fig; WNWINHIHIW.lWlllWlWWMWW

      

THS

/
c7031 .

This is to certify that the
thesis entitled

NON-INVASIVE MEASUREMENT OF TRANSPORT
EFFECTS ON TIGERS

presented by
Daniel Phillip Dembiec

has been accepted towards fulfillment
of the requirements for the

MS. degree in Zoolgy

/. / 414

Major " . essor’s Signature
July 14, 2004

Date

MSU is an Affirmative Action/Equal Opportunity Institution

 

 

—__

 

I iJeRARy
Michigan State
L University

 

 

 

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c:/C|RC/DateDue.p65-p. 15

 

 

NON-INVASIVE MEASUREMENT OF TRANSPORT
EFFECTS ON TIGERS

By

Daniel Phillip Dembiec

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE
Department of Zoology

2004

i. 1". ”Trrh’I‘rlhpiI‘rIIF

.tILI thLr . lftI at. 5.an II; .II. Ir! 1 l

 

\f-IIDIII’EL-II E‘IEEEIII‘! tut-6 fitting—{It

ABSTRACT
NON-INVASIVE MEASUREMENT OF TRANSPORT EFFECTS ON TIGERS
By

Daniel Phillip Dembiec

The transport of tigers between zoos is a common captive management practice
that may affect tiger welfare. The objectives here are to validate a simple fecal cortisol
extraction protocol for Radioimmunoassay (RIA) analysis, measure baseline effects of
transport on tigers during a controlled 30-minute transport, and measure the effects of
transport between zoos. Feces were spiked with known amounts of cortisol. After
extraction 79.4 % was consistently recovered and measurements paralleled expected
recoveries. Five tigers were crated and transported for 30-minutes. Average respiration
rates increased during transport and remained elevated 10 minutes afterwards. Average
immune-reactive fecal cortisol concentrations (IRFCC) peaked 3-6 days after transport
and returned to baseline 9-12 days afterwards. Naive tigers exhibited a higher average
increase in IRFCC and longer recovery times than the experienced tigers. Naive tigers
also performed repertoires with greater intensity. Results suggest that prior exposure to
transport may lead to habituation, thus reducing its effects. Fecal samples were collected
fiom four tigers before, during, and after transport between zoos. Using RIA, IRFCC
was measured. On average, [RF CC peaked 6-8 days afterwards and remained higher
than baseline for 15-17 days. Results indicate that variable crate durations and methods

of transport may affect tigers differently.

ACKNOWLEDGEMENTS

I extend my sincerest respect and gratitude to Dr. Richard Snider for
having the vision to create the Professional Masters Zoo & Aquarium Science
program. It is such a pertinent program to the zoo world and has the potential to
mold students into the most influential professionals in the zoo industry. I’m
looking forward to seeing it grow. I also thank Dr. Snider for having the
conviction to allow me to participate in this program. He opened my eyes to the
dynamics of the zoo industry, and continued to guide me.

I thank Dr. Aroaldo Zanella. My studies could not have been
accomplished without him welcoming me into his laboratory. My studies also
could have not been accomplished without his unique expertise in indicators of
animal welfare and without his provision of the necessary resources to
accomplish my goals. Throughout my studies he has provided me a great wealth
of knowledge on the scientific evaluation of animal welfare. I also thank him for
being a significant part of the Zoo & Aquarium Science program. His expertise
acts as a catalyst to its success.

I thank my other two committee members, Dr. Catherine Lindell and Dr.
John Giesy for providing the necessary scientific perspective from outside the
field of captive animal management and ensuring the soundness of my studies. I
thank Dr. Kirsty Laughlin for her support on almost all aspects of my academic

journey. She was an invaluable resource as she provided me her knowledge

iii

and personal support throughout my studies. I thank Cheryl Leece for all her
help in the laboratory. Without her expertise my data would not exist.

I thank the Zoo & Aquarium Science Group and the Animal Behavior and
Welfare Group for their support and input. Discussions with both of these groups
facilitated the maturation of my studies. I thank Dr. Renate Snider and the rest of
my NSC 840 groups for giving my writing the necessary attention and for making
it decipherable. I also thank Heather Huseman for giving up her time to help
complete my lab work.

I am grateful to institutions that participated in my studies. This includes
the Potter Park Zoo, the Beardsley Zoo, the Chaffee Zoo, the St. Louis Zoo, the
Miller Park Zoo, the Fort Wayne Children’s Zoo, and the Knoxville Zoo. I would
like to thank Gerry Brady at Potter Park Zoo for being so informative and helping
me find tigers to participate in my study. I would especially like to thank all
personnel at Turpentine Creek Wildlife Refuge. The work that is done there is
and will continue to be the inspiration behind my studies.

I would also like to extend a special thank you to Melissa Murphy for an
uncountable number of things. Even with 400 miles between us, she was there
at my side through all I encountered and was even willing to endure long, hot,
and nasally challenging moments. Her support was inspirational.

Finally, I cannot put into words how thankful I am for my family.
Regardless of whether they understood what I was doing they were always there
to support me. My mother and father, Dennis and Linda Dembiec, have been

role models throughout my life. I owe all my success to my father’s relentless

iv

guidance and my mother's impenetrable compassion. My brothers, Chris and
Scott Dembiec, challenged me throughout life and have set the standards in

which my goals are based. For them I am truly thankful.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vii
LIST OF FIGURES ............................................................................... viii
CHAPTER 1

INTRODUCTION TO ZOO ANIMAL WELFARE ......................................... 1
CHAPTER 2

MEASURING FECAL CORTISOL IN TIGER FECES ................................... 7
CHAPTER 3

THE EFFECTS OF TRANSPORT STRESS ON TIGER PHYSIOLOGY AND
BEHAVIOR ....................................................................................... 13
CHAPTER 4

THE EFFECTS OF TRANSPORT FROM ONE 200 TO ANOTHER ON THE
HPA—AXIS OF TIGERS ........................................................................ 34
CHAPTER 5

CONCLUSIONS ................................................................................. 46
LITERATURE CITED .......................................................................... 48

vi

LIST OF TABLES

Table 1. Characteristics of each tiger subjected to transportation. Experience
refers to whether the tiger has experienced the transportation procedure previous
to the study ........................................................................................ 18

Table 2. Activity budget of each tiger one hour before and one hour after
transport in percentage of time spent performing each state ......................... 22

Table 3. Percent of time tigers spent performing each behavior during
transport ............................................................................................ 23

Table 4. Immune reactive fecal cortisol concentrations (ng/g) for each time block
of 4 tigers. Time 0 represents time of transport. Baseline refers to three days
prior to transport .................................................................................. 25

Table 5. Characteristics of each tiger transported between zoos ................... 37

Table 6. Mean 1: SE of immune reactive fecal cortisol concentrations (ng/g) for
each time block of 4 tigers. Time 0 represents time of transport. Baseline refers
to the average fecal cortisol concentration for 6 days prior to transport. N is
variable from 2 to 3 for individual tigers and variable from 8 to 12 for all

tigers ................................................................................................ 40

vii

LIST OF FIGURES

Figure 1. General model of the relationship between zoo animal welfare and the number
of animal welfare issues that exist over time as the function of zoos has evolved from
being an institution committed to public entertainment to an institution committed to
wildlife conservation (adapted fiom: Fisher, 1967; Mench and Kreger, 1996; Norton, et
al., 1995; Shapiro, 2000) ............................................................................. 2

Figure 2. Respiration rates for 4 tigers 10 min before, during, and 10 min after
transfer .............................................................................................. 24

Figure 3. Average immune reactive fecal cortisol concentrations of experienced
(N=2) and naive tigers (N=2). Day 0 represents time of transport ................... 26

Figure 4. Average immune reactive fecal cortisol concentrations of experienced
and naive tigers combined. Day 0 represents time of transport ...................... 27

Figure 5. IR fecal cortisol concentrations of Tigers 1-4 for each 3-day time
block ................................................................................................. 41

Figure 6. Average IR fecal cortisol concentrations of all tigers ....................... 42

viii

Chapter 1: Introduction to Zoo Animal Welfare

Zoos were established in the late 1800’s, but the consideration of animal
welfare was initially driven only by economic concerns. For example, zoo
managers would improve welfare standards only when their poor practices
caused animals to die resulting in economic losses. Since then, the philosophy
of human superiority over animals and our concern for animal welfare have
evolved. Animals are no longer viewed only in the context of how they are
directly useful to humans, and zoos no longer exist for entertainment purposes
alone (Mench and Kreger, 1996).

The role of zoos in society has evolved from being purely an institution for
entertainment to an institution that supports wildlife conservation, and educates
the general public about the importance of wildlife and current conservation
issues (Conway, 2000; Margodt, 2000; Mench and Kreger, 1996; Norton, et al.,
1995; Shapiro, 2000). As the function of zoos evolve from entertainment to
conservation, animal welfare standards evolve as well (Figure 1). If a zoo exists
purely for public entertainment, then managers will primarily focus on public
needs instead of animal needs. Therefore, a zoo that exists purely to entertain
will generally have low animal welfare standards resulting in an increase in poor
welfare issues. Conversely, if a zoo exists purely to conserve wildlife and
educate the public, then it is necessary for that zoo to provide optimal welfare
standards to accomplish these objectives. In general, a zoo that exists purely for

conservation and education purposes will have high welfare standards and a low

number of poor welfare issues. This evolution has been perceived and
documented (Fisher, 1967; Mench and Kreger, 1996; Norton, et al., 1995;
Shapiro, 2000) over time. Past zoos contained poorly enriched enclosures
usually deficient in size and content. Overtime, zoos have continued to provide
more appropriately sized and enriched exhibits (Fisher, 1967; Mench and Kreger,

1996; Norton, et al., 1995; Shapiro, 2000).

Zoo Animal Welfare

Number of Animal
Welfare Issues

 

 

Entertainment Zoo Conservation Zoo

Time

Figure 1. General model of the relationship between zoo animal welfare and the
number of animal welfare issues that exist over time as the function of zoos has evolved
from being an institution committed to public entertainment to an institution committed
to wildlife conservation (adapted from: Fisher, 1967; Mench and Kreger, 1996; Norton,

et al., 1995; Shapiro, 2000).

If zoos are continually evolving to conserve and educate, then why do
welfare issues persist? The goal of conserving animals and their natural biology

can only be accomplished if zoo managers provide the most natural environment

possible for each animal, thus reinforcing the animal’s natural biology by allowing
the animal to live in simulated wild-like conditions. Similarly, the public education
goal can only be accomplished by providing the animals with as close to a
natural environment as possible. Under these conditions it is hoped that they
can perform natural behaviors for the public to see. If zoos are moving towards
these two goals, shouldn’t poor welfare issues cease to exist?

Although poor welfare issues should be decreasing, they persist in zoos
for a number of reasons. One major reason is that although the traditional
function of zoos was to entertain, this function still exists because entertaining
the public is an important means for generating funds that are needed to support
conservation and education programs. At the same time the entertainment
function attracts large numbers of people who inadvertently become educated
about the biology and ecology of animals and conservation issues. A second
major reason is the lack of resources such as space, personnel, and appropriate
substrate for enclosures. Often the lack of resources can be linked to a lack of
funds to obtain those resources furthering the need to generate revenue by
public entertainment.

A third major reason for the existence of animal welfare issues is the lack
of information about the natural history of exotic animal species, which prevents
zoo managers from providing an optimal captive environment. This lack of
knowledge further justifies the need for continual research in situ and ex situ so
that the natural history of exotic species can be better understood and an

appropriate captive environment can be provided.

A fourth reason is lack of knowledge of how an animal’s biology is affected
by captive management practices and how to measure and reduce those effects.
In many cases, common practices may alter behavior or physiology. For
example, pandas housed in traditional enclosures perform more stereotypic
behavior than pandas enclosed in more natural environments (Liu et al., 2003)
and free-ranging female elephants above the age of 50 produce calves with
greater success than captive female elephants above the age of 50 (Brown,
2000)

Transport of zoo animals from one place to another is a common practice
that can affect behavior and physiology. Studies have assessed transport effects
on farm animals to help improve agricultural practices (Lambooy, 1988 and
Bradshaw et al., 1996). These studies demonstrated that transport might result
in body temperature increase (Brundige et al, 1998), an increase in
hypothalamic-pituitary—adrenal (HPA)-axis activation (Broom and Johnson, 1993),
and a depressed immune response (Baker and Gemmell, 1999; Broom and
Johnson, 1993). Zoo animals often appear stressed during transport as well
making them susceptible to these negative effects (pers. obs.). Few studies
have assessed transport effects on 200 species, and no studies on tigers have
been rigorously documented.

The term “stress” has been applied in a wide range of contexts, and
disagreement of its meaning among researchers makes it difficult to clearly
define (Wielebnowski, 2003). Selye generally defined stress as any stimulus that

threatens or appears to threaten the homeostasis of an individual (Selye, 1984).

In the following text, the term “stress” will refer to any homeostatic changes in
tiger physiology or behavior that are perceived to occur as a result of the stressor
(transport).

Many studies have used the hormone cortisol to measure an animal’s
stress level during common management procedures (Merlot et al., 2004,
Roussel et al., 2004, Braastad et al., 1998). To assess transport effects on tiger
stress, a means to measure changes in cortisol production using non-invasive
methods must be implemented. The following chapter explores the use of feces
(which can be collected non-invasively) as a substrate from which cortisol levels
can be measured.

Feces may be the substance of choice to measure hormonal fluctuations
in tigers because feces collection is simple, safe, and noninvasive. Feces have
been shown to contain cortisol concentrations that can be linked to the amount
an animal produces. Based on this information one can learn how the physiology
of the animal has been affected by a management procedure, and appropriate
management decisions can be made. For example, the measurement of cortisol
in cheetah feces has allowed managers to identify increases in stress and its
effects on cheetah reproductive cycles so that more effective breeding decisions
can be made (Jurke, 1997).

The use of tiger feces to measure cortisol had not been documented, and
methods to measure cortisol in feces had not been perfected. Current corticoid
extraction methods (Palme et al., 1998; Brown et al., 2001) can be labor-

intensive and often alter the sample (T erio et al., 2002) resulting in skewed

measurements. It is necessary to develop other extraction methods because to
date the published methods all demonstrate specific problems that either mask or
alter the samples. Similariy, variable amounts of water content in feces can
affect resulting hormone measurements. To eliminate these effects without
altering the sample by drying (Terio et al., 2002) the use of a correction factor
was investigated.

To test the validity of tiger feces analysis and to subsequently test
transport effects, the following objectives were addressed:
Objective 1: Validate a new fecal cortisol extraction protocol for
Radioimmunoassay (RIA) analysis
Objective 2: Develop and test a correction factor that eliminates water content of
feces as a source of error
Objective 3: Determine effects of a controlled 30-minute transport on tiger
behavior and physiology
Objective 4: Measure effects of the common transport procedure from one zoo to
another on the HPA-axis of tigers and compare results with the 30-minute

controlled transport

Chapter 2: Measuring Fecal Cortisol in Tiger
Feces

INTRODUCTION

Fecal corticoid levels have been used as an effective welfare indicator for
nondomestic felids. Wielebnowski et al. (2002) reported that clouded leopards
performing more stress-related behaviors such as pacing and self-injury (i.e., fur
plucking or tail chewing) had higher fecal corticoid levels than those that
performed less stress-related behaviors. Since increases in cortisol levels were
correlated with an increase in stress, and transport is perceived to induce stress
(pers. obs.), measuring cortisol in feces is a practical way to identify physiological
effects of transportation stress. As shown by Graham and Brown (1996), when
cortisol was injected into domestic cats, feces contained 80% of adrenocorticoid
metabolites, making the feces a reliable substrate to correlate with cortisol levels
in the body. Presence of cortisol in tiger feces has not been documented, and
methods used to measure cortisol in feces have not been perfected. Current
extraction methods (Palme and Mostl, 1997; Graham and Brown, 1996) are
labor-intensive and may alter the sample (Terio et al., 2002), resulting in
inaccurate measurements. To counter these problems we devised a simple
cortisol extraction protocol that minimizes sample manipulation.

Fecal water content varies considerably between samples due to
individual differences such as gut passage time, diet, water intake, and time

exposed to air. Water content must be eliminated as a source of error so that

cortisol concentration can accurately be determined. Two possible solutions are
to either dry the feces to 0 % water content or determine the percent dry matter
from an aliquot of the sample and calculate a correction factor. Since drying an
entire sample takes time and some drying techniques may cause the breakdown
of glucocorticoids (Terio et al., 2002), normalizing samples for water content may
be quicker and yield more accurate results. This study was designed to test the
effectiveness of our extraction protocol, and to determine if normalization for
water content eliminates the effects of variation in dry matter content on cortisol

measurement.

METHODS

Validation of a new cortisol extraction protocol

Each entire fecal sample was combined with a 20% methanol, 80% distilled
water solution so that they were in a 1:4 proportion (weight by weight). The
resulting mixture was homogenized thoroughly using a tissue homogenizer
(Brinkmann Instruments, Westbury, NY) and filtered through 7 mm2 gauge wire
mesh to eliminate bones and hair. Three aliquots (1.5 mL each) were kept to
determine dry matter content. Next, 40 g of solution was transferred to a 50 mL
polypropylene tube (USA Scientific Incorporation, Ocala, FL) and centrifuged at
3,000x g for 30 min. Then, 1.5 mL supernatant was transferred to a 1.5 mL
eppendorf tube, and each tube was microcentrifuged at 13,000x g for 15 min
(these steps were performed in triplicate). One mL of the supernatant was

transferred to an RIA tube (‘25l-cortisol RIA kit; Coat-A-Count; Diagnostic

Products Corporation, Los Angeles, CA), and immediately dried in a vacuum
concentrator (Appropriate Technical Resources Inc.; Laurel, MD). Dried
supernatant was resuspended in 200 uL distilled water before RIA. Resulting RIA
values were corrected for water content as performed by Wilson et al. (2002)
using the determined proportion dry matter from the three aliquots taken.

The assay sensitivity of the RIA kit is 0.2 pg/dL. When performing an RIA,
test tubes lined with cortisol antibodies are used. To show that these antibodies
accurately bind to cortisol the antibody cross-reactivity is as follows: 100 % with
cortisol, 11.4 % with 11-deoxycortisol, 1.6 % with betamethasone, 0.98 % with
cortisone, 0.94 % with corticosterone, 0.26 % with 11-deoxycorticosterone, 0.34
% with tetrahydrocortisol, and < 0.1 % with testosterone, aldosterone,
androsterone, progesterone, estriol, estradiol, and estrone.

This method for extracting cortisol from tiger feces was tested by adding 10
uL of 3H-cortisol to 6 original samples of 40 g feces and measuring the recovered

3H-cortisol after extraction. Indicators of accuracy and parallelism were obtained

by adding 500 pg of 5 different cortisol concentrations (0, 0.012, 0.062, 0.123,

and 0.246 pg/dl) to 40 g of 5 separate aliquots of the same homogenized fecal

sample. The 0 pg/dl concentration was used as the control to which all other

concentrations were compared.

Using a mathematical correction factor to eliminate effects of water content

To measure water content effects on fecal cortisol measurements we

added 500 pl of 6 different cortisol concentrations (0, 0.012, 0.062, 0.123, 0.246,

and 0.615 ug/dl) to 40 g of 6 separate aliquots of a large, pooled sample of tiger
feces. Next, we added 2 times their weight of a 20% methanol, 80% distilled
water solution and homogenized them. Each concentration was then separated
into 5 different 5 mL treatments: the control (C), experimental control (EC),
experimental low (EL), experimental medium (EM), and experimental high (EH).
Then, each EC, EL, EM, and EH were dried completely, and water was added to
each to obtain 0, 15, 30, and 60 % water content by weight respectively. The
control was frozen throughout the treatment period. Finally, each sample was
extracted using the previously described method, and cortisol was measured in

triplicate using RIA.

Correcting for dry matter content and statistical analysis

Resulting cortisol concentrations were compared using a one-way analysis of
variance (SAS Institute Inc., Cary, North Carolina, Copyright © 2001). Then,
resulting concentrations were normalized for water content. Once corrected,
resulting concentrations were again compared using a one-way analysis of

vanance.

RESULTS
Validation of a new cortisol extraction protocol

The intra-assay coefficient of variation was 5.1 %. After extraction, the
average recovery of 3H-cortisol was 79.4 i 0.1 %. When testing for accuracy and

parallelism, we recovered 83:0.3, 92i0.1, 911:0.1, 93:0.1% of the different

10

concentrations (0.012, 0.062, 0.123, and 0.246 ug/dl) of spiked cortisol

respectively.

Using a mathematical correction factor to eliminate effects of water content
Before mathematically correcting for dry matter content, significant differences in
cortisol concentration were found between the control and all experimental
treatments and between experimental treatments (ANOVA;F4,25=7.84,p<0.001).
After mathematically correcting for dry matter content, we found no significant

differences among any treatments (ANOVA; F4,25=1.43;NS).

DISCUSSION

Based on these results, the extraction protocol measured concentration of
cortisol in tiger feces with 79 % accuracy. We also recovered cortisol with great
consistency, as the standard error was only 0.1 % between all samples.
Similarly, our test for parallelism showed that the amount of cortisol recovered
proportionately increased with an increase in cortisol concentration.

Drying is a recurring step in current extraction protocols to avoid skewed
measurements caused by a variation in dry matter content. In this study, there
were significant differences in recovered cortisol between samples from the
same pooled feces, but with different concentrations of dry matter. Normalizing
for water content eliminated the significant differences. Fecal glucocorticoids

break down as a result of some drying techniques (Terio et al., 2002). To

11

eliminate this problem we propose that samples not be dried during the
extraction protocol, and are instead normalized for water content.

Based on these results this protocol serves as an accurate method to
measure cortisol concentration in tiger feces. The next step is to
pharrnacologically validate this protocol by demonstrating that increases in
recovered fecal cortisol indicate an increase in adrenal activity. To do so an
increase in measured cortisol must be linked with a stressful event that would
stimulate adrenal activity. The study presented in the following chapter attempts

to link fecal cortisol increase with transportation stress.

12

Chapter 3

Dembiec, Daniel P., Snider, Richard J., and Zanella. Adroaldo J. 2004. The
effects of transport stress on tiger physiology and behavior. Zoo Biology 23(4):
335-346.

13

INTRODUCTION

The transport of animals from one place to another is a common practice
that can affect their behavior and physiology. Many studies have assessed
transport effects on farm animals to help improve agricultural practices.
Lambooy (1988) showed that pig activity levels decrease during transport, while
Bradshaw et al. (1996) reported that transport increased cortisol levels in pig and
sheep saliva. Few studies have assessed transport effects on the physiology
and behavior of zoo species, and no studies on tigers have been rigorously
documented. An increase in body temperature of pigs (Brundige et al., 1998) an
increase in hypothalamic-pituitary-adrenal (HPA)-axis activation (Broom and
Johnson, 1993), and a depressed immune response in brushtail possums (Baker
and Gemmell, 1999; Broom and Johnson, 1993) have been identified as effects
of transportation stress. While a depressed immune response may be viewed as
a negative effect, short-term activation of the HPA-axis may be beneficial to
adaptive processes. For example, it has been shown that HPA-axis activation in
some cases enhances Ieaming in some mammals and humans (Wolf, 2003).

It is estimated that only between 5,000 and 7,500 tigers remain in the wild
(Seidensticker, 1999). Zoos are currently playing a large role in preventing
extinction of endangered tiger subspecies through captive breeding. Transport
under these conditions is often necessary to introduce animals of the opposite
sex for mating purposes. Tigers are also transported simply to be added to a
zoo’s collection to help educate and entertain the public. Their coloration and

behavior make them effective animals to help illustrate important ecological

14

concepts such as adaptive coloration and the ethology of forest predators, and
their size (Ward et al., 1998) and beauty make them popular with the public.
Therefore, tigers are often transported between zoos for breeding purposes and
to enhance the quality of the zoo following the recommended terms in the
American Zoo and Aquarium Association's (AZA) Tiger Species Survival Plan
(SSP).

Conservation efforts are often successfully supported by in-situ studies of
animals in captivity. As wild populations continue to decline, the information we
obtain from captive animals about their basic biology becomes increasingly
important. In addition, captive animals can serve as a resource for reestablishing
endangered populations and maintaining genetic diversity. Captive breeding
programs have been very effective in some cases, but in others animals have
failed to breed for unknown reasons. For example, the giant panda is well-known

for reproducing poorly in captivity (McGeehan, et al., 2002).

When the natural biology of 200 animals is altered, they serve a less
effective role in educating the public. For example, Baxter and Plowman (2001)
showed that the lack of browsing opportunity for giraffes in captivity caused an
increase in stereotypic behavior, and Mallapur and Chellam (2002) reported that
Indian leopard activity was influenced by feeding time and differed from activity
levels in the wild. To effectively utilize zoos as ex-situ conservation and public
education facilities, any effects captive management may have on the behavior
and physiology of zoo animals must be investigated and understood.

Transportation effects is one management practice that has not been intensively

15

studied. The better understanding of zoo animal behavior and physiology
coupled with understanding of techniques used to measure these indicators of

well-being enable us to identify threats to animal welfare.

Fear may inhibit exploratory behavior. On the other hand, fear may also
stimulate flight or hiding behavior. Space limitations in a transfer cage prevent
such responses to fear, which often results in the performance of stereotypies
such as rapid circling or pacing (Manser, 1992). Stereotypies may also result

from conditions of conflict or lack of control (Dantzer, 1986; Mason, 1991 ).

Facial expressions may serve an important role in assessing an animal’s
well-being. Ears positioned back and low in domestic cats has been used in past
studies to identify a defensive or fearful response to stimuli (Yoshinobu et al.,
2001; Levine et al., 1990; Johansson et al., 1979). In general, ears positioned
back and low have been associated with defensive behavior, fear, and confusion
while ears perked up and fonrvard are associated with relaxation, alertness, and

inquisitiveness (Thome, 1992).

In feline species, cortisol has been identified as the most important
naturally occurring glucocorticoid (Goossens et al., 1995) and is a product of the
HPA-axis. Immune-reactive (IR) cortisol can then be extracted from feces
(Schatz and Palme, 2001; Graham and Brown, 1997) and used as an effective
indicator of levels in the body (Mostl and Palme, 2002). In tigers, the time

between cortisol production and excretion in feces is unknown.

This study was performed to quantitatively and qualitatively evaluate how

transportation affects the behavior and physiology of tigers. Grandin (1997)

16

stated that previous experience may influence the way an animal reacts to
transport. Therefore, a comparison between effects on experienced and

inexperienced tigers was included in this study.

METHODS
Animals and Study Site

The Michigan State University All-University Committee on Animal Use
and Care approved this study as did the AZA’s Tiger SSP. This study was
performed during the summer of 2001 at Turpentine Creek Wildlife Refuge
(T CWR) in Eureka Springs, Arkansas. Turpentine Creek Wildlife Refuge is a
non-profit wildlife refuge that provides a home to over 100 large carnivores
confiscated or otherwise received from private ownership. Five tigers were
chosen for the study (Table 1). Selected tigers were similar in age, housed
individually, and acclimated to their enclosure. Two were female, and three were
male. Tigers were also chosen based on their experience with the transport
procedure. Two tigers had experienced the transport procedure at least two
times before our study began (experienced group). Both tigers had been
transported previously to be introduced to a new enclosure. Three tigers had

never experienced this transport procedure (naive group).

17

Table 1. Characteristics of each tiger subjected to transportation. Experience
refers to whether the tiger has experienced the transportation procedure previous
to the study.

 

 

 

 

 

 

 

1193 Sub-species Age (years) Gender Experience
1 Bengal/Siberian hybrid 7 Female Yes
2 Bengal 7 Male Yes
3 Corbetti 6 Male No
4 Siberian 6 Female No
5 Bengal/Siberian hybrid 8 Male No

 

 

 

 

 

 

All tigers were exposed to the same environmental conditions and daily
diet. They were enclosed individually outdoors in wire mesh cages with concrete
flooring. Each cage contained a small concrete den for shelter and was partially

covered by a tarpaulin cover for additional shelter from the summer sun.

Transport Procedure, Sample Collection, and Behavioral Observation

The transport procedure was consistent with the recommended transport
procedure outlined by the AZA’s Tiger SSP (Tiger lnforrnation Center, 2003). To
establish baseline cortisol levels, entire stools were collected from each tiger for
6 days prior to transport and immediately frozen. On day 6 each tiger was led
into a 2.7 x 1.2 x 1.2 m wire mesh cage. The cage was already mounted on a
flat trailer attached to a 4-wheel drive vehicle. All tigers readily entered the cage
when the door was opened within 5 min of having access to it. The tiger was
driven over gravel and dirt roads to a shaded area outside of the refuge walls
approximately 0.4 km from the original enclosure. The relocation site was not in

close proximity to any other animals. Following transport, each tiger was

18

released back into its original enclosure. Each transport episode lasted 30 min
from the time the cage door was locked to the time the tiger was released back
into the original enclosure, but the average time in transit was 8.5 min. Voided
feces during transport were collected (if necessary) afterwards. Finally, feces
was collected continuously during the 12 d following transport so that IR cortisol
concentration could be determined.

Tigers were videotaped in their home enclosure for 1 h prior to transport,
during transport, and for 1 h following release from the transport cage. Behavior
was subsequently analyzed for all 5 tigers using behavioral observation recording
software (Observer Base Package for Windows Version 3.0, Noldus Information
Technology, 1996). The behavioral analysis included activity level, time spent
pacing, pace rate (turns per min), time spent investigating (sniffing transfer cage),
and ear position.

Tiger respiration rate (breaths per min) was estimated by counting rib
cage flex at 15 s intervals. The only common time respiration rate could be
observed for all tigers was approximately 10 min before, during, and
approximately 10 min after transport. For one tiger rib cage flex could not be

seen so respiration rates could only be counted for four tigers.

IR Cortisol Extraction and Radioimmunoassay
The entire fecal sample was combined with a 20% methanol, 80% distilled
water solution so that they were in a 1:4 proportion (weight by weight). The

resulting mixture was homogenized thoroughly using a tissue homogenizer

19

(Brinkmann Instruments, Westbury, NY) and filtered through 7 mm2 gauge wire
mesh to eliminate bones and hair. Three aliquots (1.5 mL each) were kept to
determine dry matter content. Next, 40 g of solution was transferred to a 50 mL
polypropylene tube (USA Scientific Incorporation, Ocala, FL) and centrifuged at
3,000x g for 30 min. Then, 1.5 mL supernatant was transferred to a 1.5 mL
eppendorf tube, and each tube was microcentrifuged at 13,000x g for 15 min
(these steps were performed in triplicate). One mL of the supernatant was
transferred to an RIA tube (125l-cortisol RIA kit; Coat-A-Count; Diagnostic
Products Corporation, Los Angeles, CA), and immediately dried in a vacuum
concentrator (Appropriate Technical Resources Inc.; Laurel, MD). Dried
supernatant was resuspended in 200 uL distilled water before RIA. Resulting RIA
values were corrected for water content as performed by Wilson et al. (2002)
using the determined proportion dry matter from the three aliquots taken.

This method for extracting glucocorticoid metabolites from tiger feces was
biochemically validated by adding 10 pL of 3H-cortisol to 6 original samples of 40
g feces. Cross-reactivity of the antibody was as follows: 100 % with cortisol, 11.4
% with 11-deoxycortisol, 1.6 % with betamethasone, 0.98 % with cortisone, 0.94
% with corticosterone, 0.26 % with 11-deoxycorticosterone, 0.34 % with
tetrahydrocortisol, and < 0.1 % with testosterone, aldosterone, androsterone,
progesterone, estriol, estradiol, and estrone. The assay sensitivity of the RIA kit

is 0.2 ug/dL. The intra-assay coefficient of variation was 5.1 %. After extraction,

the average recovery of 3H-cortisol was 79.4 i 0.1 %.

20

Indicators of accuracy and parallelism were obtained by adding 500 pg of
5 different cortisol concentrations (0, 0.012, 0.062, 0.123, and 0.246 ug/dl) to 40

g of 5 separate aliquots of the same homogenized fecal sample. After extraction,
we recovered 8110.2, 83:0.3, 92i0.1, 91:l:0.1, 93:0.1% of cortisol respectively.
Although this protocol has not been pharmacologicaly validated through
conducting an ACTH challenge, the biological response associated with
transportation stress indicates that changes in IR fecal cortisol serve as an index
of adrenal activity. Moreover, the magnitude of the response among tigers may

reflect individual differences in HPA-axis activity.

Statistical Analysis

We used a repeated measures design to compare differences in cortisol
concentration between a 3 d block before transport (-3 to 0) with each 3 d block
after transport representing time frames 0 to 3, 3 to 6, 6 to 9, and 9 to 12 d after
transport. The RIA-determined IR fecal cortisol concentration from each
defecation was averaged (Mean i SE) within 3 d blocks due to irregularity in
defecation patterns between tigers. This was to ensure that we had analogous
data for each tiger at each corresponding time block and to avoid potential
effects of circadian patterns on hormonal production. Missing data for tiger 5 for
one of the blocks resulted in this subject being omitted from the analysis.
Differences between experienced and nah/e tiger behavior and respiration rate
were determined using a one-way analysis of variance (SAS Institute Inc., Cary,

North Carolina, Copyright 9 2001).

21

RESULTS
Behavioral Effects

Response to transport varied between tigers. Time budgets for all tigers
before and after transport are given in Table 2. There were no significant
differences between any of the five tigers' behavioral time budgets before and
after transport. All tigers spent at least 5 min lying down immediately after
transport, and tigers spent an average (Mean :1: SE) of 75 j: 13 % of the hour

following transport lying.

Table 2. Activity budget of each tiger one hour before and one hour after
transport in percentage of time spent performing each state

Tiger Lay Stand Walk Pace Sit
Before After Before After Before After Before After Before After

1* 83.0 99.0 1.5 0.5 4.6 0.6 10.0 0.0 0.0 0.0

2* 100.0 84.0 0.0 4.6 0.0 5.9 0.0 5.5 0.0 0.0

3 25.2 24.6 6.1 5.3 4.5 2.8 62.5 67.3 1.7 0.0

4 72.7 87.9 7.2 2.2 6.1 4.6 8.7 5.3 5.4 0.0

5 100.0 80.3 0.0 6.1 0.0 13.6 0.0 0.0 0.0 0.0
* denotes experienced tiger

Time budgets for all tigers during transport are given in Table 3. One
experienced tiger spent almost the entire time upright (standing, slow pacing, or
propping on the side of the cage) during transport. When this tiger paced, the
average rate was 17 turns per min. The other experienced individual alternated
between standing, walking, and laying. One naive tiger paced rapidly the

majority of the time (63 %) with an average of 35 turns per min. The second

22

naive tiger sat the majority of the time (73 %), and the third laid the majority of

the time (63 %).

Table 3. Percent of time tigers spent performing each behavior during transport.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tiger Lay Stand Walk Sit Prop Pace Investigate Ears
Back
1* 47.5 20.4 30.8 0.0 1.2 0.0 11.0 8.6
2* 0.5 26.0 43.1 0.0 30.5 34.0 3.9 3.9
3 10.4 11.6 4.5 7305 0.0 0.0 2.6 78.7
4 12.7 11.6 73.9 0.0 0.6 73.9 0.6 92.5
5 63.4 11.4 19.5 5.7 0.0 2.3 0.3 74.5

 

 

* denotes experienced tiger

Na'ive tigers had their ears oriented back and low (82 :I: 5.4 %) for a
significantly greater percentage of time (P = 0.01) than experienced tigers (6 i:
2.7 %). Experienced tigers spent an average of 7.5 :l: 3.6 % of the time

investigating the transport cage, while naive tigers spent an average of

1.4i 1.0%.

Physiological Effects

For one tiger (tiger 3) rib cage flex could not be seen. Therefore, analysis
was performed for 4 out of the 5 tigers. Baseline respiration rates varied
between tigers (Figure 2). The average (Mean :5 SE) respiration rate of all tigers
10 min before transport was 56.1 i 17.9 breaths/min. During transport the
average respiration rate was 94.6 :I: 19.3 breaths/min (~169 % above baseline),

and 10 min after transport it was 132.3 i 9.5 breaths/min (~236 % above

baseline).

23

 

160
140
120

 

 

 

 

i 100 lBefore
g 80 IDuring
3 6° UAfter

40
20

 

1' 2* 4 5

Tlger
‘ denotes experienced group

 

 

 

Figure 2. Respiration rates for 4 tigers 10 min before, during, and 10 min after
transfer.

The average respiration rate of the experienced group was 166 i 57 %
above baseline during transport and 195 :I: 56 % above baseline after transport
(Figure 3). The average respiration rate of the naive group increased to 230 i 84
°/o above baseline during transport and 394 i 36 % above baseline after
transport. The differences between the experienced and naive groups were not
significant during (P = 0.66) or after (P = 0.14) transport.

The time block in which IR fecal cortisol concentration peaked varied
between the 4 tigers analyzed for IR fecal cortisol (Table 4). Peaks ranged from
the 0 to 3 d block to the 6 to 9 d block. After averaging all tigers’ IR fecal cortisol
concentrations within each block (Figure 3), IR cortisol concentrations peaked
after transport during the 3 to 6 d block. At peak levels the IR cortisol
concentration increased to 239 % above baseline concentration. Recovery time

also varied between tigers. The 0 to 3 d, 3 to 6 d, and 6 to 9 d blocks were all

24

significantly higher than baseline (P = 0.02, P < 0.01, and P = 0.01 respectively).
On average, IR cortisol concentration returned to levels comparable to baseline
during the 9 to 12 d block. Samples collected during this time block did not differ

significantly from baseline (P = 0.15).

Table 4. Immune reactive fecal cortisol concentrations (ng/g) for each time block
of 4 tigers. Time 0 represents time of transport. Baseline refers to three days
rior to transport.

 

 

 

 

 

 

 

 

 

 

 

 

Tiger Baseline 0 to 3 Days 3 to 6 Days 6 to 9 Days 9 to 12 Days
1* 35. 0 :l: 0.4 42.2 d: 1.4 79.3 i 1.3’ 52.4 i 4.1’ 47.4 :I: 1.1’
P = 0.086 P = 0.002 P = 0.003 P = 0.025
2* 88.1 :t 1.2 171.4 :I: 4.0+ 137.8 :I: 1.9+ 116.0 :I: 2.0 82.7 :I: 2.1
P < 0.001 P = 0.044 P = 0.271 P = 0.834
3 58.3 :I: 9.6 61.8 :I: 1.1 56.9 i 5.5 241.2 i 8.8’ 54.9 :I: 1.5
P = 0.241 P = 0.226 P = 0.034 P = 0.901
4 29.4 i 6.1 134.0 :I: 2.0’ 365.9 1 6.1+ 110.3 1: 6.9‘ 34.0 i 2.1’
P < 0.001 P < 0.001 P = 0.02
Ave. 65.2 i 1.4 85.8 i 1.4’ 155.5 1: 4.6 112.5 :I: 4.1+ 57.5 i 2.4
P = 0.025 P < 0.001 P = 0.011 P = 0.152

 

* denotes experienced tiger
* denotes significantly higher than baseline concentration

25

 

 

‘8“

 

S

 

 

 

 

 

 

 

 

IR Fecal Cortisol Concentratlon (no/g)

 

-3100

 

 

 

0103

 

 

 

 

3 to 6
Tlme Blocks (days)

6109

 

 

 

I Experienced Tigers
13 Naive Tlgers

 

 

 

 

 

91012

 

Figure 3. Average immune reactive fecal cortisol concentrations of experienced

(N=2) and naive tigers (N=2). Day 0 represents time of transport.

Both average experienced and naive tiger IR cortisol concentrations

peaked during the 3 to 6 d time block (Figure 3). For experienced tigers the 3 to

6 d time block was the only one significantly (P < 0.05) higher than baseline. For

naive tigers IR cortisol concentrations were significantly higher than baseline (P <

0.05) for all blocks until the 9 to 12 d block (P = 0.74) when concentrations

returned to baseline. Average IR cortisol concentration for experienced tigers

during the peak interval was 158 % above average baseline concentration.

Average IR cortisol concentration for naive tigers during the peak interval was

482 % above average baseline concentration.

26

 

 

 

180

—l_l
88

IR Fecal Cortisol
Concentration (nglg)
8

 

-3100 0t03 3106 6109 91012
TlmeBIocks(days)

 

 

 

Figure 4. Average immune reactive fecal cortisol concentrations of experienced
and naive tigers combined. Day 0 represents time of transport

DISCUSSION

As we continue to find new measures to assess zoo animal welfare, we
must use them to better understand how common management practices may
affect their behavior and physiology, ensuring that the effects of these practices
do not impede an animal’s effectiveness as a conservation and educational
animal. This is especially important with felid species because very few studies
have identified behavioral or physiological effects of management practices.
Observed behavioral responses to transport may be indicative of negative
physiological responses.

The data collected on the IR cortisol concentrations in fecal samples
provides novel information on the HPA-axis activity of tigers, which has not
previously been documented. We found that baseline fecal glucocorticoid

concentrations in tigers appear to vary greatly between individuals as is true in

27

cheetahs (Jurke et al., 1997) and clouded leopards (Wielebnowski et al., 2002).
This may be due to individual variation in past experience, response to past
experience, metabolic functioning, or to subspecies in this study.

This is also the first time the effects of transportation stress have been
quantified in tigers. Although respiration rate increased overall during transport,
it increased further during the 10 min following transport, indicating that the
effects of the stressor may continue after the physical challenge ceases. All
tigers rested in a shaded area immediately upon release into their respective
enclosures after transport. The increase in respiration rate and immediate
resting response suggests that transport can be an exhausting experience for
tigers although high summer temperatures may have contributed.

The percent increase in cortisol levels following transport varied between
individuals, indicating that individuals respond to stress differently. However, it
may also reflect differences due to age, sex, or subspecies. The timing of the
observed peaks in IR cortisol concentrations also varied between individuals,
which may indicate differences in gut passage time. Finally, behavioral
responses varied between individuals. Two tigers reacted to the stressor actively
by pacing or walking the majority of time during transport; two tigers were
inactive by laying and sitting in the same spot with little movement the majority of
the time; and one tiger shifted between positions. This indicates tigers may
exhibit either flight or hiding behavior, which often results in pacing when
subjected to a restrictive environment, or they may exhibit an inhibition of

exploratory behavior or activity during transport. Therefore, activity level alone

28

without accompanying type of behaviors performed may not serve as a sufficient
indicator of tiger well-being.

Mammals respond differently to handling and transport procedures
depending on their past experience (Grandin, 1997). In both the physiological
and behavioral assessments, naive tigers seemed to respond more strongly to
the transport procedure than experienced tigers. Naive tiger respiration rates
increased by a greater percentage both during and after transport, although the
differences were not Significant. The average IR cortisol concentration of
experienced tigers increased after transport but was significantly higher than
baseline only during the peak block (3 to 6 d), whereas the average IR cortisol
concentration of naive tigers increased to levels significantly higher than baseline
in the block immediately following transport and for every block up to the 9 to 12
(1 block. The peak fecal corticoid concentration of naive tigers was also greater
in comparison to baseline levels than the peak fecal corticoid concentration of
experienced tigers. In spite of the low replication, our data support the
hypothesis that transport caused the naive tigers’ IR cortisol concentration to be
higher than the experienced tigers’, and that naive tigers were affected longer.

Although our physiological data do support the hypotheses that transport
caused an increase in cortisol production and resulted in a greater response by
naive tigers, one must be critical when just looking at glucocorticoid response.
An increase in glucocorticoid production also can occur as a result of positive
stimuli. For example, De Jong et al. (1998) showed that pigs housed in a more

enriched environment had greater levels of salivary cortisol than did pigs housed

29

in a less enriched environment. It has also been shown that cortisol production
in horses increases during mate introduction (Colbom et al., 1991). Therefore,
behavioral data should be coupled with glucocorticoid data before determining
whether a stimulus can be considered ”good” or ”bad” stress.

The observed physiological effects correspond to similar behavioral
trends. In both the experienced and the naive group at least one tiger was
predominantly active during transport and at least one tiger was predominantly
inactive. The major difference between the naive and experienced tiger groups
was the intensity of the behaviors performed. Naive tigers tended to respond
with greater intensity than experienced tigers. The naive tiger that paced the
majority of time did so almost continuously (stopping only once to rest) and at a
much faster rate than the experienced tiger that paced. Conversely, the
experienced tiger paced more slowly and stopped pacing more often. This
stereotypy (pacing) may have been a result of the tiger’s inability to express flight
behavior. The more intense performance of pacing may be indicative of a
greater motivation to perform the behavior. Similarly, naive tigers that were
inactive (sitting or lying the majority of time) simply remained in one spot while
the experienced tigers alternated between states. This possibly indicates a
greater suppression of exploratory behavior and activity due to higher levels of
fear.

Finally, we looked at ear position and investigative behavior. In our study,
experienced tigers had their ears positioned back and low approximately 6 % of

the time, while naive tigers did approximately 82 % of the time. This suggests

3O

that the naive tigers were experiencing fear or were defensive the majority of the
time, and experienced tigers may have been either relaxed or even interested.
Naive tigers also performed less investigative behavior than experienced tigers.
This is expected if naive tigers were experiencing more fear as fear may inhibit
exploratory behavior (Manser, 1992).

Novelty can be both a strong stressor (Dantzer and Morrnede, 1983), or it
can be stimulating (Grandin, 1997), depending on how it is presented. If the
novel item or situation is threatening in any way, or if the animal has no control
over the situation, it is likely to react adversely to it. Negative past experiences
during a procedure will result in a negative association with that procedure
(Mendl et al. 2001). This will further result in either a lack of cooperation by the
animal or an increase in the adverse effects of the stressor. Therefore, it must
be ensured that experience with a stressor is not negative so that adverse effects
can be eliminated.

Animals have been able to habituate to many common procedures.
Wienker (1986) showed that giraffes could be trained to voluntarily enter a
squeeze cage. To motivate animals to cooperate, one must first present the
novel situation in a way that will not be threatening. Positive stimuli should be
provided to counteract any negative stimuli that may result from a procedure.
For example, Grandin et al. (1995) showed that nyala antelope could be trained
for blood sampling by using a food reward as positive reinforcement. In the case
of potentially stressful situations like transport, it is the manager's job to make the

procedure as positive as possible. Currently, it Is common practice for zoos to

31

crate train animals before transport. This study further supports the importance
of properly acclimating animals to transport elements through procedures such
as crate training to reduce stress. Through positive reinforcement, it may even
be possible to stimulate a positive association with transport. If this is the case,
animals may even await transport, and the negative stress previously
experienced will transform into positive stress.

This study demonstrated that stress from transport even as short as 30
min can affect a tiger physiologically for up to 9 to 12 d. We suggest that the
duration of the challenge is not the only factor contributing to the physiological
response. Other factors that may have contributed to increased release of IR
fecal glucocorticoids include confinement, forced movement, cage motion,
handling by humans as perceived by the animal, or novel sensory stimuli
experienced during transport.

We anticipate that during transport from one zoo to another, there are
even more factors that may play a role. In this study, tigers were re-released into
their original, familiar enclosure. When transported to another 200, an animal
must withstand much longer durations of confinement in the transfer cage,
become accustomed to an unfamiliar environment, and adjust to new people,
management procedures, and sensory stimuli. It is conceivable that such
extended transport could result in more extreme effects. Therefore, future
studies should focus on one of the individual factors suggested to better

understand the extent to which each factor affects tigers.

32

CONCLUSIONS

1. IR cortisol concentrations can be measured in tiger feces and may be used
as an indicator of glucocorticoid production in response to stressful stimuli.

2. Even short-terrn transportation procedures can cause significant increases in
IR cortisol concentration of tigers.

3. Response repertoires vary from active to inactive between individual tigers
during transport.

4. Previous exposure to transport correlated with less adverse behaviors. Naive
tigers were more extreme in their activity level time budget. They also
investigated less and had their ears oriented back for a much greater percentage
of time. Such repertoires may be indicative of poor tiger welfare.

5. Effects of transport on glucocorticoid production may be reduced by

increasing tiger experience with elements of the management procedure.

33

Chapter 4: The effects of transport from one 200
to another on the HPA-axis of tigers

INTRODUCTION

An entire transport procedure can be divided into three major stages each
of which may induce a stress response in tigers (Kranz, 1996). The first stage is
preparation for transport. This stage Includes all novel stimuli a tiger may
experience before transport such as crate training or changes in daily husbandry.
The second stage is the transportation method. This stage includes the method
used to crate the tiger, the method used to move the crate, the stimuli the tiger
experiences while in the crate, and the method used to release the tiger. The
third stage is the introduction into a new enclosure. Included in this stage are all
stimuli the tiger experiences after it is released into its new enclosure such as the
physical structure of a novel environment and novel management practices.

The 30-min controlled study in the previous chapter was designed to test
only the effects of two factors common to all transport procedures, crating a tiger
and relocating it. Since these factors are always a part of transport procedures,
the effects measured in that study could be considered the minimum or baseline
effect of the transport procedure. When tigers are transported between zoos,
many more factors contribute to transportation stress. For example, the duration
the tiger is crated is always longer than 30 min. Depending on the distance
between facilities, tigers may be crated for as little as three hours or as much as

one week according to Felid Taxon Advisory Group (TAG) standards

34

(Shoemaker, 2003). The effect crate duration has on the stress axis of tigers is
currently unknown.

The method each 200 uses to crate a tiger may vary. Sometimes a tiger
must be anesthetized because either the facility‘s structure does not allow for
simple transfer into a crate or because the tiger is uncooperative, while other
times facilities have the structure and are able to crate train, making the use of
anesthesia unnecessary. Similarly, the method of transport once the tiger is
crated may vary. If the distance between Institutions can be driven, usually a
specialized van or truck will be used. For long journeys, or journeys over—seas, a
tiger may spend time in an airplane or a vessel. The effect that different transport
methods have on tigers has not yet been quantified.

Finally, factors exist that may affect a tiger after arrival at a new facility.
First, the tiger is exposed to a completely new environment. In the previous
study, tigers were released into the same enclosure where they originally
dwelled, therefore eliminating a novel environment as a factor that contributed to
the stress response. In AZA-accredited zoos, tigers are required to be
transported to a quarantine holding area for 30 days before being allowed on
exhibit (Shoemaker, 2004). As a result, tigers are often being moved from a
familiar, large, and highly enriched area to an unfamiliar, small, and poorly
enriched area. Holding areas seldom provide for adequate flight or hiding
behaviors and because individuals may respond to novelty with fear, stereotypic

behaviors may be performed (Manser, 1992).

35

Included in a new environment are novel sensory stimuli. For example, a
tiger may experience for the first time loud noise from construction that is being
performed near its new enclosure, or it may experience novel smells caused by
different surrounding plants. How an individual responds to novelty can vary
both behaviorally and hormonally (Birke, 1979) and can often be influenced by
past experience (Joseph and Gallagher, 1980) as was seen in the 30-minute
controlled study.

Similarly, the tiger is exposed to novel management procedures. Due to
the dynamic differences between each zoo, no two zoos manage an animal in
exactly the same way. For example, the 200 that the tiger arrived at may feed at
a different time of day or may feed a different type of diet than the 200 from which
the tiger came. A tiger also must adjust to being managed by new individuals.
Often animals may react more adversely to unfamiliar individuals. Boivin, et al.
(1998) reported that calves were more likely to allow a familiar person to touch
them more quickly than an unfamiliar person.

The ultimate goal of understanding the effects of transportation stress on
tigers is to understand how each one of these factors contributes to stimulating
the stress response. While the previous chapter dealt with understanding the
baseline response to transport, one goal of this chapter is to assess the effects of
common transport procedures between zoos on the hypothalamus-pituitary-
adrenal (HPA) axis of 4 different tigers during 4 different transportation episodes.
Since there were differences between the tigers and the transportation episodes,

another goal of this study is to compare the effect different factors involved with

36

different transportation procedures have on tigers. A common transport
procedure includes all factors previously discussed and therefore, can be
described as the common response to a transport procedure. The final goal of

this study is to compare the baseline response with the common response.

METHODS

The Michigan State University All-University Committee on Animal Use
and Care approved this study as did the AZA’s Tiger SSP. Included in this study
were 4 tigers. Characteristics of each tiger are given in Table 5. All tigers were
housed individually during the study, fed a consistent daily diet, and housed on
exhibit for a month before transport. All tiger were healthy for the month before
transport. After transport no medical problems or major behavioral problems
were observed. The tigers were again fed a consistent diet and were housed
individually in quarantine for the 30 days after transport. Tiger 4 was exposed to
construction noise after transport and zookeepers observed an increase in

pacing while noise from construction was loudest.

Table 5. Characteristics of each tiger transported between zoos.

 

 

 

 

 

 

Tiger Sub- Age Gender Duration Transport Transport
species (yrs) Crated (hr) Method Experience
1 Amur 9 Male 14 Van, Twice
Anesthetized previously
to be crated
2 Amur 3 Female 9 Van None
3 Sumatran 12 Male 4 Van, Three times
Anesthetized previously
to be crated
4 Corbetti 3 Female ~12 Airplane, Once
Anesthetized previously
for release

 

 

 

 

 

 

 

 

37

The transport procedures were consistent with the recommended
transport procedures outlined by the AZA’s Tiger SSP [Tiger lnforrnation Center,
2003]. Entire stools were collected from each tiger for 6 days prior to transport
and immediately frozen. Voided feces were collected from each tiger during
transport (if necessary) and continuously for 28 days following transport so that
Immune Reactive (IR) cortisol concentration could be determined. Occasionally,
fecal samples could not be collected daily because the tiger was not being
cooperative (especially immediately after transport) or because the tiger did not
defecate. For comparison, 8 mo after transport feces was collected every third
day from Tiger 1 (control) for one month to ensure that there was not a monthly
spike in cortisol that coincided with spikes assumed to result from transport.
Cortisol was extracted using the procedure outlined in Chapter 2 and measured

using Radioimmunoassay (RIA).

Statistical analysis

Resulting IR fecal cortisol concentrations from samples collected before transport
were averaged for each tiger individually and for all tigers collectively. All IR
fecal cortisol concentrations derived from samples after transport were averaged
into 3-day blocks. Then, a repeated measures test was used to compare the
average IR fecal cortisol concentration before transport with each average
concentration within each 3-day block after transport for each tiger individually

and for all tigers collectively.

38

RESULTS

Each tiger showed a significant spike in cortisol following transport (Table 6,
Figure 5.). Tiger 1’s cortisol level peaked during the 3 to 5-day block to 434 °/o
greater than the baseline value. The IR fecal cortisol concentration of this tiger
did not return to baseline until the 15 to 17-day block. The IR fecal cortisol level
of Tiger 2 reached a maximum during the 6 to 8-day block to 494 % greater than
baseline. Her cortisol level did not return to baseline until the 12 to 14-day block.
Tiger 3’s cortisol level peaked on the 0 to 2-day block to 516 % above baseline.
His cortisol level did not return to baseline until the 6 to 8-day block. Finally,
Tiger 4’s cortisol level peaked during the 6 to 8-day block to 341 % above
baseline. Although Tiger 4’s cortisol levels never significantly returned to

baseline, it did level off during the 15 to 17-day block.

39

Table 6. Mean 1 SE of immune reactive fecal cortisol concentrations (ng/g) for
each time block of 4 tigers. Time 0 represents time of transport. Baseline refers
to the average fecal cortisol concentration for 6 days prior to transport. N is

variable from 2 to 3 for individual tiggs and variable from 8 to 12 for all tigers.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time Tiger 1 Tiger 2 Tiger 3 Tiger 4 All Tigers
Block
Baseline 7.5 1 0,7 6.2 1 0,5 5.3 1 0.5 16.1 1: 1.0 8.9 1 0.6
0 to 2 24.3 1 3.5+ N/A 27.5 1 2.1’ 38.0 1 1.2’ 29.6 1 25’
Days P < 0.001 P < 0.001 P < 0.001 P < 0.001
3 to 5 32.6 1 19.8+ 19.8 1 3.5’ 20.4 1 4.3’ 50.4 1 7.8+ 26.9 1 3.6’
Days P = 0.014 P < 0.001 P < 0.001 P < 0.001 P = 0.014
6 to 8 15.0 1 5.8 30.9 1 1.8+ 6.3 1 1.8 54.9 1 2.5’ 28.7 1 3.7
Days P = 0.206 P < 0.001 P = 0.596 P < 0.001 P = 0.206
9 to 11 27.6 1 5.8’ 15.3 1 1.9 9.4 1 1.5’ 40.0 1 3.0’ 21.0 1 26’
Days P = 0.001 P = 0.214 P = 0.011 P < 0.001 P = 0.001
12 to 14 18.0 1 3.2’ 7.0 1 0.4 24.7 1 6.2’ 35.3 1 0.8’ 21.6 1 28’
Days P = 0.002 P = 0.128 P = 0.003 P < 0.001 P = 0.002
15 to 17 5.5 1 0.3’ 10.0 1 2.4+ 4.6 1: 0.9 30.3 1 1.6’ 12.4 1 24*
Days P = 0.013 P < 0.001 P = 0.459 P < 0.001 P = 0.014
18 to 20 8.4 1 1.2 11.3 1 0.4‘ 3.7 1 0.3’ 32.6 1 1.5’ 15.2 1 2.2
Days P = 0.579 P < 0.001 P = 0.010 P < 0.001 P = 0.579
21 to 23 17.2 1 3.7’ 11.3 1 0.8’ 4.8 1 0.6 49.5 1 3.0’ 15.1 1 31’
Days P = 0.013 P = 0.030 P = 0.458 P < 0.001 P = 0.013
24 to 26 19.5 1 4.2’ 24.1 1 8.0 2.11 1 0.1‘ 53.2 1 0.1’ 23.2 1 42*
Days P = 0.006 P = 0.200 P < 0.001 P < 0.001 P = 0.006
> 27 12.6 1 3.1 7.5 1 0.8 N/A N/A N/A
Days P = 0.115 P = 0.085

 

* denotes significantly higher than baseline concentration

40

 

 

Tiger 1

Tiller 1
Gantrol

nela
88888

 

0

before 0102 3105 6108 91011121014151017101020211023241026 27+
Time Blocks (days)

 

 

Tiger 2

 

before 0102 3105 6108 91011 121014151017131020 211023 241026 27+
TTme Blocks (clays)

 

 

 

 

Tiger 3

 

before 0102 3105 6103 91011 121014 151017 181020 211023 241026
Time Blocks (days)

 

 

 

Tiger 4

“9’9

 

before 0102 3105 6108 91011 121014 151017 181020 211023 241026
Tlme Blocks (days)

 

 

 

Figure 5. IR fecal cortisol concentrations of Tigers 1-4 for each 3-day time block.

41

All tigers appeared to have a second spike in IR fecal cortisol
concentration at least one week after levels returned to baseline. Each one of
these spikes was significantly higher than baseline, but not as high as the initial
spike. Finally, after averaging all tigers' IR fecal cortisol concentrations into 3-
day blocks (Figure 6.), a peak occurred during the 6 to 8-day block to 333 %
above baseline and remained elevated until the 15 to 17-day block. The second

spike occurred during the 24 to 26-day block.

 

 

 

888

 

nelo

 

 

     

 

 

 

‘ 1.. ' f. 2 -:

- '._. 1 r _., ".".~...i£=r .—-"' .1. I. .‘.'I -.-.Z :31 1.111 :3

before 0102 3105 6108 91011 121014 151017 181020 211023 241026
Tlme Blocks (days)

a
CO

 

 

 

 

 

Figure 6. Average IR fecal cortisol concentrations of all tigers.

DISCUSSION

Based on the 30-minute controlled experiment, simply crating a tiger for 1/2 hour
caused IR fecal cortisol levels to increase to 239 % above baseline on average.
It also caused cortisol levels to remain elevated for an average of 9 to 12 days.
Since a greater number of stressful factors (longer duration crated, introduction
into a new environment, etc.) are experienced during all common transport

procedures between zoos, it is expected that during transport the average tiger’s

42

cortisol level will increase by at least this baseline factor of 2.39, and tigers will
be affected for at least 9 days. In this study, the average spike in IR fecal cortisol
was 333 % above baseline, and levels returned to baseline during the 15 to 17-
day block showing that tigers experienced a higher spike as well as a longer
duration of increased IR fecal cortisol concentration when transported between
zoos.

With the knowledge that simply crating and moving each tiger stimulates a
239 % spike in IR fecal cortisol concentration and it takes 9 to 12 days to
recover, how do the other factors of transport duration, method of transport, and
introduction into a novel environment contribute to the remaining increase in
cortisol production and recovery time? For example, how does anesthetizing a
tiger for crating or release affect cortisol production? Is stress influenced by a
novel environment responsible for the prolonged recovery time, or does the
duration of transport have a greater effect on recovery time?

Although the low sample size in this study does not allow definitive
statements about how these factors contribute to transportation stress, trends
can be identified. For example, the tiger with the most transport experience and
the shortest crate duration (Tiger 3) recovered more quickly than tigers with less
experience and longer crate durations. These results parallel results from the
study in Chapter 3 in which individuals with transport experience recover more
quickly than individuals with no transport experience. Braastad et al. (1998)
reported that short stressors such as loud noise and short-term human exposure

(20 seconds) did not affect silver fox vixen (Vulpes vulpes) body temperature and

43

activity, while long-term stressors such as human handling for 5 minutes or
human presence for 90 minutes caused significant increases in both body
temperature and activity. Therefore, shorter crate durations may reduce
recovery time.

It is recommended that every mammal be housed under conditions that
allow shifting and crating without the use of anesthesia (Bush, 1996).
Unfortunately, all facilities are not yet appropriately structured for simple shifting
into a crate, and sometimes tigers are difficult to crate train. In this study, three
tigers (Tiger 1, Tiger 3, and Tiger 4) were anesthetized either during crating or
while being removed from the crate. Anesthesia has been shown to reduce
cortisol production during surgical procedures (Graf and Senn, 1999, and Dalin et
al., 1993). Dalin et al. (1993) also showed that significant increases in cortisol
production were delayed until recovery from anesthesia. Both anesthetized
tigers experienced lower spikes in cortisol, but it took them longer to recover from
transport than the tigers not anesthetized. A delay in recovery may be due to an
anesthesia-induced delay in initial increased adrenal activity.

Just as the 30-minute controlled study provided data quantifying a
baseline stress response to transport, this study provided data quantifying the
average response of tigers to transport between zoos. This study along with the
previous 30-minute controlled study lay the foundation for future studies to tease
apart the effects each potential stressor (crate duration, transport method used,
and introduction into a new environment) has on tiger behavior and physiology.

This study also lays the foundation for other future studies. For example, all

tigers in this study experienced a second spike in cortisol 6 to 12 days after
recovery. The long-term effects of transport on cortisol production and behavior
should also be explored as additional spikes in cortisol after transport may affect

tiger behavior and ultimately tiger welfare.

45

Chapter 5. Conclusions

To continue improving welfare standards, we must expand our understanding of
the effects captive management practices have on 200 animals by devising ways
to measure these effects. The fecal cortisol extraction protocol used in the
previous studies effectively measures long-term effects of stressful events on the
tiger HPA-axis. Fecal cortisol may also be used to measure short-term effects
grouped into 3-day blocks. Before fecal cortisol can be used to assess fluxes in
adrenal activity during time periods shorter than three days, we must improve our
understanding of the lag time between when cortisol is produced and when it
shows up in feces. The data presented here suggest that lag time may vary
between individuals.

In tigers, behavioral indicators of stress before, during, or after transport
may not be obvious. Therefore, physiological indicators may be more important
in detecting stress-related effects. Tiger IR fecal cortisol concentrations increase
as a result of transport. To reduce stress, steps may be taken such as crate
training and introduction to transport components and other novelties a tiger may
encounter. During transport between zoos the method used and the duration
crated may have a significant impact on tiger stress levels. Further studies
should explore the effects of increased crate duration and different transport
methods.

Although tigers displayed a second spike in IR fecal cortisol concentration
after transport, signifying that there is a longer-term effect than just one spike in

cortisol production, chronic effects were not observed. Therefore, crating or even

46

transport may serve as an effective means to stimulate a short-term stress
response. Stimulating short-term stress may be an effective way to exercise
natural behaviors in tigers such as mating or flight behavior. The implications of

utilizing stress as a means to exercise natural behavior should be explored

further.

47

LITERATURE CITED

Baker, M. L., and Gemmell, R. T. 1999. Physiological changes in the brushtail
possum (Tn'chosurus vulpecula) following relocation from Arrnidale to
Brisbane, Australia. Journal of Experimental Zoology. 284(1): 42-49.

Baxter, E. and Plowman, A. B. 2001. The effect of increasing dietary fibre on
feeding, rumination, and oral stereotypies in captive giraffes (Giraffe
camelopardalis) Animal Welfare. 1 0(3): 281 -290.

Berkeley, E. V., Kirkpatrick, J. F., Schaffer, N. E., Bryant, W. M., and Threlfall, W.
R. 1997. Serum and fecal steroid analysis of ovulation, pregnancy, and
parturition in the black rhinoceros (Diceros bicomis). Zoo Biology. 16: 121-
132.

Birke, L. I. A. 1979. Object investigation by the oestrous rat and guinea-pig: the
oestrous cycle and the effects of oestrogen and progesterone. Animal
Behaviour. 27:350-358.

Boivin, X., Garel, J. P., Mante, A., and Le Neindre, P. 1998. Beef calves react
differently to different handlers according to the test situation and their
previous interactions with their caretaker. Applied Animal Behaviour
Science. 55(3-4): 245-257.

Braastad, B. 0., Osadchuk, L. V., Lund, G., and Bakken, M. 1998. Effects of
prenatal handling stress on adrenal weight and function and behaviour in

novel situations in blue fox cubs (Alopex Iagopus). Applied Animal
Behaviour Science. 57 (1 -2): 1 57-169.

Bradshaw, R. H., Hall, S. J. G., and Broom, D. M. 1996. Behavioural and cortisol
response of pigs and sheep during transport. Veterinary Record. 138: 233-
234.

Broom, D. M. and Johnson, K. G. 1993. Stress and Animal Welfare. London:
Chapman & l-lall Inc. 95 p.

Brown, Janine L. 2000. Reproductive monitoring of elephants: An essential tool
for assisting captive management. Zoo Biology. 19(5): 347-367.

Brundige, L., Oleas, T., Doumit, M. and Zanella. A.J. (1998) Loading techniques
and their effect on behavioral and physiological responses of market
weight pigs. Journal of Animal Science, 76, supp. 1.:99.

Bush, Mitchell. 1996. Methods of Capture, Handling, and Anesthesia. In: Wild
Mammals in Captivity. The University of Chicago Press. Chicago, Illinois.

48

Colbom, D. R., Thompson, D. L., Roth, T. L., Capehart, J. S., and White K. L.
1991. Responses of cortisol and prolactin to sexual excitement and stress
in stallions and geldings. Journal of Animal Science. 69(6): 2556-2562.

Conway, William. January 2000. The Changing Role of Zoos and Aquariums in
the 21"t Century. Communique. 11-12, 47.

Dantzer, R., Momiede, P. 1983. Stress in farm animals: a need for reevaluation.
Journal of Animal Science. 57: (1 ): 6-18

De Jong, l. C., Ekkel, E. D., Van de Burgwal, J. A., Lambooy, E., Korte, S. M.,
Ruis, M. A. W., Koolhaas, J. M. and Blokhuis, H. J. 1998. Effects of
strawbedding on physiological responses to stressors and behavior in
growing pigs. Physiology and Behavior. 64(3): 303-310.

Fisher, James. 1967. Zoos of the World: The Story of Animals in Captivity. The
Natural History Press. Garden City, New York.

Goossens, M. M. C., Meyer, H. P., Voorhout, G., and Sprang, E. P. M. 1995.
Urinary excretion of glucocorticoids in the diagnosis of
hyperadrenocorticism in cats. Domestic Animal Endocrinology. 12(4): 355-
362.

Graf, Bruno and Senn, Markus. 1999. Behavioural and physiological responses
of calves to dehoming by heat cauterization with or without local
anesthesia. Applied Animal Behaviour Science. 62(2-3): 153-171.

Graham, Laura H. and Brown, Janine L. 1996. Cortisol metabolism in the
domestic cat and implications for non-invasive monitoring of
adrenocortical function in endangered felids. Zoo Biology. 15 (1): 71-82.

Grandin, T. 1997. Assessment of stress during handling and transport. Joumal of
Animal Science. 75: 249-257.

Grandin, T., Rooney M. 3., Phillips, M., Cambre, R. C., lrlbeck, N. A., and
Graffam, W. 1995. Conditioning of nyala (Tragelaphus engasr) to blood
sampling in a crate with positive reinforcement. Zoo Biology. 14: 261.

Johansson, Gunnar G., Rosset, Seija, Sandstroem, Marita, and Hannu, Soini.
1979. Increased fear reactions in cats after carbachol administered
intraventriculariy. Physiology & Behavior. 22(5): 803-807.

Joseph, R., and Gallagher, R. E. 1980. Gender and early environmental

influences on activity, overresponsiveness, and exploration.
Developmental Psychobiology. 13:527-544.

49

Jurke M. H., Czekala, N. M., Lindburg, D. G., and Millard, S. E. 1997. Fecal
corticoid metabolite measurement in cheetah (Acinonyxjubatus). Zoo
Biology. 16: 133-147.

Kranz, Karl R. 1996. Introduction, Socialization, and Crate Training Techniques.
ln: Wild Mammals in Captivity. The University of Chicago Press. Chicago,
llinois.

Lambooy, E. 1988. Road transport of pigs over a long distance: Some aspects of
behavior, temperature, and humidity during transport and some effects of
the last two factors. Animal Production. 46 (2): 257-264.

Levine, Eric S., Litto, Joseph W., and Jacobs, Barry L. 1990. Activity of cat locus
coeruleus noradrenergic neurons during the defense reaction. Brain
Research. 531 (1-2): 189-195.

Liu, Dingzhen, Wang, Zhipeng, Tian, Hong, Yu Changqing, Zhang, Guiquan,
Wei, Rongping, and Zhang, Heming. 2003. Behavior of giant pandas
(Ailuropoda melanoleuca) in captive conditions: Gender differences and
enclosure effects. Zoo Biology. 22(1): 77-82.

Mallapur, Avanti, and Chellam, Ravi. 2002. Environmental influences on
stereotypy and the activity budget of Indian leopards (Panthera pardus) in
four zoos in Southern India. Zoo Biology. 21 (6): 597-605.

Mason, G. J. 1991. Stereotypy: a critical review. Animal Behavior. 41: 1015-
1038.

Manser, Caroline E. 1992. The Assessment of Stress in Laboratory Animals.
London, UK: RSPCA, Harper Collins. Pp. 20-21.

Margodt, Koen. 2000. The Welfare Ark: Suggestions for a Renewed Policy in
Zoos. VUBPRESS. Brussels.

McGeehan, Laura, Li, Xuebing, Jackintell, Lori, Huang, Shiqiang, Wang, Aiping,
and Czekala, Nancy. 2002. Hormonal and behavioral correlates of estrus
in captive giant pandas. Zoo Biology. 21 (5): 449-466.

Mench, Joy A. and Kreger. 2000. Ethical and Welfare Issues Associated with
Keeping Wild Mammals in Captivity. In Kleiman et al. Wild Mammals in
Captivity: Principles and Techniques. The University of Chicago Press.
Chicago and London.

Mendl, M., Burman, 0., Laughlin, K., and Paul, E. 2001. Animal Memory and
Animal Welfare. Animal Welfare. 10: $141-$159.

50

Meriot, E., Meunier-Salaun, M., and Prunier, A. 2004. Behavioural, endocrine,
and immune consequences of mixing in weaned piglets. Applied Animal
Behaviour Science. 85(3-4): 247-257.

Mostl E. and Palme, R. 2002. Hormones as indicators of stress. Domestic Animal
Endocrinology. 23: 67-74.

Norton, Bryan G., Hutchins, Michael, Stevens, Elizabeth F., and Maple, Terry L.
1995. Ethics on the Ark: Zoos, Animal Welfare, and Wildlife Conservation.
Smithsonian Institution Press. Washington and London.

Roussel, S., Hemsworth, P. H., Boissy, A., and Duvaux—Ponter, c.2004. Effects
of repeated stress during pregnancy in ewes on the behavioural and
physiological resonse to stressful events and birth weight of their offspring.
Applied Animal Behaviour Science. 85 (3-4): 259-276.

Schatz, S. and Palme, R. 2001. Measurement of faecal cortisol metabolites in
cats and dogs: A non-invasive method for evaluating adrenocortical
function. Vet. Res. Commun. 25 , 271-287.

Seidensticker, J., Christie, 8., and Jackson, P. (eds.). 1999. Riding the
TigerzTiger Conservation in Human-dominated Landscapes. Cambridge
University Press.

Selye, H. 1984. The Stress of Life. McGraw—Hill Book Co., New York, NY.

Shapiro, Leland. 2000. Applied Animal Ethics. Delmar Thomson Learning.
Albany, NY.

Shoemaker, Alan H. 2004.200 Standards for Keeping Large Felids in Captivity.
American Zoo and Aquarium Felid Taxon Advisory Group website.
http://www.felidtag.orq.

Terio, Karen A., Brown, Janine L., Moreland, Rachel, and Munson, Linda. 2002.
Comparison of different drying and storage methods on quantifiable
concentrations of fecal steroids in the cheetah. Zoo Biology. 21: 215-222.

Thome, C. 1992. The Waltham Book of Dog and Cat Behavior.New
YorkzPergamon Press. 84-84 Pp.

The Tiger lnfonnation Center. 2003. httmflwwwfitkgscrg.
Ward, Paul l., Mosberger, Nicole, Kistler,Claudia, and Fischer, Oliver. 1998. The

relationship between popularity and body size in 200 animals.
Conservation Biology 12 (6): 1408-1411.

51

Wielebnowski, Nadja. 2003. Stress and distress: evaluating their impact for the
well-being of 200 animals. JA VMA. 223 (7): 973-976.

Wielebnowski, Nadja C., Fletchall, Norah, Carlstead, Kathy, Busso, Juan M., and
Brown, Janine L. 2002. Noninvasive assessment of adrenal activity
associated with husbandry and behavioral factors in the North American
clouded leopard population. Zoo Biology. 21: 77-98.

Wienker, W. R. 1986. Giraffe squeeze cage procedures. Zoo Biology. 5: 371.

Wilson, E., Dembiec, D. P., Laughlin, K., and Zanella. A. J. 2002. (poster) Dry
matter content and its effects on fecal cortisol measurement. Proceedings
of the 2002 International Society of Applied Ethology Regional
Conference. Quebec, Canada.

Wolf, OT 2003. HPA axis and memory. Best Practice & Research Clinical
Endocrinology & Metabolism. 17(2): 287-299.

Yoshinobu, Mori, Jingyi, Ma, Sansei, Tanaka, Kyoji, Kojima, Koji, Mizobe,
Chiharu, Kubo, and Nobutada, Tashiro. 2001. Hypothalamically induced
emotional behavior and immunological changes in the cat. Psychiatry and
Clinical Neurosciences. 55(4): 325-332.

52