
 
2012
 
Suzuki
 &
 
Komaba,,
 2012
 
(UP)
 

Amersham
 Pty.
 
Ltd
 

0.4
 

11
 

0.2
 

5.5
 

0.01
 

0.28
 


 


 


 


 

0.04
 

1
 

L.A.,
 Cali
 

0.2
 

5.5
 

France
 
Ann
 Arbor,
 
USA
 

0.24
 

6.6
 


 

CIS
 
Biointernational
 
Caymen
 
Chemicals
 

0.0035
 

0.1
 

RIA
 

Immulite
 

DPC*
 

L.A.,
 Cali
 

0.2
 

5.5
 

plasma
 

RIA
 

0.2
 

5.5
 

RIA
 

DPC*
 
Seimens
 
Healthcare
 
Diagnostics
 

L.A.,
 Cali
 

serum
 

Deerfield,
 Ill
 

0.15
 

4.2
 

serum
 

RIA
 

Bio
 International
 

France
 

0.24
 

6.6
 

serum
 

RIA
 

Immulite
 
TKCO1
 
(Coat-­‐a-­‐
count)
 
I^125,Cort
 
CT2
 
I^125,Cort
 
CT2
 

Bio
 International
 

France
 

0.24
 

6.6
 

serum
 

TR-­‐FIA
 

DELFIA
 
system
 

PerkinElmer
 

Waltham,
 
Mass.
 

0.02
 

0.55
 

Intermedico*
 
Cosmo
 Bio
 

Appendix C: Killer Whale Cortisol Studies, Assay information. Author (s) of original study on cortisol level, sample type,
assay method, assay producer, location of where the assay was produced, analytical assay sensitivity in µg/dl and nmol/L.

Author
Ayres et al.,
2012
Suzuki et al.,
1998
Suzuki et al.,
2003
Lyamin et al.,
2005
Lyamin et al.,
2005

Sample
type
fecal GC
metabolites

Assay
Method

Serum

RIA

Serum

RIA

Assay
System/Kit
I 125
corticosterone
FKA404
antibody
FKA404
antibody

Plasma

RIA

I 125 cortisol

Plasma

RIA

I 125 cortisol

RIA

Producer
Biomedic
als
Cosmo
Bio
Cosmo
Bio
Biomedic
als
Biomedic
als

Location
Costa
Mesa, CA
Tokyo,
Japan
Tokyo,
Japan
Costa
Mesa, CA
Costa
Mesa, CA

Sensitivity
µg/dl

Sensitivity
nmol/L

0.8

22.1

0.01

0.3

0.01

0.3

0.17

4.7

0.17

4.7

Appendix D: Beluga Whale Cortisol Studies, Assay information. Author (s) of original study on cortisol level, sample type,
assay method, assay producer, location of where the assay was produced, analytical assay sensitivity in µg/dl and nmol/L.

Author
 

Sample
 
type
 

Assay
 
Metho
d
 

Assay
 
System/Kit
 

Producer
 

Location
 

Sensitivi
ty
 μg/dl
 

Sensitivit
y
 nmol/L
 

172

 

St.
 Aubin
 &
 
Geraci,
 1989
 
St.
 Aubin
 &
 
Geraci,
 1992
 
St.
 Aubin
 et
 al.,
 
2001
 
Tryland
 et
 al.,
 
2006
 
Orlov
 et
 al.,
 
1991
 
Schmitt
 et
 al.,
 
2010
 
Spoon
 &
 
Romano,
 2012*
 

Plasma
 

RIA
 

I^125
 

Plasma
 

RIA
 

Immulite
 

Plasma
 

RIA
 

Serum
 

CLEIA
 

Immulite
 
Immulite
 
1000
 System
 

serum*
 

RIA
 

serum/pl
asma
 

CLEIA
 

Kort,
 I^125,
 
 
IMMULITE
 
Coat-­‐A-­‐
Count
 

Serum
 

CLEIA
 

Immulite
 

New
 
England
 
Nuclear
 
Intermedic
o*
 
Intermedic
o*
 

Boston,
 MA
 
Willowdale,
 
Ontario
 
Willowdale,
 
Ontario
 

DPC*
 

L.A.,
 CA
 

IBOKH
 

Sorin,
 France
 

DPC*
 

L.A.,
 CA
 

0.2
 

5.5
 

Siemens
 

L.A.,
 CA
 

0.2
 

5.5
 

0.36
 

10
 

0.2
 

5.5
 

0.2
 

5.5
 

0.2
 

5.5
 


 


 

Appendix E: Confounding Variables Information Table, Abbreviation Key. Assistance in understanding the “Confounding
Variables” tables for Atlantic bottlenose dolphins, killer whales, and beluga whales.

Captive
 Environment
 

Abbreviation
 

Indoor
 enclosure
 

ID
 

Outdoor
 enclosure
 

OD
 

Origin
 

Abbreviation
 

Wild
 caught
 

WC
 

Captive
 born
 

CB
 

Appendix F: Atlantic Bottlenose Dolphin Cortisol Studies, Confounding Variables. Author (s) of original study on cortisol
levels, mean cortisol levels in nmol/L and µg/dl, season or month the study was conducted in (months in numeric form),
age of the animal (s) sampled, sex of the animal (s) sampled, if the captive animals were kept in an indoor (ID) or outdoor
(OD) enclosure, time it took for sample to be collected (in wild studies from initiation of chase until sample collection),
time spend in captivity, origin of captive animals (wild caught or captive born), time of day when samples were collected,
any significant notes listed in each study.

Author
 
Thomson
 &
 
Geraci,
 1986
 

Mean
 
F
 
nmol/
L
 

Mean
 
F
 
μg/dl
 

Season
 

100
 

3.6
 

10
 

Age
 


 
St.
 Aubin
 et
 al.,
 
1996
 

77.3
 

2.8
 

66.2
 

2.4
 


 


 

 


 
18
M:1
8F
 
M1
7:F
14
 
3M:
5F
 

35
 

1.3
 

June
 

4
 

F
 

71.7
 

2.6
 

year
 
round
 


 
Ortiz
 &
 Worthy,
 
2000
 
Medway
 et
 al.,
 
1970
 
Thomson
 &
 
Geraci,
 1986
 

Sex
 


 

ID
/O
D
 

Time
 
to
 
Sampl
e
 
up
 to
 5
 
hours
 


 

Time
 in
 
Captivity
 

Origin
 

AM:
 
PM
 

Notes
 


 


 


 


 


 


 


 


 


 

 


 


 


 

 


 
NH
 

3
 

WC
 

both
 

lifted
 

23-­‐260
 
mins
 

 
<20-­‐40
 
mins
 

 
ID
 
O
D
 


 
w/in
 
10
 
mins
 

173

 

Orlov
 et
 al.,
 1988
 
Orlov
 et
 al.,
 1991
 

Copland
 &
 
Needham,
 1992
 
St.
 Aubin
 et
 al.,
 
1996
 
Suzuki
 et
 al.,
 
1998
 
Reidarson
 &
 
McBain,
 1999
 
Reidarson
 &
 
McBain,
 1999
 
Noda
 et
 al.,
 2006
 
Ridgeway
 et
 al.,
 
2006
 
Pedernera-­‐
Romano
 et
 al.,
 
2006
 
Naka
 et
 al.,
 2007
 
Ridgeway
 et
 al.,
 
2009
 

90.34
 
90.3
 

3.3
 

year
 
round
 

3.3
 

year
 
round
 


 


 

 

90
 

3.3
 


 


 
6
 
mos-­‐
>10
 
yrs
 

52.4
 

1.9
 

year
 
round
 

4
 -­‐33
 

10.5
 

0.38
 

9-­‐12
 

63.5
 

2.3
 


 

69
 

2.5
 


 

Matu
re
 
12-­‐
18
 
10,
 
13
 

4M:
2F
 
18M:
18F
 

M:F
 

 
M
 


 
O
D
 
O
D
 
O
D
 
O
D
 

160
 

5.8
 


 

mature
 


 

14.3
 

0.52
 


 

21
 

M
 


 
19.7
 


 
0.7
 


 


 
4-­‐11
 


 

10.2
 

0.37
 


 

4-­‐22
 


 

44.1
 

1.6
 

3
 

26
 
17
 ,
 
23
 

F
 


 
O
D
 
O
D
 

M
 


 

39,
 
35
 

1M:
1F
 

Ortiz
 et
 al.,
 2010
 

u.d.
 

u.d.
 

summ
er
 

Houser
 et
 al.,
 
2011
 

15.49
 

0.56
 

4-­‐6
 

15.5
 

0.56
 


 


 
16.3
 


 
0.59
 


 

5-­‐
~20
 

 
5-­‐
~20
 

35.87
 

1.3
 


 


 

Blasio
 et
 al.,
 
2012
 

 
Blasio
 et
 al.,
 
2012
 
Suzuki
 &
 
Komaba,
 2012
 
(UP)
 

O
D
 
O
D
 


 
O
D
 


 


 


 


 

lifted
 


 


 


 


 

lifted
 


 

lifted
 and
 
diazapa
m
 

both
 


 

before
 
transp
ort
 


 


 

varied
 


 
WC
 
and
 
CB
 


 

15
 &
 11
 

WC
 

AM
 


 

life
 

CB
 

both
 


 
clinically
 
normal
 


 

life
 

CB
 

both
 


 

2-­‐3
 hours
 
after
 
 
drained
 

>5
 years
 

WC
 


 

raised
 to
 get
 
sample
 


 


 


 

both
 


 


 
1-­‐5
 years
 


 


 


 


 

both
 


 

 
3
 not
 
husbandr
y
 


 


 


 


 


 

both
 


 


 


 

WC
 


 

O
D
 


 

>18
 
years
 

WC
 

2M:
4F
 

O
D
 


 

5-­‐~20
 

5
 WC
 

2M:
2F
 


 
ID
 


 


 
5-­‐~20
 

2
 WC
 


 
750
-­‐10
 
A
 
9-­‐
10
 
AM
 
9-­‐
10
 
AM
 

F
 

O
D
 


 

>1
 

WC
 


 


 


 


 


 


 

Appendix G: Killer Whale Cortisol Studies, Confounding Variables. . Author (s) of original study on cortisol levels, mean
cortisol levels in nmol/L and µg/dl, season or month the study was conducted in (months in numeric form), age of the
animal (s) sampled, sex of the animal (s) sampled, if the captive animals were kept in an indoor (ID) or outdoor (OD)
enclosure, time it took for sample to be collected (in wild studies from initiation of chase until sample collection), time
spend in captivity, origin of captive animals (wild caught or captive born), time of day when samples were collected, any
significant notes listed in each study.

Author
 
Ayres
 et
 
al.,
 2012
 
Suzuki
 et
 
al.,
 1998
 
Suzuki
 et
 
al.,
 2003
 
Lyamin
 et
 

Mean
 F
 
nmol/L
 

Mean
 F
 
μg/dl
 


 
5.52
 


 
0.2
 

5.52
 

0.2
 

7.17
 

0.26
 

Seaso
n
 

Age
 


 
2
 mat:1
 
unmat
 
11,
 12,
 
&
 14
 

Sex
 
M72
:F66
 
2M:
1F
 
2M:
1F
 

11
 ,23
 

3F
 

5-­‐10
 
9-­‐12
 
year
 
round
 

 

ID/
OD
 

Time
 
to
 
samp
le
 
n/a
 


 
OD
 


 

OD
 
OD
 


 

 

Time
 in
 
captivity
 
n/a
 
≥10
 
years
 
≥10
 
years
 
many
 

Origin
 

AM
:P
M
 


 
WC
 


 
bot
h
 

WC
 

bot
h
 

1WC:
 


 

Notes
 


 

 
pregnan
t
 
pregnan

174

 

al.,
 2005
 
Lyamin
 et
 
al.,
 2005
 

9.66
 

0.35
 

11,
 23
 

3F
 

OD
 


 

years
 

1CB
 

t
 

many
 
years
 

1WC:
 
1CB
 

post-­‐
partum
 


 


 

Appendix H: Beluga Whale Cortisol Studies, Confounding Variables. Author (s) of original study on cortisol levels, mean
cortisol levels in nmol/L and µg/dl, season or month the study was conducted in (months in numeric form), age of the
animal (s) sampled, sex of the animal (s) sampled, if the captive animals were kept in an indoor (ID) or outdoor (OD)
enclosure, time it took for sample to be collected (in wild studies from initiation of chase until sample collection), time
spend in captivity, origin of captive animals (wild caught or captive born), time of day when samples were collected, any
significant notes listed in each study.

Author
 
St.
 Aubin
 &
 
Geraci,
 1989
 
St.
 Aubin
 &
 
Geraci,
 1992
 
St.
 Aubin
 et
 
al.,
 2001
 
Tryland
 et
 al.,
 
2006
 
Orlov
 et
 al.,
 
1991
 

Mean
 F
 
nmol/L
 

Mean
 
F
 μg/dl
 

Season
 

90
 

3.3
 

July
 

110
 

89.1
 

4
 

3.2
 

125.1
 

4.5
 

82.6
 

2.9
 

July
 
Summe
r/fall
 
Octobe
r
 
Spring
 
and
 Fall
 

Age
 
Sub-­‐
adult
 
juvenil
e
 

>2
 
calves-­‐
adults
 


 

Time
 to
 
sample
 
w/in
 1
 
hour
 
w/in
 1
 
hour
 

49.7
 

1.8
 

Fall
 

19
 


 
2M:1
F
 

27.6
 

~1
 

Fall
 

9-­‐28
 

2M:5
F
 

Time
 
in
 
captivi
ty
 

Orig
in
 

n/a
 

n/a
 

n/a
 

n/a
 


 
<1
 hour
 

n/a
 

n/a
 

<1
 hour
 

n/a
 

n/a
 

1
 year
 

yes
 


 

 
OD
 


 
Schmitt
 et
 
al.,
 2010
 
Spoon
 &
 
Romano,
 
2012*
 

Sex
 
34M:
8F
 
8M:2
F
 
118
M:60
F
 
14M:
7F
 

ID
/O
D
 


 
OD
 
ID/
OD
 


 


 

AM
:P
M
 

Notes
 


 


 


 


 


 


 


 


 
shallow
 
sample
 
not
 
repro
 
active
 


 

~18
 

yes
 


 
bot
h
 


 


 


 

175

 

Bibliography
Ayres, K. L., Booth, R. K., Hempelmann, J. A., Koski, K. L., Emmons, C. K., Baird, R. W., BalcombBartok, K. (2012). Distinguishing the Impacts of Inadequate Prey and Vessel Traffic on an
Endangered Killer Whale (Orcinus orca) Population. PLoS ONE, 7(6), e36842.
Axelrod, J. & Resine, T. (1984) Stress Hormones: Their Interaction and Regulation. Science, 224, 452459.
Babic, N., Kiang-Teck, J. & Weiss, R.E. (2011). Endocrine testing protocols: Hypothalamic pituitary
adrenal axis. Endotext. Retrieved from
http://www.endotext.org/protocols/protocols1/protocolsframe1.htm.
Balcomb, K. 2012 The Center for Whale Research retrieved from www.whalereserach.com
Balm, P.H.M. (Ed.), 1999. Stress Physiology in Animals. CRC Press, Boca Raton, Florida
Barrett‐Lennard, L., Smith, T. G., & Ellis, G. M. (1996). A Cetacean Biopsy System Using
Lightweight Pneumatic Darts, and Its Effect on the Behavior of Killer Whales. Marine Mammal
Science, 12(1), 14-27.
Bayazit, V. (2009). Evaluation of Cortiosl and Stress in Captive Animals. Australian Journal of Basic
and Applied Sciences, 3(2), 1022-1031.
Bearzi, G. (2000). First report of a common dolphin (Delphinus delphis) death following penetration of
a biopsy dart. Journal of Cetacean Research and Management 2:217–221.
Berrow, S. D., B. McHugh, D. Glynn, E. McGovern, K. M. Parsons, R. W. Baird and S. K. Hooker.
(2002). Organochlorine concentrations in resident bottlenose dolphins (Tursiops truncatus) in the
Shannon Estuary, Ireland. Marine Pollution Bulletin 44:1296–1313.
Berta, A., Sumich, J. L., & Kovacs, K. M. (2006). Sound Production for Communication, Echolocation,
and Prey Capture. Marine Mammals: Evolutionary Biology (2nd ed.). (270-305) Burlington, MA,
San Diego, CA, London, UK: Academic Press.
Best, P. B., D. Reeb, M. B. Rew, P. J. Palsbøll, C. Schaeff and A. Brandão. (2005). Biopsying southern
right whales: Their reactions and effects on reproduction. Journal of Wildlife Management
69:1171–1180.
Blasio, A. L., Pardo, M. R., Pérez, R. V., Maldonado, F. G. (2011). Maintenance behavior and cortisol
levels in bottlenose dolphins (Tursiops truncates) in closed and open facilities. Vet. Méx, 43(2),
103-112.
Bruce-Alllen, L.J. & J. R. Geraci. (1985). Wound healing in the bottlenose dolphin (Tursiops
truncatus). Canadian Journal of Fisheries and Aquatic Science. 42:216–228.
Buholzer, L., Desportes, G., Siebert, U., Vossen, A., Anderson, K., Larsen, F., Teilmann, J., Dietz, R. &
Shephard, G. (2004). Cortisol levels in captive and wild harbor porpoises (Phocoena phocoena)
and effect of handling methods. European Research on Cetaceans. 15:364-367
Castellote, M. & Fossa, F. (2006). Measuring acoustic activity as a method to evaluate welfare in
captive beluga whales (Delphinapterus leucas). Aquatic Mammals. 32(3), 325-333

176

 

Cetabase. (2011). Retrieved April 1, 2013 from http://www.ceta-base.com.
Chrousos, G. P., & Gold, P. W. (1992). The concepts of stress and stress system disorders. Overview of
physical and behavioral homeostasis. JAMA: the journal of the American Medical Association,
267(9), 1244–1252.
Clark, L. S., Cowan, D. F., & Pfeiffer, D. C. (2006). Morphological Changes in the Atlantic Bottlenose
Dolphin (Tursiops truncatus) Adrenal Gland Associated with Chronic Stress. Journal of
Comparative Pathology, 135(4), 208-216.
Corkeron, P. (2008). Captivity. In Perrin, W.F., Würsig, B. & Thewissen, J.G.M. (Eds.), Encyclopedia
of Marine Mammals 2nd Edition (218-223). Burlington, MA: Academic Press.
Copland, M.D. & Needham, D.J. (1992). Hematological and Biochemical changes associated with
transport of dolphins (Tursiops truncatus). AAAM Conferences, 18-22 May. Hong Kong, 23.
Cowen, D. & Curry, B. (2002). Histopathological assessment of dolphins necropsied onboard vessels in
the eastern tropical Pacific tuna fishery. NOAA Southwest Fisheries Science Center.
Administrative report: LJ-02-24C
Curry, B. E. (1999). Stress in mammals: the potential influence of fishery-induced stress on dolphins in
the eastern tropical Pacific Ocean. NOAA Technical Memorandum, NMFS, NOAA-TM-NMFSSWFSC-260. Southwest Fisheries Science Center, La Jolla, CA, 21 pp.
Dantzer, R. & Mormede, P. (1995). Psychoneuroimmunology of stress, in Stress, the Immune System
and Psychiatry, Leonard, B., and Miller, D. (Eds.), John Wiley & Sons, West Sussex, U.K., 47-53
Dellman, H. D. & J. Eurell. 1998. Textbook of veterinary histology. 5th edition. Lippincott Williams
and Wilkins, Baltimore , MD
Dierauf, L.A. (1990). CRC handbook of marine mammal medicine: health, disease, and rehabilitation.
Boca Raton, FL:CRC Press. 733 pp.
Edwards, E.F., & Perkins, P. C. (1998). Estimated tuna discard from dolphin, school, and log sets in the
eastern tropical Pacific Ocean, 1989-1992. FISHERY BULLETIN-NATIONAL OCEANIC AND
ATMOSPHERIC ADMINISTRATION, 96, 210–222.
Esch, C. H., Sayigh, L.S., Blum, J.E., &Wells, R.S. (2009). Whistles as potential indicators of stress in
bottlenose dolphins (Tursiops truncatus). Journal of Mammalogy, 90(3):638-650
Fair, P. A., & Becker, P. R. (2000). Review of stress in marine mammals. Journal of Aquatic Ecosystem
Stress and Recovery, 7(4), 335-354.
Ford, J.K.B. (2008). Killer Whales. In Perrin, W.F., Würsig, B. & Thewissen, J.G.M. (Eds.),
Encyclopedia of Marine Mammals 2nd Edition (685-691). Burlington, MA: Academic Press.
Ford, J., Ellis, G., Balcomb, K. (2000). Killer Whales; The Natural History and Genealogy or Orcinus
orca in British Columbia and Washington (12-99). Vancouver, BC, Canada: UBC Press.
Fossi, C.,Marsili, M., Leonzio, L., Notarbartolo, C., Di Sciara, G., Zanardelli, M., & Focardi, S. (1992).
The use of non-destructive biomarker in Mediterranean cetaceans: Preliminary data on MFO

177

 

activity in skin biopsy. Marine Pollution Bulletin, 24(9), 459-461. doi:10.1016/0025326X(92)90346-8
Gröschl, M. (2008). Current Status of Salivary Hormone Analysis. Clinical Chemistry, 54(11), 1759 1769. doi:10.1373/clinchem.2008.108910
Hammond, P., & Tsai, K. T. (1983). Dolphin mortality incidental to purse-seining for tunas in the
eastern Pacific Ocean, 1979-1981. Rep. Int. Whal. Comm, 33, 589–597.
Hogg, C. J., Rogers, T. L., Shorter, A., Barton, K., Miller, P. J. O., & Nowacek, D. (2009).
Determination of steroid hormones in whale blow: It is possible. Marine Mammal Science, 25(3),
605-618. doi:10.1111/j.1748-7692.2008.00277.x
Houser, D. S., Yeates, L. C., & Crocker, D. E. (2011). Cold stress induces an adrenocortical response in
bottlenose dolphins (Tursiops truncatus). Journal of zoo and wildlife medicine: official publication
of the American Association of Zoo Veterinarians, 42(4), 565–571.
Houser, D.S., Wasser, S., Cockrem, J.F., Kellar, N. & Romano, T. (n.d.) Variability of hormonal stress
markers collected from a managed dolphin population. Distribution statement. Award number:
N000141110436
Hunt, Kathleen E., Rolland, R. M., Kraus, S. D., & Wasser, S. K. (2006). Analysis of fecal
glucocorticoids in the North Atlantic right whale (Eubalaena glacialis). General and Comparative
Endocrinology, 148(2), 260-272. doi:10.1016/j.ygcen.2006.03.012
Jefferson, Thomas (2012). The Most Endangered Cetacean Species. Retrieved from
http://www.savethewhales.org/mostendangered.html
Krahn, M.M., Wade, P.R., Kalinowski, S.T., Dahlheim, M.E., Taylor, B.L., Hanson, M.B., Ylitalo,
GmM., Angliss, R.P., Stein, J.E.& Waples, R.S. (2002). Status review of southern resident killer
whales (Orcinus orca) under the endangered species act. NOAA technical memorandum NMFSNWFSC-54, National Oceanic and Atmospheric Administration (NOAA), National Marine
Fisheries Service, Seattle, WA. Retrieved from
http://www.nwfsc.noaa.gov/assets/25/4243_06162004_130449_tm54.pdf
Krahn, M. M., Hanson, M. B., Baird, R. W., Boyer, R. H., Burrows, D. G., Emmons, C. K., Ford, J. K.
B., et al. (2007). Persistent organic pollutants and stable isotopes in biopsy samples (2004/2006)
from Southern Resident killer whales. Marine Pollution Bulletin, 54(12), 1903-1911.
doi:10.1016/j.marpolbul.2007.08.015
Kravitz, L., Maglione-Garves, C., Suzanne, S. (2005). Cortisol Connection: Tips on Managing Stress
and Weight. ACSM’s heath and Fitness Journal: September/October 2005-Volune 9-Issue 5-pp
20-23.
Kricka, L.J., Thorpe, G.H.G. & Stott, R.A.W. (1987). Enhanced chemiluminescence enzyme
immunoassay. Pure and Applied Chemistry. 59(5), 651-654.
Lackey, R. T. (2000). Restoring wild salmon to the Pacific Northwest: chasing an illusion? In Koss, P.
& Katz, M. (Eds). What we don’t know about pacific northwest fishruns---an inquiry into
decision-making (91-143). Portland, OR: Portland State University.

178

 

Lair, S., Béland, P., De Guise, S., & Martineau, D. (1997). Adrenal hyperplastic and degenerative
changes in beluga whales. Journal of Wildlife Diseases 33(3):430-437
Larkin, I. & Donnelly, K. (Eds.). (2008). Comparing Fecal Cortisol Levels Among Captive West Indian
Manatees: Who’s More Stressed? Paper presented at Florida Marine Mammal Health Conference
III, St. Augustine, Florida, 13.
Liu, J., Chen, Y., Guo, L., Gu, B., Liu, H., Hou, A., Liu, X., et al. (2006). Stereotypic behavior and fecal
cortisol level in captive giant pandas in relation to environmental enrichment. Zoo Biology, 25(6),
445–459.
Lyamin, O., Pryaslova, J., Lance, V., & Siegel, J. (2005). Animal behaviour: Continuous activity in
cetaceans after birth. Nature, 435(7046), 1177–1177.
Mancia, A., Warr, G.W., & Chapman, R.W. (2008). A transcriptomic analysis of the stress induced by
capture-release health assessment studies in wild dolphins (Tursiops truncatus). 17(11):2581-2589
Martineau, D. (2007). Potential Synergism between Stress and Contaminants in Free-ranging Cetaceans.
International Journal of Comparative Psychology, 20(2). Retrieved from
http://escholarship.org/uc/item/866341xp
Medway, W., Geraci, J. R., & Klein, L.V. (1970). Hematologic response to administration of
corticosteroid in the bottle-nosed dolphin (Tursiops truncatus). American Journal of Physiology.
157:563-565
Medway, W. (1980). Some bacteria and mycotic diseases of marine mammals. Journal of American
Veterinary Medical Association, 177, 831-834.
Morin, P. A., LeDuc, R. G., Robertson, K. M., Hedrick, N. M., Perrin, W. F., Etnier, M., Wade, P., et al.
(2006). Genetic Analysis of Killer Whale (orcinus Orca) Historical Bone and Tooth Samples to
Identify Western U.s. Ecotypes. Marine Mammal Science, 22(4), 897–909. doi:10.1111/j.17487692.2006.00070.x
Morisaka, T., Konshima, S., Yoskioka, M., Suzuki, M., & Nakahara, F. (2010). Recent Studies on
Captive Cetaceans in Japan: Working in Tandem with Studies on Cetaceans in the Wild.
International Journal of Comparative Psychology. 23, 644-663.
Möstl, E., & Palme, R. (n.d.). Hormones as indicators of stress. Domestic Animal Endocrinology, 23(1),
67-74.
Naka, T., Katsumata, E., Sasaki, K., Minamino, N., Yoshioka, M., & Takei, Y. (2007). Natriuretic
Peptides in Cetaceans: Identification, Molecular Characterization and Changes in Plasma
Concentration. Zoological Science, 24(6), 577-587.
National Oceanic and Atmospheric Administration. (2005). Amendment to Scientific Research Permit
774-1714-03. La Jolla, CA: Southwest Fisheries Science Center.
Niessen, W. M. A. (2006). Liquid chromatography--mass spectrometry. Boca Raton, FL: CRC Press.
Noda, K., Aoki, M., Akiyoshi, H., Asaki, H., Ogata, T., Yamauchi, K., Shimada, T., et al. (2006). Effect
of bovine lactoferrin on the immune responses of captive bottlenosed dolphins (Tursiops
truncatus) being transported over long distances. Veterinary Record, 159(26), 885–888.

179

 

Noren, D. P., & Mocklin, J. A. (2011). Review of cetacean biopsy techniques: Factors contributing to
successful sample collection and physiological and behavioral impacts. Marine Mammal Science,
28(1), 154-199. doi:10.1111/j.1748-7692.2011.00469.x
Norris, K. S., & Dohl, T. P. (1980). Behavior of the Hawaiian spinner dolphin, Stenella longirostris.
Fishery bulletin, 77(4), 821–849.
O’corry-Crowe, G.M. (2008). Beluga Whales. In Perrin, W.F., Würsig, B. & Thewissen, J.G.M. (Eds.),
Encyclopedia of Marine Mammals 2nd Edition (143-146). Burlington, MA: Academic Press.
Orcas in captivity, a look at killer whales in aquariums and parks. (2013). Retrieved June 1, 2013 from
http://www.orcahome.de/orcastat.htm.
Orlov, M., Mukhlya, A., & Kulikov, N. (1988). Hormonal indices in the bottle-nosed dolphin Tursiops
truncatus in the norm and in the dynamics of experimental stress. Journal of Evolutionary
Biochemistry and Physiology, 24(4), 431-436.
Orlov, M., Mukhlya, A.M., & Kuz’min, A. A. (1991) Hormonal and electrolyte changes in cetacean
blood after capture and during experimental stress. Journal of Evolutionary Biochemistry and
Physiology. 27, 197-205
Ortiz, R.M., Long, B., Casper, D., Ortiz, C.L., & Williams, T.M. (2010). Biochemical and hormonal
changes during acuate fasting and re-feeding in bottlenose dolphins (Tursiops truncatus). Marine
Mammal Science. 26(2), 409-419
Ortiz, R. M., & Worthy, G. A. (2000). Effects of capture on adrenal steroid and vasopressin
concentrations in free-ranging bottlenose dolphins (Tursiops truncatus). Comparative
Biochemistry and Physiology - Part A: Molecular & Integrative Physiology, 125(3), 317-324.
Ortiz, R.M., Wade, C.E., & Ortiz, C.L. (2001). Effects of prolonged fasting on plasma cortisol and TH
in postweaned northern elephant seal pups. American Journal of Physiology. 280(3), 790-795.
Ortiz, R.M., Wade, C.E., Ortiz, C.L. & Talamantes, F. (2003) Acutely elevated vasopressin increases
circulating concentrations of cortisol and aldosterone in fasting northern elephant seal (Mirounga
angustirostris) pups. The Journal of Experimental Biology. 206, 2795-2802
Parsons, K. M.,Durban, J. W. & Claridge, D. E. (2003). Comparing two alternative methods for
sampling small cetaceans for molecular analysis. Marine Mammal Science 19:224–231.
Pedernra-Romano, C., Valdez, R.A., Singh, S., Chiappa, X., Romano, M. C., & Galindo, F. (2006).
Salivary cortisol in captive dolphins (Tursiops truncates): a non-invasive technique. Animal
Welfare, 1(5), (359-362).
Peterson, R.E., Karrer, A., & Guerra, S.L. (1957). Evaluation of the Silber-Porter procedure for
determination of plasma hydrocortisone. Analytical Chemistry. 29, 144.
Porstmann, T., & Kiessig, S. T. (1992). Enzyme immunoassay techniques an overview. Journal of
Immunological Methods, 150(1–2), 5-21. doi:10.1016/0022-1759(92)90061-W
Rangel-Negrín, A., Alfaro, J. L., Valdez, R. A., Romano, M. C., & Serio-Silva, J. C. (2009). Stress in
Yucatan spider monkeys: effects of environmental conditions on fecal cortisol levels in wild and
captive populations. Animal Conservation, 12(5), 496–502.

180

 

Raubenheimer, P.J., Young, E.A., Andrew, R. & Seckl, J.R. (2006). The role of corticosterone in human
hypothalamic-pituitary-adrenal axis feedback. Clinical Endocrinology. 65(1):22-26
Reidarson, T. H., & McBain, J. F. (1999). Hematologic, Biochemical, and Endocrine Effects of
Dexamethasone on Bottlenose Dolphins (Tursiops truncatus). Journal of Zoo and Wildlife
Medicine, 30(2), 310-312.
Reiss, D., & Marino, L. (2001). Mirror self-recognition in the bottlenose dolphin: A case of cognitive
convergence. Proceedings of the National Academy of Sciences, 98(10), 5937–5942.
doi:10.1073/pnas.101086398
Ridgway, S., Carder, D., Finneran, J., Keogh, M., Kamolnick, T., Todd, M. & Goldblatt, A. (2006).
Dolphin continuous auditory vigilance for five days. The Journal of experimental Biology,
209:3621-3628.
Ridgway, S., Keogh, M., Carder, D., Finneran, J., Kamolnick, T., Tood, M., & Goldblatt, A. (2009).
Dolphins maintain cognitive performance during 72 and 120 hours of continuous auditory
vigilance. The Journal of Experimental Biology. 212:1519-1527
Rolland, R. M., Hunt, K. E., Kraus, S. D., & Wasser, S. K. (2005). Assessing reproductive status of
right whales (Eubalaena glacialis) using fecal hormone metabolites. General and Comparative
Endocrinology, 142(3), 308-317.
Rolland, R., Parks, S., Hunt, K., Castellote, M., Corkeron, P., Nowacek, S., Wasser S. & Kraus, S.
(2012). Evidence that ship noise incrases stress in right whales. Proceedings of the Royal Soceity
B: Biological Sciences. 279(1737), 2363-2368.
Romano, T. A., Keogh, M. J., Kelly, C., Feng, P., Berk, L., Schlundt, C. E., & Carder, D. A. (2004).
Anthropogenic sound and marine mammal health: measures of the nervous and immune systems
before and after intense sound exposure. Canadian Journal of Fisheries and Aquatic Sciences,
61(7), 1124-1134. doi:10.1139/f04-055
Rose, N. (2011). Killer Controversy, Why orcas should no longer be kept in captivity. The Humane
Society International/The Humane Society of the United States. Retrieved February 1, 2012 from
http://www.hsi.org/news/press_releases/2011/09/orcas_captivity_report_091911.html
Ross, P. S., Ellis, G.M., Ikonomou, M. G., Barrett-Lennard, L. G., Addison, R. F. (2000). High PCB
concerntations in free-ranging Pacific killer whales, Orcinus orca: Effects of age, sex and dietary
preference. Marine Pollution Bulletin. 40(6), 504-512.
Schmitt, T. L., St. Aubin, D. J., Schaefer, A. M., & Dunn, J. L. (2010). Baseline, diurnal variations, and
stress‐induced changes of stress hormones in three captive beluga whales, Delphinapterus leucas.
Marine Mammal Science, 26(3), 635-647. doi:10.1111/j.1748-7692.2009.00366.x
Schwarzenberger, F., Möstl, F., Bamber, E., Pammer, J., & Schmehlik, O. (1991). Concentrations of
progestagens and oestrogens in the faeces of pregnant Lipizzan, Trotter and Thoroughbred mares.
Journal of Reproduction and Fertility Supplement. 44:489–499.
Schwarzenberger, F. (2007). The many uses of non‐invasive faecal steroid monitoring in zoo and
wildlife species. International Zoo Yearbook, 41(1), 52-74. doi:10.1111/j.17481090.2007.00017.x

181

 

Sheriff, M. J., Dantzer, B., Delehanty, B., Palme, R., & Boonstra, R. (2011). Measuring stress in
wildlife: techniques for quantifying glucocorticoids. Oecologia, 166(4), 869-887.
doi:10.1007/s00442-011-1943-y
Singh, A.K., Jiang, Y., White, T., & Spassova, D. (1997). Validation of nonradioactive
chemiluminescent immunoassay methods for the analysis of thyroxine and cortisol in blood
samples obtained from dogs, cats, and horses. Journal of Veterinary Diagnostic Investigation.
9:261-268
Spoon, T. R., & Romano, T. A. (2012). Neuroimmunological response of beluga whales
(Delphinapterus leucas) to translocation and a novel social environment. Brain, Behavior, and
Immunity, 26(1), 122–131.
St. Aubin, D. J. & Dierauf, D. A. (2001). Stress and Marine Mammals in Dierauf, L.& Gulland, M.
(eds.), CRC handbook of marine mammal medicine 2nd ed (253-270). Boca Raton, FL: CRC Press
LLC.
St. Aubin, D. J. (2001). Endocrinology in Dierauf, L. & Gulland, M. (eds.), CRC handbook of marine
mammal medicine 2nd ed (165-192). Boca Raton, FL: CRC Press LLC.
St. Aubin, D. J, Ridgway, S. H., Wells, R. S., & Rhinehart, H. (1996). Dolphin Thyroid and Adrenal
Hormones: Circulating Levels in Wild and Semidomesticated Tursiops Truncatus, and Influence
of Sex, Age, and Season. Marine Mammal Science, 12(1), 1-13. doi:10.1111/j.17487692.1996.tb00301.x
St. Aubin, D. J., & Geraci, J. R. (1989). Adaptive Changes in Hematologic and Plasma Chemical
Constituents in Captive Beluga Whales, Delphinapterus leucas. Canadian Journal of Fisheries
and Aquatic Sciences, 46(5), 796-803. doi:10.1139/f89-099
St. Aubin D.J. & Geraci, J.R. (1992). Thyroid Hormone Balance in Beluga Whales, Delphinapterus
leucas: Dynamics after Capture and Influence of Thyrotorpin. Canadian Journal of Veterinary
Research. 56: 1-5.
St. Aubin, D. J. (2002a). Further assessment of the potential fishery-induced stress on dolphins in the
eastern tropical Pacific. Administrative Report LJ-02-23C, NMFS, Southwest Fisheries Science
Center, La Jolla , CA . 13 pp
St. Aubin, D. J., Forney, K. A., Chivers, S.J., Scott, M. D., Danil, K., Romano, T. A., Wells, R. S., &
Gulland, F. (2013) Hematological, serum, and plasma chemical constituents in pantropical spotted
dolphins (Stenella attenuate). Marine Mammal Science. 29(1), 14-35.
Stead, S.K., Meltzer, D.G.A., & Palme, R. (2000). The measurement of glucocorticoid concentrations
in the serum and faeces of captive African elephants (Loxodonta africana). After ACTH
stimulation: research communication. Journal of the South African Veterinary Association. 71(3),
192-196.
Suzuki, M., Tobayama, T., Katsumata, E., Yoskioka, M., & Aida, K. (1998). Serum Cortisol Levels in
Catpive Killer Whale and Bottlenose Dolphin. Fisheries Science, 64(4), 643-647.
Suzuki, M., Uchida, S., Ueda, K., Tobayama, T., Katsumata, E., Yoshioka, M., & Aida, K. (2003).
Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins

182

 

Tursiops aduncus and killer whales Orcinus orca. General and Comparative Endocrinology,
132(3), 427-433.
Sweeney, J.C. (1990). Surgery. Handbook of Marine Mammal Medicine: Health, Disease, and
Rehabilitation. Dierauf, L.A. (Ed.), Bca Raton, FL:CRC Press, 215-234.
Terio, K.A. & Munson, M.L. (2004). Evidence for chronic stress in captive but not free-ranging
cheetahs (Acinoyx jubataus) based on adrenal morphology and function. Journal of Wildlife
Diseases, 40(2):259-266
Thomson, C. A., & Geraci, J. R. (1986). Cortisol, Aldosterone, and Leucocytes in the Stress Response
of Bottlenose Dolphins, Tursiops truncatus. Canadian Journal of Fisheries and Aquatic Sciences,
43(5), 1010-1016. doi:10.1139/f86-125
Tryland, M., Thoresen, S. I., Kovacs, K.M., & Lydersen, C. (2006). Serum chemistry of free-ranging
white whales (Delphinapterus leucas) in Svalbard. Veterinary Clinical Pathology 35(2):199-203
Tsigos, C. & Chrousos, G. (2002) Hypothatlamic-Pituitary-Adrenal Axis, Neuroendocrine Factors and
Stress. Journal of Psychosomatic Research, 53, 865-871.
Turban, S., Wang, X.Y., & Knepper, M.A. (2003). Regulation of NHE3, NKCC2, and NCC abundance
in kidney during aldosterone escape phenomenon: role of NO. American Journal of PhysiologyRenal Physiology. 285(5), F843-51. doi: 10.1152/ajprenal.00110.2003
Wade, P. R., Watters, G. M., Gerrodette, T., & Reilly, S. B. (2007). Depletion of spotted and spinner
dolphins in the eastern tropical Pacific: modeling hypotheses for their lack of recovery. Marine
Ecology Progress Series, 343, 1–14.
Waples, K. A., & Gales, N. J. (2002). Evaluating and minimising social stress in the care of captive
bottlenose dolphins (Tursiops aduncus). Zoo Biology, 21(1), 5-26. doi:10.1002/zoo.10004
Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, C.M., Bechert, U., Millspaugh, J.J.,
Larson, S., Monfort, S.L. (2000). A generalized fecal glucocorticoid assay for use in a diverse
array of nondomestic mammalian and avian species. General and Comparative Endocrinology.
120, 260–275.
Wasser, S.K., Papageorge, S., Foley, C., Brown, J.L., (1996). Excretory fate of estradiol and
progesterone in the African elephant (Loxodonta africana) and patterns of fecal steroid
concentrations throughout the estrous cycle. General and Comparative Endocrinology. 102, 255–
262.
Wasser, S.K., Monfort, S.L., Southers, J. & Wildt, D.E. (1994). Excretion rates and metabolites of
estradiol and progesterone in baboon (Papio cynocephalus cynocephalus) feces. Journal of
Reproduction and Fertility, 101, 213-220.
Wasser, S., (2012) Causes of Decline among Southern Resident Killer Whales. Retrieved January 26,
2012 from The University of Washington, School of Conservation Biology website:
http://conservationbiology.net/research-programs/killer-whales-2/
Weller, D. W., V. G. Cockcroft, B. Würsig, S. K. Lynn and D. Fertl. (1997). Behavioral responses of
bottlenose dolphins to remote biopsy sampling and observations of surgical biopsy wound healing.
Aquatic Mammals 23:49–58.

183

 

Wells, R.S. (n.d.). The bottlenose dolphin (Tursiops truncatus) as a model to understand variation in
stress and reproductive hormone measures in relation to sampling matrix, demographics, and
environmental factors. Distribution statement: Award Number N00014111052.
Wells, R. & Schwacke, L. (January, 2012). Understanding stress in bottlenose dolphins. Retried from
http://sarasotadolphin.org/2012/01/17/5389/.
Wells, R.S. & Scott, M.D. (2008). Common Bottlenose Dolphin. In Perrin, W.F., Würsig, B. &
Thewissen, J.G.M. (Eds.), Encyclopedia of Marine Mammals 2nd Edition (143-146). Burlington,
MA: Academic Press.

184

 

185