The Influence of the Sound Environment on the Welfare Of

Home , Mico (genus), Pied tamarin

THE INFLUENCE OF THE SOUND ENVIRONMENT ON THE WELFARE OF

ZOO-HOUSED CALLITRICHINE MONKEYS

JASON D. WARK

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Kristen E. Lukas

Department of Biology CASE WESTERN RESERVE UNIVERSITY

August 2015

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of Jason D. Wark, candidate for the degree of Doctor of Philosophy*.

Signed:

______Mark A. Willis, Ph.D. (chair of the committee)

______Kristen E. Lukas, Ph.D.

______Mandi W. Schook, Ph.D.

______Christopher W. Kuhar, Ph.D.

______Charles T. Snowdon, Ph.D.

Date: 21 May 2015 * We also certify that written approval has been obtained for any proprietary material contained within.

This work is dedicated to:

Frances Chung, my partner and best friend; my parents, Brenda and Lee Wark, for instilling their love of animals;

and my friends, both human and animal, that have enriched my life.

Table of Contents

Acknowledgements ...... vii

Abstract ...... x

Chapter One: Introduction: Sound and its significance ...... 1

Chapter Two: Fecal glucocorticoid metabolite responses to management stressors and social change in four species of callitrichine monkeys ...... 32

Chapter Three: The influence of waterfall sounds and access to off-exhibit areas on the behavior and exhibit use of three species of callitrichine monkeys ...... 58

Chapter Four: Do zoo animals use off-exhibit areas to avoid noise? A case

study exploring the influence of sound on the behavior, physiology, and exhibit

use of two pied tamarins (Saguinus bicolor) ...... 81

Chapter Five: The response of callitrichine monkeys to auditory stimuli and the

importance of exhibit choices ...... 98

Chapter Six: A pilot investigation on the use of startle reactivity as a measure of

affect in callitrichine monkeys ...... 114

Chapter Seven: General discussion and future outlook ...... 134

References ...... 143

LIST OF TABLES

Chapter Two:

Table 1. Demographic background of study subjects...... 35

Table 2. Assessment of fecal markers fed to four species of callitrichine monkeys:

golden lion tamarin (GLT); callimico (CA), pied tamarin (PT); white-fronted

marmoset (WFM)...... 41

Table 3. Summary of fecal glucocorticoid metabolite response of four

callitrichine species after veterinary exams...... 43

Chapter Three:

Table 1. Demographic background of study subjects...... 61

Table 2. Overview of experimental design...... 62

Table 3. Ethogram of behaviors considered in this study...... 63

Table 4. Sound levels (median ± interquartile range) in the study exhibits with

the waterfall feature on and off...... 69

Table 5. The median (± IQR) number of visitor groupsa per observation at

exhibits across study conditions...... 70

iii

Chapter Four:

Table 1. Ethogram of select behaviors considered in this study...... 85

Table 2. The equivalent continuous (Leq) and median (L50) sound level indices

(median ± IQR) for the exhibit area when a waterfall feature was on and off and

for the off-exhibit area when a speaker broadcast white noise volume matched to

the waterfall noise on exhibit...... 90

Chapter Five:

Table 1. Description of sound conditions...... 101

Table 2. The duration, frequency, and tempo of playback stimuli...... 102

Chapter Six:

Table 1. Description of sound conditions...... 118

Table 2. Ethogram of general behaviors considered in this study...... 120

Table 3. Behavior of tamarins and marmosets before, during, and after exposure

to sound conditions and acoustic startle event...... 124

Table 4. Summary of startle response measures after an acoustic startle event

following sound conditions...... 129

LIST OF FIGURES

Chapter Two:

Figure 1. Fecal glucocorticoid levels of four golden lion tamarins (A: LF1; B:

LF2; C: LM1; D: LM2) after capture and anesthesia during a routine veterinary exam (time 0 h)...... 45

Figure 2. Fecal glucocorticoid metabolite concentrations in a female (A) and male (B) callimico before and after a routine veterinary exam (time 0 h)...... 47

Figure 3. Fecal glucocorticoid concentrations of a female pied tamarin (A) and her adult male offspring (B) before and after the death of the breeding male and veterinary exam (time 0 h)...... 48

Chapter Three:

Figure 1. The least squares means from GLMM behavior models for each species showing the effects of the waterfall (a-f) and access (g-l) conditions...... 72

Figure 2. The influence of waterfall noise on monitoring the visitor area when monkeys had no access off-exhibit and when they had access off-exhibit...... 73

Figure 3. The least squares means from GLMM exhibit use models for each species showing the effects of the waterfall...... 74

Chapter Four:

Figure 1. The percent of time spent off exhibit during sound conditions involving

experimental modifications of a waterfall feature and white noise played from a

speaker in the off-exhibit area...... 91

Figure 2. Fecal glucocorticoid metabolite concentrations (ng/g) for a male (grey

circles) and female (black Xs) pied tamarin across experimental sound conditions...... 93

Chapter Five:

Figure 1. Spectrograms of playback stimuli used in this study (A,B: Mozart; C,D:

Rainforest Sounds; E,F: Affiliation-based Tamarin Music; G,H: Fear/Threat-

based Tamarin Music)...... 103

Figure 2. Diagram of experimental playback procedure...... 105

Figure 3. The mean percent of time three callitrichine groups (pied tamarin,

golden lion tamarin, and callimico) spent in the location a speaker was placed

during auditory conditions...... 107

Figure 4. The mean group rate (occurrences/min visible) of behavior for three

callitrichine species (A: pied tamarin; B: golden lion tamarin; C: callimico) during

auditory conditions...... 109

Figure 5. The mean group rate (occurrences/min visible) of behavior for three

callitrichine species (A: pied tamarin; B: golden lion tamarin; C: callimico) during

periods with access to off-exhibit areas (Phase 1) and no access (Phase 2)...... 110

Chapter Six:

Figure 1. The percent of visible time marmosets spent inactive and locomoting

during baseline and playback periods for each of the seven sound conditions...... 125

Figure 2. The percent of visible time pied tamarins spent engaged in self-directed behavior during the 80 dB White Noise startle trial...... 127

vii

Acknowledgements

I am eternally grateful for the love, support, and guidance I have received from so many during this journey. First and foremost, I want to thank Franke Chung for not breaking up with me when I moved away to Cleveland and, even better, joining me on this adventure. From our first apartment, where we found a cat companion, to our current one, she has made the beautiful and relaxing retreat I am fortunate to call home. She has shown endless patience and support during my periods of endless stress for that I am thankful. Her attention to detail and standards of excellence for her own work have pushed me to be the most rigorous scientist I can be. This work would not be possible without her love and support.

I would not be here if it were not for the passion for animals and science that my parents, Lee and Brenda Wark, instilled in me. Our frequent trips to the Philadelphia Zoo and their enrolling me in summer zoo camp, as well as our trips to the San Diego Zoo and

Chicago Field Museum, inspired a curiosity of the natural world that will stay with me forever.

I have to thank Dr. Kristen Lukas, my academic advisor, for shaping that curiosity into a career. From our first meeting, Kristen has always been much more than an advisor and I cannot thank her enough for her friendship and guidance through these years. She has taught me to keep having fun, no matter how tough life gets. Also, I am especially grateful for her support of my frequent and diverse research interests, which surprisingly have yet to include gorillas. Kristen, I will never forget that you made this all possible for me and will always look to honor this opportunity by paying it forward.

viii

My entire dissertation committee has had a profound impact on my academic and professional development and I thank you all for sharing your time to make this effort possible. I am grateful for the personal mentorship Dr. Mandi Schook has given me and for always pushing me to do the best science I can, even though it did mean learning advanced statistics. The experimental nature of this project would not have been possible without the strong support from Dr. Chris Kuhar. Chris has always challenged me to endeavor for the highest standards of scientific rigor and practicality in my research designs and for that I am thankful. I am grateful for Dr. Mark Willis’s support of the zoo program, guidance through my initial years, and never letting my research thoughts wander too far from a hypothesis and predictions. My dissertation project has benefited greatly from Dr. Charles Snowdon’s extensive experience and invaluable insight into callitrichine species—I truly could not have found a better addition to my dissertation committee. Also, I am very appreciative of Dr. Snowdon’s near immediate email responsiveness and patience with our technology conferencing challenges. Finally, Dr.

Pam Dennis, although a casualty of a last minute dissertation rule decree, has been a part of this process from day one. Her cheerful attitude and passionate care for all species, big and small, will always be an inspiration to me.

I have been especially lucky to have shared an office with a fantastic team of people during my time at Cleveland. To my academic big sisters, Drs. Elena Less and

Grace Fuller: I thank you both for all the help and support you’ve offered me over the years and am grateful for the many laughs we’ve shared. I would like to thank Christine

Cassella for her passionate care for all life and those vegetarian nudges along the way. I am thankful for Jason Wagman’s fellowship and easygoing personality. As for my final

office buddies, the awesome team of Austin Leeds and Bonnie Baird, I am very grateful

for all your help and, of course, our friendship.

I am also extremely grateful for all the zoo staff and volunteers that helped make this research possible. First, I have to thank Laura Amendolagine, Cleveland’s Wildlife

Endocrinology Lab manager, for her patience and always helping my assays do a good job. I am also grateful for all the support of Cleveland Metroparks Zoo’s animal managers for this project, particularly Andi Kornak, Tad Schoffner, and Lynn Koscielny.

They have welcomed my views throughout and their leadership has left an indelible mark on my professional development. I thank the RainForest keepers Joe Ropelewski, Scott

Parish, Terri Rhyner, and Chris Gertiser for their facilitation of this research. This project would not have been possible without the help of many research volunteers. In particular, I would like to thank Christine Olle, Devyn Riley, Amanda Barabas, Ingrid

Rinker, Elaine Leickly, Suzy Peoples and Jen Avondet for their help in data collection and entry.

I would like to extend a special thank you to David Teie. A talented composer,

Mr. Teie’s interest in the origins of music led to his collaboration with Dr. Snowdon and their development of tamarin-specific music. I am grateful for his permission to have incorporated this unique music into my research project.

Lastly, I am fortunate to have known and had the opportunity to work closely with a very special person, Joan Rog. Joan was simply an amazing woman. She had a fervent love of family and critters and tenacity for her research that left its mark on all around her. For Joan and the furry friends I lost along this journey, I will cherish our memories and am forever grateful for our time together.

The Influence of the Sound Environment on the Welfare of Zoo-housed

Callitrichine Monkeys

JASON WARK

Abstract

Animals in the zoo environment are exposed to a multitude of sounds. Some sounds, such as those from the visiting public, are inexorable components of the sound

environment of a zoo and may, in some cases, have a negative impact on the behavior of

animals. Auditory masking of visitor noise, such as from waterfall features, may

alleviate adverse effects of noise but this has not yet been evaluated. Other sounds, such

as music or habitat sounds, may be introduced in an attempt to enrich the animals but

their utility is questionable. This project investigated the influence of the sound

environment on four species of callitrichine monkeys: pied tamarin, white-fronted

marmoset, golden lion tamarin, and callimico. The goal of this research was to identify

enriching and adverse sound environments and, in the case of the latter, evaluate

strategies to ameliorate this effect and improve welfare. The general hypothesis was that

sounds experienced in the zoo setting can affect the welfare of zoo-housed callitrichine

monkeys. This project focused on sound from a nearby waterfall feature as well as music

and other sounds that may be viewed by some as auditory enrichment. The aims were

fourfold. First, hormonal and cognitive methods for assessing welfare were evaluated.

Second, the potential of sound from a nearby waterfall feature to provide beneficial

auditory masking was assessed. Third, rainforest sounds and music were assessed as potential forms of auditory enrichment. Lastly, the influence of providing access to quiet off-exhibit areas was examined. Experimental manipulations of the waterfall feature did not identify beneficial auditory masking effects and indicated waterfall noise may instead be aversive. Playing rainforest sounds or music did not elicit behavioral benefits.

Providing access to off-exhibit areas was beneficial and appeared to ameliorate some stressful effects of the waterfall noise. These results indicate the sound environment did influence the welfare of callitrichine monkeys, albeit minimally, and suggest quieter sound environments may be beneficial for these species. Providing access to quiet areas is an important strategy for promoting environmental choices for zoo animals and opportunities to cope with noise.

Chapter One: Introduction: Sound and its significance

Sound plays a central role in the lives of many animals, from signaling early warning of danger, allowing detection of prey, or communication between individuals.

However, not all sounds provide reliable unambiguous information. A rustle of leaves could be a squirrel searching for food or a predator ready to pounce. Noise, generally defined as unwanted sounds, may elicit a maladaptive response or potentially hamper an animal’s ability to detect reliable sounds (Wright et al., 2007). Thus, an animal’s ability to distinguish meaningful sounds from noise and promote appropriate responses will likely enhance the fitness of that individual.

For animals living in a captive setting—with no risk of predation and no need to hunt or locate mates—many sounds no longer convey biological meaning and much of the sound environment may be perceived as noise. However, maladaptive responses to noise may persist and represent an important concern for animal caretakers. These responses may be short-term and have little consequence, but they may also affect psychological and physiological processes. Sounds can have a strong impact on human emotions and may similarly affect wild animals, both positively and negatively.

Research on multiple species have identified direct links from auditory pathways to the amygdala, a brain region associated with emotion (LeDoux, 2000). As sound is pervasive, managing noise, unlike other aspects of the physical environment, is particularly challenging. On top of that, how we experience the sound environment is shaped by our hearing range and perception of sounds. Many species can hear sounds we cannot detect and our knowledge of how animals perceive different sounds—whether as

potential threats, distractions, or as a sources of interest—is limited. These concerns

highlight the importance of carefully considering the effects of the sound environment on

the welfare of zoo-housed animals.

Animal Welfare

Defining welfare

The Animal Welfare Act of 1966 and its subsequent amendments established a

legal mandate in the United States for providing proper care and management of animals.

Although a clear definition of welfare has proven challenging, a growing number of studies have concluded that welfare is a multifaceted concept and likely varies along a continuum (Boissy et al., 2007; Mason and Mendl, 1993; Yeates and Main, 2008). This project utilizes the definition of animal welfare proposed by the Association of Zoos and

Aquariums Animal Welfare Committee (2015): “Animal welfare refers to an animal’s collective physical, mental, and emotional states over a period of time, and is measured on a continuum of good to poor.”

Welfare, emotion, and affective states

Concern for animal welfare is often based in the assumption that animals are sentient beings capable of experiencing pain and suffering (Mendl et al., 2009).

However, this acknowledgement does not signify that animals experience humanlike emotions, which also feature a strong cognitive component. To differentiate between these concepts, I will refer to “affective states” when discussing the positive and negative subjective experiences of animals (i.e. valence) that may or may not involve a cognitive and conscious component, and “emotion” when specifically referring to those states

3 experienced by humans. However, as some authors do attribute emotional states to animals (e.g. fear-potentiated startle response), some overlap is inevitable.

Two models of emotion have been debated: the dimensional and discrete models.

Briefly, the dimensional model developed by Russell (1980) posits that our emotional experience is built from two primary dimensions: valence (i.e. how attractive or aversive an experience is) and arousal (i.e. how stimulating an experience is). These dimensions are typically modeled as a two-dimensional affective space, with four possible affective endpoints. Proponents for discrete models, on the other hand, argue that discrete emotions form the basis of our subjective experience. For instance, Eckman and

Friesen’s (1971) cross-cultural study of facial expressions identified six discrete emotions: happiness, anger, sadness, disgust, surprise, and fear. Although these models may be complementary (Mendl et al., 2010), this project will draw from the dimensional model of emotion for several reasons. First, the measurement of specific dimensions thought to underlie affective states is more practical for zoo research than the approach often employed in studies of discrete emotions, in which individuals are often experimentally subjected to events thought to induce particular emotional states (e.g. forced swim test and depression; Porsolt et al., 1977). Second, one of the measures in the current project, startle reactivity, was developed to reveal underlying moods, or background emotional states. The concept of mood is an important feature of dimensional models of emotion, in which mood states are thought to combine with emotion-inducing events to form the current affective state of an individual.

Behavioral and physiological indicators of psychological well-being:

Philosophical and historical constraints have hampered discussion of animal affect (Fraser, 2009). The main challenge to studying the subjective experience of animals was famously articulated by Nagel (1974), specifically that we can never truly know what an animal experiences. Although Nagel’s point is valid, indirect measures may be available that can offer us a glimpse of the subjective experience of animals.

Changes in behavior or the expression of specific behaviors may be one possible indirect indicator of animal affective state. Some examples include: self-directed behavior in primates (Maestripieri et al., 1992), types of vocalizations (Boinski et al.,

1999; Burman et al., 2007), social behaviors (Videan et al., 2007), general activity levels

(Markowitz et al., 1995; Shepherdson et al., 1989), stereotypies (Wells and Irwin, 2008), or self-injurious behavior (Lutz et al., 2003). These measures are often ideal choices for zoo research as they can be typically collected non-invasively and with relatively simple equipment. Although identifying affective states are not always the direct purpose of these behavior studies, some authors have suggested that positing affective states can offer a more parsimonious explanation of behavior (Fraser, 2009).

Preference tests, although not directly a measure of welfare, may offer additional insight into aspects of the environment or husbandry that can influence welfare.

Although other behavior measures could only offer a correlative means of assessing welfare, preference tests allow researchers to more directly “ask” the animal what it perceives as best. In preference testing, the animal is presented with alternatives and the motivational difference for one alternative over the other is used to determine their preference (Kirkden and Pajor, 2006). Motivation describes the strength of the animal’s

5 willingness to either obtain a reward (positive motivation) or avoid an aversive stimulus

(negative motivation). The two main forms of preference tests are choice tests, in which the animal is provided with various options and their response is measured, and operant tests, whereby the animal is given control over some stimulus through the use of an operant device (e.g. lever/button pressing). However, it should be noted that although preference tests offers an additional means of assessing welfare, its underlying assumption, that animals prefer things beneficial to them, may not always be the case as is evident in animal models of drug addiction or obesity.

Relying on behavior to assess animal welfare has limitations as animals may not always express their affective state overtly. Assessment of physiological measures may provide additional insight of into an animal’s current welfare state (Möstl and Palme,

2002). One common approach is to measure activity of the hypothalamic-pituitary- adrenal (HPA) axis, also known as the stress pathway. After recognition of a stressor, defined as any real or perceived threat to the behavioral or physiological homeostasis of an individual (Moberg and Mench, 2000), corticotropin-releasing hormone is produced by the hypothalamus which stimulates adrenocoticotropic hormone synthesis in the anterior pituitary and promotes the release of glucocorticoids by the adrenal glands.

Glucocorticoids are a class of steroid hormones that serve to liberate stored energy and, in the case of a stressor, adaptively respond through fight or flight behaviors. These hormones, typically cortisol or corticosterone, are excreted from the body through urine and feces, allowing non-invasive measurement of HPA activity (Möstl and Palme, 2002).

Unfortunately, there are several key limitations to the use of cortisol as a measure of welfare (Mormède et al., 2007). First, HPA activity may follow a circadian rhythm for

some species, making the time of cortisol measurement important (Sousa and Ziegler,

1998). Also, cortisol is not only secreted in response to negative situations but also in pleasurable situations as well (Colborn et al., 1991). Under chronic stress, the reactivity of the HPA axis can be reduced, making low glucocorticoid levels hard to interpret

(Mizoguchi et al., 2003). Finally, there may be differences of HPA activity based on individual factors such as sex, species, and social rank (Touma and Palme, 2005). These challenges highlight the importance of validating the measurement of glucocorticoids for use in a given species (Touma and Palme, 2005).

Choice and control

It has been argued that providing animals with a degree of choice or control may be important for promoting positive welfare (Dawkins, 2004). Although there may be some inherent psychological benefit of new choices, additional options also allow animals to seek features that are beneficial and avoid ones that are not. As mentioned previously, one solution has been to incorporate operant devices, allowing the animals to directly control the stimulus (Hanson et al., 1976; Novak and Drewsen, 1989). However, providing active control over the environment may not always be practical in zoos (e.g. visual presence and noise of zoo visitors). In these situations, it may be more feasible to provide zoo animals with a passive form of control by allowing access to off-exhibit areas (Owen et al., 2005; Ross, 2006).

When giant pandas were given access to off-exhibit areas, they exhibited lower levels of behaviors indicative of agitation and had lower urinary cortisol concentrations, compared to baseline periods with no access (Owen et al., 2005). When allowed access, pandas spent a mean of 32.8% of their time in off-exhibit areas, although this varied

considerably between individuals (5-63%). As the authors note, levels of agitation-

related behaviors were relatively low during baseline conditions (3.5%) compared to

other studies (Swaisgood et al., 2001), suggesting that their exhibits met most of their

needs. Although the authors discuss many possible motivations individuals may have

had for using off-exhibit areas (e.g. avoid noise and proximity to visitors, regulate

thermal stress, etc.), the actual reasons individuals used these areas remains unclear.

Also, given the large individual differences in the amount of time spent off exhibit, these

motivations may have been greater for some individuals than others.

Access to off-exhibit areas also appeared to improve the welfare of a pair of zoo-

housed polar bears (Ross, 2006). Both bears reduced levels of pacing and other

stereotypic behaviors. Unlike the giant pandas discussed previously, pacing and other

stereotypic behaviors under baseline conditions accounted for a moderate portion of the

activity budget of these polar bears. Surprisingly, the bears did not spend much time off-

exhibit, with the percentage of time not visible increasing from 2.1% in the baseline

condition to only 4.3% when given access to off-exhibit areas. The number of visitors at

the exhibit and ambient temperatures were consistent across conditions, suggesting these

factors did not motivate the behavioral change. These results suggest that, for these

animals, the choice to access off-exhibit areas may have been more important than spending time in those areas. As Ross (2006) points out, off-exhibit areas were less enriched than the exhibit space but may have still provided retreat opportunities that benefitted the polar bears.

Although no study has yet investigated the effect of off-exhibit access for zoo- housed callitrichines, Badihi (2006) did find that when laboratory-housed common

8 marmosets were given free access to an outdoor space their use of this space was dependent on the temperature and weather outside. These results suggest that, if provided with a choice, features of the physical environment, may play a role in motivating animals to use specific areas.

Affective startle modification

Modulation of the startle reflex may represent an additional but largely unexplored measure of welfare (Paul et al., 2005). In both people and animals, emotional processes have been shown to be involved in the startle response. For example, it is well documented that fear can increase the startle response of people and animals (“fear- potentiated startle effect; Lang et al., 2000). Environmental stimuli may also be able to modulate the affective state and thus the startle responsiveness of people and animals.

Nature sounds and music have been shown to modify the startle response of people, with unpleasant sounds causing an increased startle response to a loud startle sound compared to pleasant sounds (Bradley and Lang, 2000; Roy et al., 2009). Furthermore, affective startle modification has been shown to depend on the valence of the environmental stimuli, a phenomenon called “affect matching” (Lang et al., 1990). For instance, participants that viewed a gunshot victim had an increased startle response compared to subjects that viewed an image of an attractive nude (Vrana et al., 1988).

Much less work has been done on the modification of the startle response of animals to environmental stimuli. Hoffman and Fleshler (1963) found that when they introduced a continuous white noise background to their startle experiments, rats showed a heightened startle response. Subsequent research has shown that startle sensitivity follows an inverted-U pattern with the amplitude of the background sound, an effect that

has been attributed to increasing arousal from increasing background sound intensities

eventually being countered by masking/ attenuation of the startle stimulus under very

loud background sound intensities (Ison and Hammond, 1971).

Although it has not been used directly as an indicator of animal welfare, startle

reactions are measured in studies of predator recognition and anti-predator behavior. For

example, Searcy and Caine (2003) found that white-fronted marmosets were more

startled by the playback of hawk calls compared to the playback of raven calls and the

sound of a power drill. In another study, the duration of freezing in response to hawk

calls was compared between right- and left-handed marmosets (Braccini and Caine,

2009). These authors found that left-handed marmosets displayed a prolonged freezing

response compared to right-handed individuals, suggesting that hemispheric

specialization of the brain may influence aspects of temperament. Taken together, these

studies suggest that if exposure to sounds or other stimuli in the zoo environment

influenced an animal’s affective state, this should modify their startle response.

Specifically, individuals experiencing a negative affective state would be expected to

have an exaggerated startle reaction whereas individuals experiencing a positive affective

state would have a blunted startle reaction and quicker return to baseline behaviors,

although this remains to be tested.

Sound as Enrichment

Overview of environmental enrichment

Environmental enrichment is an important method for improving the welfare of captive animals, and is legally mandated for the psychological well-being of captive

primates by the Animal Welfare Act. Environmental enrichment can be defined as

modifications that increase environmental complexity and confer psychological benefits

(Carlstead and Shepherdson, 2000). This complexity can occur in the physical, temporal,

or social environment (Carlstead and Shepherdson, 2000). Thus, a well-provisioned

environment should allow animals various opportunities, be it interacting with another

animal, investigating objects, or just by offering a complex exhibit space to explore.

Sensory enrichment

One method of increasing the physical complexity of the environment is through

sensory enrichment. This form of enrichment often involves incorporating auditory,

olfactory, or visual stimuli. Lutz and Novak (2005) term this “passive” enrichment, to differentiate the level of interaction from the “active” enrichment of toys and foraging devices that require manipulation. As this difference in interaction levels suggest, behavioral changes may be more difficult to observe. Thus, Schapiro and Bloomsmith

(1995) compared physical, feeding, and sensory (video) enrichment and concluded that sensory enrichment had little impact on the behavior of singly-housed rhesus macaques.

Although no behavioral effect of sensory enrichment was observed, it is hard to conclude

that this enrichment has little utility as it may effect a more subtle psychological appraisal

of the environment that might not be detected through overt behavioral sampling.

Animal welfare is complex and evaluating the influence of sensory enrichment on

welfare may benefit from psychological indicators of efficacy.

In a review of sensory enrichment, Wells (2009) found generally positive

behavioral effects of auditory, olfactory, and visual stimulation. Common methods of

auditory stimulation involve music or natural sounds from ambient habitats or animal

vocalizations. Olfactory stimuli have included biologically-relevant scents such as from

a predator, prey, or pheromones from a conspecific animal. Less relevant scents have

also been employed, such as lavender, essential oils, or spices. Finally, visual enrichment

studies have also investigated biologically-relevant images such as that of conspecifics in addition to less relevant images such as heterospecific animals that may not be encountered in the wild.

As positive results have been documented in both biologically-relevant and non- relevant sensory enrichment studies, it may be unlikely that biological relevance is important for enrichment success (Wells, 2009). Although biological relevance may not be critical, some evidence does suggest that sensory enrichment that stimulates the dominant senses that an animal uses has greater potential for positive welfare benefits.

For instance, olfactory stimulation of primates, whose dominant senses are vision and audition, has shown little success in enhancing animal well-being (Wells et al., 2007).

Finally, most studies have only been over short time periods and the long-term benefits of sensory enrichment, specifically auditory and olfactory enrichment, remain unresolved

(Wells, 2009).

Humans

Before discussing how auditory enrichment can benefit captive animals, it is important to briefly consider the effect music and other sounds can have on us. Music is known to have existed at least 40,000 years ago and is shared throughout all human cultures (Fitch, 2006). Although the evolutionary origins for music are unclear, its ability to profoundly affect us is certain. Upon hearing music, the emotional response is almost immediate, with music excerpts of a second or less sufficient to cause an emotional

12 response (Bigand et al., 2005; Peretz, 2001). Although some studies have found effects of music experience or culture, the reflexive effect of music on our emotions suggests some processes may be innate. Research on infants supports this claim, with studies showing that infants are able to perceive some aspects of music similar to adults (Trehub and Hannon, 2006).

In Western cultures, differential effects of music genre have been noted, with heavy metal and rock music typically increasing anxiety and arousal and classical music lowering arousal (Kemper and Danhauer, 2005). These genre differences have been correlated to some properties of the sounds. Specifically, tempo and rhythm have been implicated in modulating arousal levels (Gomez and Danuser, 2007).

Some of the beneficial effects of positive music genres include reductions in subjective measures of anxiety and decreased sympathetic nervous system activation

(Kemper and Danhauer, 2005). Knight and Rickard (2001) explored the ability of music to mediate the anxiety of undergraduates preparing for an oral presentation. In their study, exposure to Pachelbel’s Canon in D major reduced heart rate, blood pressure, and salivary cortisol measures in addition to decreasing anxiety as measured through the

State-Trait Anxiety Inventory. Khalfa et al. (2003) also documented a decrease in salivary cortisol during a similar procedure. The beneficial effects of music are so widely recognized that they now serve an important role in medical therapy programs (Evans,

2002; Kemper and Danhauer, 2005). Music therapy has been used to treat a variety of both acute and chronic medical illnesses (e.g. Updike and Charles, 1987; Palakanis et al.,

1994; Koger et al, 1999). Especially relevant to the present project is the use of music in laboratory-based mood induction procedures (Gerrards‐Hesse et al., 1994; Westermann et

al., 1996). Although music is not as successful as other mood induction procedures, it is

one of the most simple, as most procedures require some type of instruction with some

featuring complex cognitive appraisals (Westermann et al., 1996).

Compared to music, our knowledge of the effect of nature sounds is limited.

Gomez and Danuser (2004) documented that exposure to the sounds of a stream with birds elicited similar levels of arousal as classical music pieces. Furthermore, nature sounds have also demonstrated utility in therapy applications (Diette et al., 2003).

Beneficial effects of aesthetically pleasing nature scenes have also been documented

(Kweon et al., 2008; Ulrich et al., 1991).

Music as auditory enrichment for animals

Given the benefits to humans, it comes as no surprise that there has been considerable interest in whether music can be used to benefit captive animals. Indeed, numerous studies have documented positive behavioral and physiological effects of music in a diverse array of taxa. Classical music exposure, in particular, has shown potential for auditory enrichment. For instance, Wells et al. have documented positive effects of classical music stimulation for diverse species. Classical music correlated with decreased barking and increased resting in kennel dogs (Wells et al., 2002), a non- significant trend to increased resting and decreased abnormal behaviors in zoo-housed western lowland gorillas (Wells et al., 2006), and a decrease in stereotypic behavior of zoo-housed Asian elephants (Wells and Irwin, 2008). A decrease in abnormal behaviors was also observed in rhesus macaques exposed to classical music (O'Neill, 1989). Also,

Howell et al. (2003) reported an anecdotal decrease in aggressive displays in one laboratory housed chimpanzee during classical music exposure. These results support an

overall calming effect of classical music. Classical music has also been shown to

stimulate positive physiological effects on fish and rats (Papoutsoglou et al., 2007; 2008;

2010; Akiyama and Sutoo, 2011). Interestingly, several studies have documented greater

benefits from classical music of the common practice period, including the Baroque,

Classical, and Romantic eras, as compared to music of the modern and contemporary era

(Lemmer, 2008; Watanabe and Nemoto, 1998). This suggests that musical characteristics

of different eras may be important and classical music should not be viewed as a single

monotypic genre. For simplicity, I will use “classical music” throughout to refer to music

from the common practice period.

Other musical genres have also occasionally shown potential for positively

influencing captive animals. Country music has shown some success for enriching dairy

cows (Uetake et al., 1997). Radio music has also shown some potential for enrichment.

Brent and Weaver (1996) reported that a radio tuned to an oldies station lowered the heart

rate of laboratory housed olive baboons but showed no behavioral effect.

In two studies on laboratory housed rhesus macaques, animals were given active

control over the playback of musical stimuli. In a study by Markowitz and Line (1989),

five female rhesus macaques housed in colony rooms were given a lever that could

activate or deactivate radio music (no genre specified). The rhesus continued to play

radio music throughout the 20 month study duration. Also, the macaques played the

radio music for an average of 50% of the day. Novak and Drewsen (1989) reported similar results with laboratory-housed rhesus macaques given control over jazz music.

Over the course of the 10 week study, the monkeys played the jazz music for roughly

50% of the time provided (two hour periods). Furthermore, Novak and Drewsen

documented an increase in affiliative behavior but no effect on urinary cortisol measures.

These studies have highlighted behavioral and physiological benefits of music

exposure for animals, similar to what has been reported for humans. However, a note of

caution needs to be made regarding the musical preferences of animals. As may be

expected from this discussion, some of the mechanisms underlying music perception are

not unique to humans and shared with a variety of animals. For instance, rhesus

macaques, but not songbirds, have demonstrated the ability to generalize octaves (Wright

et al., 2000). In addition, a wide variety of species have demonstrated the ability to

distinguish between consonance and dissonance, or harmonically pleasing and

displeasing sounds to humans (e.g. classical music vs. metal music; McDermott and

Hauser, 2005). Humans show clear preferences for consonant sounds over dissonant

ones. However, in a y-maze preference task, cotton-top tamarins showed no preference for consonance, although presumably they could distinguish consonance and dissonance

(McDermott and Hauser, 2004). This questions the degree to which musical preferences, but not necessarily perception, are shared among humans and animals. An additional feature of music that has been implicated in animal preferences is the tempo of the music.

Specifically, Videan et al. (2007) reported a greater decrease in agonism in laboratory- housed chimpanzees exposed to slower tempo music (easy-listening) as opposed to faster tempo music (classical). McDermott and Hauser (2007) reported a similar preference in tamarins and marmosets for slower tempo music (lullabies) over fast tempo music

(techno) using a y-maze preference task. In addition, the authors also observed this preference using artificial audio streams of clicks, thus reducing the possibility of the

16 monkeys responding to other musical features. However, when humans, tamarins, and marmosets were given the choice of slow tempo music (flute lullaby, sung lullaby,

Mozart) or silence, monkeys, unlike the human subjects, preferred silence over all three musical pieces. A similar preference for silence over music has been reported for rats

(Krohn et al., 2011).

Furthermore, music has in some cases been shown to cause stressful reactions.

For example, laying hens were more fearful when played classical music compared to hens exposed to normal barn noises, as indicated by an increase in tonic immobility for the classical music condition (Campo et al., 2005). Loud radio music (70 – 80 dB) has been shown to increase the salivary cortisol of marmosets (Pines et al., 2004). Although it is unclear if the animals in these studies found the specific sounds stressful or were reacting to a louder environment, these results do urge caution and highlight the need for additional research before applying auditory enrichment in captive settings (Patterson-

Kane and Farnworth, 2006).

In summary, music has been shown to benefit the well-being of animals as evidenced by behavioral and physiological markers. For many species, musical preferences and positive effects of music on animals appears similar to that seen in humans. Generally, classical music has stimulated beneficial influences on welfare.

However, our understanding of the mechanisms underlying musical preferences and effects are incomplete. As species-specific differences have been observed in response to music, future research is needed to explore additional sounds for the potential of auditory enrichment. In addition, the effects of music are not always straight forward, as some studies have reported unforeseen negative effects. Music may represent a potential form

of auditory enrichment but research should be conducted before its application into the

captive environment (Patterson-Kane and Farnworth, 2006).

The sounds of nature

In contrast to music, the effects of ambient habitat sounds have not been commonly explored (Lutz and Novak, 2005). Three studies have investigated the effect of African rainforest sounds on western lowland gorillas (Ogden et al., 1994; Robbins and Margulis, 2014; Wells et al., 2006). Ogden et al. (1994) observed increased activity in adult gorillas when exposed to the audio playback of rainforest sounds recorded in

Cameroon. As the level was greater than expected from studies of wild gorillas, the authors interpreted this response as being indicative of agitation. Surprisingly, infants demonstrated lower levels of clinging, suggesting that they may have been calmed by the rainforest sounds. However, these results are difficult to interpret as decreased clinging by the infants may have been a reflection of the increased activity of adults.

Alternatively, the change in the infant’s behavior may have spurred the noted behavioral changes in the adults.

Wells et al. (2006) assessed the effects of African rainforest sounds, along with classical music (various artists), and a no audio control condition on gorillas. No behavioral changes were observed under each sound treatment although the authors anecdotally reported the gorillas first reacted to the rainforest sounds with a brief fear response.

Most recently, Robbins and Margulis (2014) exposed three gorillas at the Buffalo

Zoo to rainforest sounds from a commercial CD, classical music (Chopin), rock music

(Muse), and a no audio control period. Playback of rainforest sounds did cause a trend of decreased regurgitation and reingestion but overall was generally similar to the no audio control period. Interestingly, several individuals reacted aversively to classical and rock music exposure, with increased hair plucking and regurgitation and reingestion. Taken together, these three studies on gorillas paint a confusing picture and highlight the inconsistent responses to sounds intended as auditory enrichment.

Rainforest sounds have also been evaluated as enrichment for several nocturnal species housed together (Clark and Melfi, 2012). Clark and Melfi observed decreased species-typical foraging behaviors in bush babies and sloths. Alarmingly, the bush babies spent more than 90% of their time in nest boxes when the rainforest sounds were playing, which the authors interpreted as a hiding response possibly reflecting neophobia. Clark and Melfi suggest that the sounds may not have been representative of nocturnal periods in the wild and defend the use of auditory enrichment to potentially mask visitor noise.

However, the biological relevance of nature sounds to animals born in captivity is questionable as these sounds are likely novel (Newberry, 1995). Habitat sounds are often incorporated into zoo buildings to enhance the immersion experience of guests and additional research on the impact of these sounds on zoo animals is warranted.

In addition to habitat sounds, the playback of animal vocalizations may represent an additional form of auditory enrichment. Positive effects have been documented for both conspecific (Rukstalis and French, 2005; Shepherdson et al., 1989) and heterospecific (Markowitz et al., 1995) vocalizations. For example, playing temporarily isolated marmosets the vocalizations of their social partner correlated with lower urinary cortisol values (Rukstalis and French, 2005). However, although animal vocalizations

are likely to cause a behavioral change, repeated exposure to the same vocalizations will

likely cause the sounds to lose biological meaning for the animals and reduce its potential

effectiveness as auditory enrichment.

Species-specific music

These inconsistencies of music and habitat sounds as auditory enrichment

highlight the need for identifying more meaningful sounds for animals. One promising

form of auditory enrichment that has recently been developed is species-specific music

(Snowdon and Teie, 2010; Snowdon et al., 2015). Species-specific music is composed using characteristics of the species such as heart rate, hearing range, and features of vocalizations thought to convey affect. Snowdon and Teie first demonstrated the potential of species-specific music by developing music of cotton-top tamarins. Spectral and temporal features of affiliative and fear or threat vocalizations were used to compose two types of musical pieces. For example, long, tonal, pure-tone notes observed in affiliation vocalizations and rapid, staccato notes observed in fear/threat vocalizations were used in the composing the tamarin music. The final pieces were then performed using a cello and singing by David Teie and the final recordings were then shifted to higher frequencies to reflect the hearing sensitivity of tamarins.

Seven pairs of cotton-top tamarins were observed before and after presentation of a 30 s excerpt of affiliation- and fear/threat-based tamarin music and equivalent human music. In the five min after exposure to the fear/threat-based tamarin music, tamarins moved more and displayed more anxious behaviors (e.g. piloerection, urination, scent marking, head shaking, and stretching) compared to the affiliation-based music, which the authors interpreted as a negative reaction. Snowdon and Teie also observed increased

social behaviors (e.g. grooming, huddling, and sex) following fear/threat-based tamarin

music and, although this was not predicted, the authors suggest this may have indicated

social comforting following a threatening stimulus. Compared to baseline measures,

tamarins exposed to the affiliation-based tamarin music moved less, foraged more, and

showed a trend to decreased social behavior. Tamarins oriented to the speakers more

after exposure to the fear/threat-based tamarin music compared to baseline conditions,

possibly reflecting heightened vigilance. Tamarins moved less following the human fear/threat-based music and displayed less anxious behavior following the human affiliation-based music. These results suggest that tamarin-specific music had greater effects than human music and that specific components of vocalizations can be used in species-specific music to modulate the affective response of the animals. More recently,

Snowdon et al. (2015) demonstrated that domestic cats were more attentive to cat- specific music compared to human music.

Mechanism of auditory enrichment

It is important to consider the mechanisms by which auditory enrichment, natural or otherwise, exert an enriching effect on captive animals. The two common mechanisms discussed in the animal literature are masking effects (Ogden et al., 1994; Tromborg,

1999) and neurophysiological effects (Wells, 2009). The masking hypothesis suggests that auditory enrichment may be beneficial by masking, or decreasing the perceptibility, of startling sounds arising in the animals’ environments, such as from zoo visitors or machinery (Ogden et al., 1994; Patterson-Kane and Farnworth, 2006; Wells, 2009). This suggestion likely stems from the successful application of masking sounds for humans.

For example, white noise and water sounds have been used in open-plan office settings to

reduce distracting sounds (Haapakangas et al., 2011; Loewen and Suedfeld, 1992).

Water sounds (e.g. fountains, waterfalls) have also been shown to be successful at

masking traffic noise (De Coensel et al., 2011; Galbrun and Ali, 2013; Jeon et al., 2010;

Nilsson et al., 2010). Interestingly, De Coensel et al. (2011) found that participants rated

fountain sounds better at masking steady traffic noise but bird sounds were better for

variable traffic noise. The authors suggest that variable sounds may provide a better

masking sound as they may draw attention away from the noise. This finding suggests an

alternative hypothesis – that auditory enrichment may be beneficial by simply distracting

attention from variable sources of noise.

Currently, no study has demonstrated positive effects of auditory masking for

animals. The study by Ogden et al. (1994) evaluated the potential of the rainforest

sounds to mask sounds of caretakers or bonobo vocalizations, with masking sounds

causing agitation in the gorillas. Tromborg (1999) explored the effects of various

background sounds on laboratory-housed California ground squirrels as part of his dissertation research. He identified a longer latency to emerge from nest boxes when exposed to thunderstorm sounds, a response he argued was the result of auditory masking promoting cautious behavior in the squirrels. As discussed above, when researchers

studying the startle response of animals applied white noise in the background of their

experiments in an attempt to mask exogenous sounds, they found the startle response was

enhanced and auditory masking was only achieved under very loud background sounds

(>80 dB; Hoffman and Flesher, 1963, Ison and Hammond, 1971). Additionally, as Wells

(2009) notes, the masking hypothesis cannot explain the differential effects of music

genres that have been reported, as one genre is not more clearly masking than another.

Additional research is needed to determine whether auditory masking can benefit

animals.

An additional hypothesis on the benefits of auditory enrichment is that of a

specific neurophysiological effect of audio exposure (Wells, 2009). The underlying

assumption of this hypothesis is that some general perceptual mechanisms may be shared

between animals and humans and lead to the positive effects of music found across

species. Neurophysiological effects of music have been described in mice. After

exposure to music, brain-derived neurotrophic factor (BDNF) increases and this has been

linked to the anxiolytic effects of music observed in mice with a BDNF polymorphism

(Angelucci et al., 2007; Li et al., 2010). Importantly, these changes were not observed

when mice were exposed to white noise, suggesting the anxiolytic effect is based on

characteristics of the sound and not just on auditory stimulation (Li et al., 2010).

Neurophysiological reactions to specific features of sound may be important for identifying both danger and safety and have an innate neurological basis. The brain stem

is important for rapidly responding to specific features of sound that may signal danger,

such as loudness, suddenness, low pitch, and high levels of dissonance and this has been

implicated as one potential primal mechanism underlying the emotional response of

humans to music (Juslin and Västfjäll, 2008). For example, in developing warning

sounds for use in various applications, researchers have found fast temporal patterns are

important for signaling urgency to listeners (Suied et al., 2008). The innate response to

these features may have an ultimate explanation in how affect is conveyed through

conspecific vocalizations, which is the mechanistic basis for species-specific music

(Juslin and Laukka, 2003; Snowdon and Teie, 2013). Aspects of predator vocalizations

may also trigger an innate response, although some degree of learning may be necessary

to correctly identify potential predators (Friant et al., 2008; Searcy and Caine, 2003).

Although Lenti Boero and Bottoni (2008) speculated that there may be an evolutionary preference for certain habitat sounds, response to these sounds may also require experience and thus, not carry biological meaning for captive-reared individuals. The response of animals to auditory enrichment is complex and beneficial effects will likely depend on acoustic features of the sounds and/ or their ability to mask aversive noise.

Noise and its impact on animals

How anthropogenic noise affects free-ranging wildlife

With an ever-expanding human population, the impact of noise from human- related activities, or anthropogenic noise, on wildlife has become an emerging conservation concern (Barber et al., 2010; Francis and Barber, 2013). For example, extensive research has shown that noise may interfere with an animal’s ability to detect the vocalizations of conspecifics (Bee and Swanson, 2007), causing many species to shift aspects of their calls to adapt (Slabbekoorn and Ripmeester, 2008). However, some species may be limited in their ability to adapt their communication systems to noise (Hu and Cardoso, 2010). Although the long-term fitness consequences of vocal adjustments have not been fully explored, some evidence suggests these changes may influence female mate choice and male-male competition (for review of this topic for avian species, see Patricelli and Blickley, 2006).

In addition, noise may interfere with the ability to detect predators, promoting increased vigilance and anti-predator behaviors (Quinn et al., 2006). For example,

ground squirrels living near a wind turbine exhibited elevated levels of vigilance and

increased likelihood of returning to their burrows in response to the playback of

conspecific alarm calls compared to ground squirrels living in a quiet area (Rabin et al.,

2006). Noise may also adversely affect the hunting ability of predators to locate prey

(Siemers and Schaub, 2011).

One response to these potential challenges is to avoid noisy areas. For example,

greater sage grouse have been shown to avoid breeding lek areas during the experimental

playback of natural gas drilling noise, and the animals that remained demonstrated

elevated fecal glucocorticoid levels, an effect the authors attributed to masking of

communication or increase perception of predation risk (Blickley et al., 2012; Blickley

and Patricelli, 2012). Duarte et al. (2011) observed marmosets living in an urban park to

avoid the noisy periphery of the park, including areas with high food availability. This

finding also raises questions about the degree to which animals habituate to frequent

noise exposure.

Francis and Barber (2013) outlined a framework for understanding the effects of

noise, the disturbance-interference continuum. In their view, the impact of noise varies along a temporal gradient, with sudden, infrequent, and unpredictable sounds being perceived as a threat and causing a startle response and anti-predation behaviors (risk- disturbance hypothesis, see Frid and Dill, 2002), whereas chronic noise is suggested to cause interference of cues through auditory masking. They propose the severity of the response may depend on temporal, intensity, and frequency characteristics of the sound.

Alternatively, Chan et al. (2010) have shown evidence that noise may just act as a distraction (distracted prey hypothesis). Although these hypotheses are based on the

25 responses of free-ranging wildlife, they may also provide a valuable framework for understanding the impacts of noise on wildlife in a zoo setting.

Comparing the sound environment in the wild and captivity

The potentially negative effects of noise on animals is concerning for zoos, which may be louder than wild habitats. In a study of sound levels in natural habitats, Waser and Brown (1986) found that rainforest habitats (Kakamega Reserve, Kenya and Kibale

Reserve, Uganda) ranged from 27 dB to 40 dB. Sound levels recorded in zoos are typically much greater than those wild estimates. Ogden et al. (1994) reported background sound levels near a gorilla exhibit was approximately 40 dB, however when the ventilation system was activated sounds exceeded 58 dB. Sound levels ranging 62 dB to 72 dB, with an average of 70 dB, were recorded at two California zoos, with the higher values relating to visitor activity (San Francisco Zoo and Sacramento Zoo, Tromborg and

Coss, 1995). More recent studies of noise in zoos have generally documented sound levels ranging between 55 – 70 dB (Cooke and Schillaci, 2007; Owen et al., 2004; Powell et al., 2006; Quadros et al., 2014).

Noise in zoos

A growing body of research has identified adverse effects of noise in the zoo environment. Owen et al. (2004) were the first to quantitatively describe the impact of noise on zoo animals. They measured the behavioral and hormonal responses to ambient noise by two zoo-housed giant pandas housed at the San Diego Zoo. The pandas displayed increased door scratching, locomotion, stress-related vocalizations, and urinary cortisol levels on days categorized as loud compared to quiet days. Powell et al. (2006)

26 documented a similar response to construction noise by pandas housed at the National

Zoo. Most recently, Quadros et al. (2014) performed a detailed observational study of noise at the Belo Horizonte Zoo in Brazil. They found sound levels in exhibits were influenced by increasing numbers of visitors, the popularity of the species, and the style of the exhibit with open circular exhibits that permitted the greatest number of visitors being the loudest. Although no behavioral differences were observed in relation to noise, the authors did document increased vigilance in response to visitors for multiple species.

Several studies have evaluated visitor noise experimentally. Birke (2002) manipulated visitor noise by asking volunteers standing in front of an orangutan exhibit to either watch quietly or talk loudly. In the loud visitor condition, the orangutans looked at the public more and infants increased the time spent holding onto parents, suggesting that loud visitors were a stressor. Larsen et al. (2014) investigated the effect of visitors on koalas under standard conditions as well as during audio playback of visitor noise.

The koalas responded to both increasing visitor numbers and louder noise treatments with heightened vigilance.

Noise is an inevitable part of the zoo environment and an unavoidable stimulus for the animals housed within. These results suggest that noise can negatively impact the lives of zoo animals and solutions to this dilemma are unclear. Noise, at suitable levels, may beneficially increase the complexity of the zoo environment. Although technically challenging, control over noise may ameliorate its negative influence. Using sounds to mask zoo noise may be attractive but has not yet been documented. A better understanding of the effect of the sound environment on the welfare of zoo animals is needed, particularly for sensitive species such as callitrichine monkeys.

Callitrichines

Natural history of callitrichine monkeys

Callitrichine monkeys are a group of small-bodied New World primates native to

Central and South America. Although their taxonomy has been disputed, the subfamily

Callitrichinae is currently recognized to include six genera: the tamarins (Saguinus), pygmy marmosets (Cebuella), Amazonian marmosets (Mico), and monophyletic

callimico (Callimico) residing in the Amazonian basin; and the lion tamarins

(Leontopithecus) and Atlantic marmosets (Callithrix) native to the Atlantic forests

(Schneider and Sampaio, 2015).

These species are distinguished for a number of key characteristics. Most notable

is their small body size, being the smallest anthropoids in the world. Although as a group

callitrichines are diminutive, body sizes can range six-fold within Callitrichinae – from

the pygmy marmoset (Cebuella pygmaea) at a little over 100 g to the golden-headed lion

tamarin at almost 700 g (Leontopithecus chrysomelas; Garber, 1992). In conjunction

with small body size, callitrichines have clawlike nails (tengulae) on all digits except the

hallux – adaptations that reflect their arboreal lifestyle. Callitrichines are also remarkable

for their obligate birth of twins, occurring in all species except callimico, and cooperative

rearing of young, the only primate beyond humans to exhibit this trait (Fernandez-Duque

et al., 2012).

Social groups in the wild are generally small (3 to 12 animals) and have a flexible

social organization typically involving one female with one or more males and offspring.

Mating systems are variable and monogamy, polyandry, and polygamy have all been

reported (see Fernandez-Duque et al., 2012 for review). In captivity, callitrichines are

housed as monogamous breeding pairs (Anzenberger and Falk, 2012).

Why study callitrichines?

There are a number of reasons to study the effects of sounds on callitrichine

monkeys. First, these species generally live in dense rainforest habitats. As vision may

be limited in these environments, this may make these species more reliant on sounds.

Second, callitrichines are the smallest anthropoids and thus, prey to numerous

predators (Caine, 1993). This predation pressure may make them particularly sensitive to

humans. Research in zoos has suggested a negative effect of zoo visitors on these

monkeys. Glaston et al. (1984) compared cotton-top tamarins on display and off exhibit.

They found reduced social behaviors in tamarins housed on display, including after the

two groups were switched housing arrangements. Armstrong and Santymire (2013)

similarly compared a pair of pied tamarins housed on display with an off-exhibit pair.

They observed decreased grooming and foraging and increased locomotion, use of nest boxes, and fecal glucocorticoid levels in the pair housed on display. This group was also noted to perform stereotypic behaviors. Wormell et al. (1996) also investigated the visitor effect on pied tamarins and found a group that experienced frequent visitation to perform more threat displays, piloerection, and approaches to the front of their cage than groups with lower levels of visitation. A similar pattern has also been observed in laboratory conditions comparing weekdays with frequent human activity to quiet weekend periods (Barbosa and Mota, 2009). Common marmosets were more frequently scent marking and moving, and performed autogrooming for longer periods of time during the busy weekdays vs. weekends. Although Barbosa and Mota (2009) did not

identify an effect of human activity on fecal glucocorticoid measures, Cross et al. (2004) has reported increased saliva cortisol in laboratory-housed common marmosets following a disturbance period. The presence of a human observer has even been utilized as a procedure to study fear and anxiety in marmosets (Human Threat test; Costall et al.,

1992).

Third, callitrichine species have shown a unique response to sounds. This has included an indifference to classical music (McDermott and Hauser, 2007), no preference

for consonant (i.e. harmonious) over dissonant sounds (McDermott and Hauser, 2004),

and no avoidance of screeching sounds described as being similar to nails on a chalk

board (McDermott and Hauser, 2004). In addition, species-specific music has been

developed for the cotton-top tamarin (Snowdon and Teie, 2010) and may represent a

potential form of auditory enrichment for other callitrichines.

Lastly, it is important to consider that callitrichines, not unlike other species, are

living in increasingly close proximity to humans in the wild and, consequentially, greater

exposure to anthropogenic noise. For example, the critically endangered pied tamarin has

one of the most restricted ranges of any primate and is only found within and near

Manaus, Brazil. A greater understanding of how a species responds to sounds in a zoo

setting may have insights for their conservation in the wild.

Thesis Objectives

The goal of this research was to identify enriching and adverse sound

environments for zoo-housed callitrichine monkeys, and in the case of the latter, evaluate

methods to ameliorate this effect and improve welfare. The general hypothesis was that

sounds commonly experienced in the zoo setting can affect the welfare of zoo-housed

callitrichine monkeys. My aims were fourfold. First, I sought to develop and validate

additional methods for assessing the welfare of these species through hormonal and

cognitive assessments. Second, I evaluated whether sound from a nearby waterfall

feature provided beneficial auditory masking. Third, I investigated the potential of

sounds as auditory enrichment for these monkeys. Fourth, I explored the effect of exhibit

choices on the response to the sound environment.

Chapter Two validates fecal glucocorticoid measures as an indicator of stress in

pied tamarins, white-fronted marmosets, golden lion tamarins, and callimico. Fecal

samples were assessed before and after a routine veterinary exam to document increased

cortisol production from handling and anesthesia. In addition, we evaluated the impact of

social changes on fecal glucocorticoid levels.

Chapters Three and Four investigate the effect of the waterfall feature on

callitrichine monkeys. In Chapter Three, the waterfall feature and access to off-exhibit areas was experimentally manipulated in multiple phases for pied tamarins, white-fronted

marmosets, and callimicos. Chapter Four expands on this investigation by carefully

manipulating the sound levels in both the exhibit and off-exhibit areas for the pied

tamarin group.

Chapters Five and Six explore the potential of sounds as auditory enrichment for

callitrichines. Chapter Five observed the behavioral response to 30 min exposure of

sounds during a quiet period for the pied tamarin, golden lion tamarin, and callimico

groups. In addition, access to off-exhibit areas was manipulated during the study to

provide a measure of preference and evaluate the effect of exhibit choices on the response

31 to sounds. Chapter Six investigates whether exposure to sounds can modify the startle response of pied tamarins and white-fronted marmosets. This study exposed animals to a brief (5 min) playback of sounds that was immediately followed by an acoustic startle event. This methodology was validated through exposure to loud white noise, a stimuli known to be aversive. This investigation represents a novel method for assessing the affective state of zoo-housed animals.

Chapter Two: Fecal glucocorticoid metabolite responses to management stressors and social change in four species of callitrichine monkeys

Introduction

Measuring glucocorticoids can provide valuable insight into the health and welfare of animals. Metabolites of these hormones, typically cortisol or corticosterone, can be measured non-invasively through feces using enzyme immunoassay (EIA), providing a valuable tool for monitoring sensitive species with minimal disruption (Keay et al., 2006; Schwarzenberger, 2007). However, as significant variation exists in the metabolism and excretion time of glucocorticoids between species, as well as possible individual differences in hormone production within a species, it is necessary to validate the EIA assay using an event known to activate the HPA axis (Touma and Palme, 2005).

One common method involves the administration of adrenocorticotropic hormone

(ACTH), a precursor in the HPA axis known to stimulate glucocorticoid production

(Heistermann et al., 2006). Although this represents a gold standard, ACTH challenges are not always possible or ideal in some situations. Alternatively, assessing glucocorticoids before and after stressful events, such as capture, restraint, and anesthesia during veterinary exams (Armstrong and Santymire, 2013) or translocation between institutions (Heistermann et al., 2006), can indicate the biological validity of an assay.

Also, demonstrating circadian variation of glucocorticoids (Sousa and Ziegler, 1998) or increases in relation to parturition (Murray et al., 2013) may provide an additional means to validate an assay.

Fecal glucocorticoid metabolites (FGM) have been previously assessed in some

species of the family Callitrichinae, a group of arboreal, small-bodied New World monkeys (common marmoset, Callithrix jacchus: Heistermann et al., 2006; Raminelli et al., 2001; Sousa and Ziegler, 1998; golden lion tamarin, Leontopithecus rosalia: Bales et al., 2005, 2006; pied tamarin, Saguinus bicolor: Armstrong and Santymire, 2013; cotton- top tamarin, Saguinus oedipus: Fontani et al., 2014; Ziegler and Sousa, 2002; mustached tamarin, Saguinus mystax: Huck et al., 2005). Although previous reports largely describe similar patterns of cortisol metabolite excretion, some discrepancies have been noted.

For example, in a study of female common marmosets, Sousa and Ziegler (1998) demonstrated diurnal variation of cortisol, with higher concentrations detected in feces during the afternoon versus morning. Previous studies on marmosets have documented diurnal variation in both urine (Wied’s tufted ear marmoset; Smith and French, 1997) and saliva (common marmoset; Cross and Rogers, 2004). However, Raminelli et al. (2001) identified elevated FGM during the afternoon in female common marmosets, but not in males. Huck et al. (2005) also observed no diurnal variation of FGM in mustached tamarins. Similarly, elevated FGM concentrations during pregnancy have been reported in golden lion tamarins (Bales et al., 2005) and common marmosets (Ziegler and Sousa,

2002) but not for mustached tamarins (Huck et al., 2005). These differences highlight the need for careful validation of FGM measures for each species, even when dealing with closely related species.

Social changes are a common occurrence in these monkeys and these events influence HPA activity. For example, periods of social instability, such as during pair bond formation or separation from a natal group, have been shown to increase cortisol

levels (Johnson et al., 1996; Smith et al., 2011; Ziegler et al., 1995). In the wild, these

periods are typically brief and resolved through immigration and emigration between

groups (Lazaro-Perea et al., 2000). However, in a zoo setting, replacement of individuals within a group after deaths and/or separations is not always immediately possible.

Understanding the impact of these social changes is important for animal managers as social instability may impact welfare and the formation of new social bonds (Johnson et al., 1996; Smith et al., 2011).

The aims of this investigation were two-fold. The first aim was to validate methods of individual fecal identification and measurement of FGM concentrations in four species of callitrichine monkeys: golden lion tamarin (L. rosalia), callimico

(Callimico goeldii), pied tamarin (S. bicolor), and white-fronted marmoset (Callithrix geoffroyi). This is the first study to measure FGM concentrations in white-fronted marmosets and callimicos and the first to compare FGM responses among callitrichine species. Biological validations were conducted using the stress response to veterinary exams. Second, we evaluated the impact of deaths of group members on FGM levels in the pied tamarin and white-fronted marmoset groups. We hypothesized that the death of breeding animals would lead to social instability and thus, alter FGM levels.

Methods

Subjects and housing

This study involved 16 monkeys from four species: golden lion tamarin (n=7), callimico (n=2), pied tamarin (n=3), and white-fronted marmoset (n=4; Table 1).

Animals were housed in similar single-species exhibits in the RainForest building of

Cleveland Metroparks Zoo. The monkeys were fed in the morning (10 AM) and late afternoon (4:30 PM) a diet consisting of canned marmoset diet (Mazuri, St. Louis, MO),

New World primate biscuit (Mazuri, St. Louis, MO), vegetables, fruit, and mealworms.

The pied tamarins also received a calcium supplement and the white-fronted marmoset diet included gum arabic. Water was available ad-libitum.

Table 1. Demographic background of study subjects. Species ID Sex Age (yr)a Golden lion tamarin LF1 Female 6.2 (L. rosalia) LF2 Female 1.2 LF3 Female 0.3 LM1 Male 12.0 LM2 Male 3.1 LM3 Male 1.2 LM4 Male 0.3

Callimico CF1 Female 3.7 (C. goeldii) CM1 Male 10.5

Pied tamarin TF1 Female 6.2 (S. bicolor) TM1b Male 17.5 TM2 Male 3.6

White-fronted marmoset MF1b Female 6.3 (C. geoffroyi) MM1 Male 8.3 MM2 Male 3.2 MM3b Male 2.9 a Age was calculated at the time of the veterinary exam for all individuals except MM3, who died prior to the exam. For this individual, age at death is provided. b Individuals that died prior to or during a veterinary exam.

Fecal marker assessment

Four indigestible items were qualitatively evaluated as fecal markers (Table 2): food dye (McCormick and Company, Inc., Hunt Valley, MD), food coloring paste

(Wilton Industries, Woodridge, IL), 2 mm glass jewelry beads (Darice, Strongsville,

OH), and non-toxic glitter (Advantus Corporation, Jacksonville, FL). Fecal markers were hand-fed in banana pieces on Monday and Wednesdays afternoons (1-3PM) and first morning void samples were evaluated for evidence of the marker for 48 h. During fecal marker evaluation, one marker type was fed per day for all species and all individuals in a group received the same marker color.

Fecal Sample Collection and Processing

First morning fecal voids were collected between 7 AM and 9 AM before and after veterinary exams. Based on the results of the fecal marker study, fecal samples were individually identified using glitter for the golden lion tamarins, pied tamarins, and white-fronted marmosets. No fecal marker was used with the callimicos and individual samples were visually identified by an observer as the animals defecated in the morning.

In addition, immediately after exams, all fresh fecal samples observed throughout the day were collected for a period of 48-72 h for all species except the white-fronted marmoset.

Fecal collection for this species was extended to 7 days because of small fecal size and difficulties collecting first morning voids. Fecal markers were not fed to the golden lion tamarins on the morning of the exam, thus fecal samples collected that afternoon that were not witnessed were not individually identifiable and are shown for reference. In addition, on the morning after the exam, fecal samples from the callimicos were present before the lights were turned on and these individually unidentifiable samples were

collected and shown for reference. Fecal samples were stored in Whirl-pak fecal bags at

-20°C. Feces were lyophilized (FreeZone 4.5 liter freeze dry system, Model: #7751020,

Labconco Corporation, Kansas City, MO, USA), pulverized, and sifted to remove food particles and glitter. Hormonal metabolites were extracted using a method adapted from

Brown (2008): 5 ml of methanol were added to 0.2 g dry feces, briefly hand vortexed, mixed for 1 h (large capacity mixer, Model: #099A LC1012, Glas-Col, Terre Haute, IN,

USA), and centrifuged at 2500 RPM for 20 min (Sorvall Legend RT+). The supernatant was poured off and stored in a -20°C freezer prior to analysis and assayed within six months.

Hormonal analysis

Fecal glucocorticoid metabolites were analyzed via the enzyme immunoassay

(EIA) protocol of Brown (2008) using polyclonal cortisol antiserum (R4866, 1:300,000 dilution) provided by Coralie Munro (University of California, Davis, CA). The EIA was validated for each species by demonstrating: 1) parallelism of the binding inhibition curve of pooled fecal extract dilutions (1:1 to 1:1024) with the cortisol standard

(R2>94%), and 2) significant recovery (> 92.0%) of exogenous cortisol from diluted fecal

samples spiked with the quality control high (200 pg/well) and low (5 pg/well). The

mean inter-assay coefficient of variation (CV) of standards and high- and low-value

quality controls was 7.2%. Intra-assay CVs were less than 10%. Microtiter plates were

read using a spectrophotometer (Epoch, Bio-Tek, Winooski, VT, USA) at 450 nm

wavelength through the Gen5 software (v2.03, Bio-Tek, Winooski, VT, USA).

Biological validation

Fecal samples were collected before and after a routine veterinary exam for the golden lion tamarins and callimicos. Samples were collected 6 days prior to and 10 days post exam for the golden lion tamarins and 5 days prior to and 7 days post exam for the callimicos. Prior to the exam, the golden lion tamarins could not be reliably identified by researchers or staff and a pooled fecal sample from the group was collected each morning. During the exam, the golden lion tamarins were marked (Flouro Tell Tail

Animal Marker, Fil, Mount Maunganui, New Zealand), allowing for individual hand feeding of fecal markers and identification of samples post exam.

In addition, fecal samples were collected from the pied tamarins and white- fronted marmosets opportunistically after veterinary exams prompted by the death of a social partner. For the pied tamarins, the entire group (TF1, TM1, and TM2) was captured and transported to the veterinary hospital for an exam on the breeding male

(TM1). During this exam, the male (TM1) was euthanized as a result of an age-related health decline, and the female received an exam and injection of Depo-Provera (Pfizer

Inc., New York, NY, USA) for contraception. The male offspring (TM2) did not receive an exam and remained in a transport box for the duration of the exam (≈1 h). As a result of elevated FGM levels in afternoon samples from the pied tamarins immediately after the exam, we assessed the potential diurnal variability of FGM concentrations by collecting an additional week of morning (7-8 AM) and afternoon (3-5 PM) fecal samples. Diurnal variation was observed and, therefore, afternoon samples following the veterinary exam were excluded from analysis.

The breeding female white-fronted marmoset (MF1) died unexpectedly and a 2 week-old neonate was temporarily removed from the group for hand rearing. The

breeding male (MM1) received a physical exam the day after this event; the male

offspring (MM2) did not receive an exam and remained in the marmoset exhibit during

MM1’s exam. Two days after this exam, the neonate was returned to the holding area for

hand rearing procedures. Baseline data were available from 50 days prior to this event.

Fecal samples from the marmosets were collected for 10 days post exam. During this

time, the neonate was housed in an incubator within audiovisual contact of the marmoset

holding area. Staff entered the holding area between 6 AM and 12 AM to feed the

neonate. Additional data collection began 19 days after this initial collection period, at

which point the neonate was housed in a wire mesh cage attached to the holding cage

which allowed for physical contact between the neonate and adult marmosets (MM1 and

MM2). Staff entered the holding area between 6 AM-6 PM for hand rearing procedures.

Two additional deaths occurred in the white-fronted marmoset group from

unrelated causes. An adult male offspring (MM3) died 89 days prior to the death of the

breeding female. The breeding male marmoset (MM1) was euthanized 40 days after the

death of the breeding female (MF1).

Statistical analysis

The baseline FGM concentration was calculated for each individual using an

iterative process whereby all baseline samples greater than the mean plus 2 standard

deviations (SD) were removed, and this statistic was recalculated until no additional

samples were greater than this value, at which point the mean was designated as the

individual baseline (Brown et al., 1999). A sample’s FGM concentration was considered

40 elevated if it exceeded 2 SD of the individual’s baseline value. For the female callimico

(CF1), one elevated sample that occurred prior to the exam was excluded from baseline calculations to generate a representative baseline for this individual but this value was retained in this individual’s validation graph.

Baseline FGM levels were compared between the male and female callimicos and pied tamarins using distribution-free confidence intervals (95% CIdf) around the median.

As the male pied tamarin’s (TM2) FGM levels appeared to increase following the death of his father (TM1), the male and female’s FGM levels were again compared using baseline data from a research project that began 98 days after the death of the breeding male. We considered a difference significant if confidence intervals did not overlap.

Diurnal variation was assessed in the male (TM2) and female (TF1) pied tamarins using descriptive statistics because of small sample sizes. Data were analyzed using SAS

University Edition (SAS Institute Inc., Cary, NC).

Results

Assessment of Fecal Markers

Fecal markers were fed in the afternoon (1-3PM) and all were observable by the following morning voids (Table 2). Although this time frame was successful for most species, when the pied tamarins were fed in early afternoon (1PM), evidence of the food coloring marker was observed prior to first morning voids, indicating the marker was excreted before the tamarins entered their nest box for the evening (5-6PM). In addition, when the tamarins were fed glitter, fecal samples would occasionally contain glitter colors fed to different individuals, even though food sharing did not occur during hand

41 feeding sessions. Although not witnessed during this project, coprophagy has been observed in this group. For the pied tamarins, feeding fecal markers later in the afternoon

(3PM) was found to be more successful.

Consumption of the fecal markers differed drastically between species. The golden lion tamarins and pied tamarins readily consumed all fecal markers, whereas the callimicos and white-fronted marmosets reacted aversively to the food coloring and food paste, often dropping the banana pieces, and would eject the beads out of the side of the mouth. As glitter was the only fecal marker readily consumed by all species, this marker was chosen and subsequently used for the remainder of the study.

Table 2. Assessment of fecal markers fed to four species of callitrichine monkeys: golden lion tamarin (GLT); callimico (CA), pied tamarin (PT); white-fronted marmoset (WFM). Fecal Amount Colors Consumption Identificationa Marker GLT CA PT WFM Food 0.1 mL Red, Yes No Yes No Yellow not coloring green, observed, other blue, colors identifiable. yellow

Food 0.1 mL Red, Yes No Yes No All colors coloring green, identifiable. paste blue

Beads 5-10 per White, Yes No Yes No Roughly 10% animal pink, (WFM and GM) to orange, 40% (PT and GLT) yellow of beads recovered.

Glitter 0.35 g Green, Yes Yes Yes Yes All colors gold identifiable. a Fecal markers were fed in the afternoon (1-3PM) and assessed the following morning (7-9AM).

Changes in FGM levels in relation to veterinary exams and animal deaths

In all four species, we observed a significant increase in FGM concentrations 24-

48 h after a veterinary exam (Table 3). As the magnitude and pattern of increase differed across species, these results are presented separately for each group.

Table 3. Summary of fecal glucocorticoid metabolite response of four callitrichine species after veterinary exams. Speciesa ID Baseline FGM Peak FGM b, d Magnitude c, d Elevated Peak b (ng/g) (ng/g) Increase Lag (h) Lag (h) Golden lion tamarin LF1 481.5 2383.2 [13639.1] 4.9 [28.3] 31.7 31.8 (L. rosalia) LF2 380.6 4087.0 [13639.1] 10.7 [35.8] 22.3 26.8 LF3 1041.8 3688.8 [13639.1] 3.5 [13.1] 25.6 25.6 LM1 751.3 5748.7 [13639.1] 7.7 [18.2] 6.0 6.0 LM2 218.6 1132.9 [13639.1] 5.2 [62.4] 23.8 23.8 LM3 397.5 9576.5 [13639.1] 24.1 [34.3] 4.4 4.4 LM4 791.2 6837.4 [13639.1] 8.6 [17.2] 24.4 24.4

Callimico GF1 492.9 1550.7 [2649.5] 3.1 [5.4] 19.5 20.3 (C. goeldii) GM1 189.4 600.4 [2649.5] 3.2 [14.0] 19.7 21.3

Pied tamarin TF1 123.5 2627.9 21.3 20.2 20.2 (S. bicolor) TM1 62.9 266.7 4.2 20.2 20.2

White-fronted marmoset MM1 32.7 4822.7 143.1 24.3 238.3 (C. geoffroyi) MM2 40.8 798.3 19.6 48.3 165.3 a Pied tamarin and white-fronted marmoset also experienced social changes that included the death of a breeding individual and for the white-fronted marmoset, hand-rearing of a neonate in a nearby area. b Data represent the initial FGM peak. c Magnitude represents the fold increase between baseline and peak FGM levels following veterinary exam. d FGM concentrations of individually unidentifiable fecal samples collected 5-20 hrs following the exam are shown in brackets for reference.

Golden lion tamarin

After capture and anesthesia during a routine veterinary exam, all golden lion tamarins demonstrated elevated FGM levels within 48 h post-exam (Fig. 1; representative individuals). In addition, all individually unidentifiable samples collected the afternoon of the exam were elevated above all individuals’ baseline FGM values. A secondary elevation in FGM levels, occurring at roughly 48-72 h post-exam, was observed in multiple individuals (LF1, LF2, LM2 and LM3; see Fig. 1). The magnitude of the primary FGM increases varied considerably across individuals (~4 to 24-fold increase compared to baseline), with a mean increase for the group of 9-fold above baseline.

Individual FGM had returned to baseline levels by 24-48 h.

Figure 1. Fecal glucocorticoid levels of four golden lion tamarins (A: LF1; B: LF2; C: LM1; D: LM2) after capture and anesthesia during a routine veterinary exam (time 0 h). Pooled samples from the group collected before the exam (grey circles) are shown for reference but were not used in individual baseline calculations. Baseline and baseline + 2SD are indicated by solid and dashed lines, respectively. Individually unidentifiable samples collected the afternoon of the exam are shown for reference and denoted with an X (see Methods).

Callimico

Both the male and female callimico showed a peak FGM level roughly 20 h post exam (Fig. 2). The magnitude of this peak was similar for both animals: 3.2 and 3.1 times greater than baseline for the male and female, respectively. By 24 h post exam,

FGM had temporarily returned to baseline concentrations for both individuals.

Secondary peaks of comparable magnitude to the primary peak were also observed

(Male: 43 h and 68 h post exam; Female: 68 h post exam).

Figure 2. Fecal glucocorticoid metabolite concentrations in a female (A) and male (B) callimico before and after a routine veterinary exam (time 0 h). Baseline and baseline + 2SD are indicated by solid and dashed lines, respectively. Individually unidentifiable samples collected the morning after the exam are shown for reference and denoted with an X (see Methods).

Pied tamarin

After a veterinary exam on the breeding male (TM1) and female (TF1) pied

tamarins, during which the breeding male was euthanized due to age-related health declines, the breeding female pied tamarin and her adult male offspring (TM2) demonstrated elevated FGM concentrations post exam, with peak concentrations

48 occurring at 20 h for the breeding female and her male offspring (Fig. 3). The breeding female’s FGM peak after the exam was 21.3 times greater than baseline. The male offspring, who was transported to the veterinary hospital but not examined, exhibited a lower magnitude FGM increase of 4.3 times greater than baseline. Both individuals demonstrated a return to baseline FGM levels within 48 h.

Figure 3. Fecal glucocorticoid concentrations of a female pied tamarin (A) and her adult male offspring (B) before and after the death of the breeding male and veterinary exam (time 0 h). Baseline and baseline + 2SD are indicated by solid and dashed lines, respectively.

White-fronted marmoset

Fecal samples from both male marmosets displayed a progressive increase in

FGM levels following a veterinary exam on the breeding male (MM1) prompted by the death of the breeding female (MF1) and temporary removal of a neonate (Fig. 4). The neonate was returned to the marmoset holding area 2 days after the exam and housed in an incubator for hand rearing procedures. The progressive FGM increase reached a peak magnitude for individual MM1 that was 143 times greater than baseline (11 days post exam) and 19.6 times greater than baseline for individual MM2 (8 days post exam).

Elevated samples began 24 h post exam for the breeding male and 48 h for the male offspring, who did not receive an exam but did experience a capture event. Fecal samples 27 days post exam showed a return to near baseline FGM levels, although a consistent return to baseline levels was not observed for either individual. No FGM increase was observed after the death of an adult male offspring (MM3) or after the death of the breeding male (MM1).

Figure 4. Fecal glucocorticoid concentrations of two male white-fronted marmosets (A: MM1; B: MM2) before and after the death of the breeding female, temporary removal of a neonate, and subsequent veterinary exam (arrow at time 0 h). The neonate was returned to the off-exhibit holding area after the veterinary exam (arrow at time 48 h). An adult offspring died before the veterinary exam validation and is shown for reference (arrow at time -2124 h). Individual MM1 died during data collection and this event is noted for MM2 (B; arrow at time 924 h). Baseline and baseline + 2SD are indicated by solid and dashed lines, respectively.

Factors influencing fecal glucocorticoid metabolite levels

We observed significantly higher FGM values in the female callimico

(Mdn=402.5 ng/g, 95% CIdf=281.3-762.1) compared to the male (Mdn=156.5 ng/g, 95%

CIdf=125.8-264.8). In the pied tamarin group, the breeding female exhibited higher FGM levels (Mdn=120.0 ng/g, 95% CIdf=78.9-170.1) than both the breeding male (TM1:

Mdn=43.6 ng/g, 95% CIdf=31.0-48.9) and their male offspring (TM2: Mdn=58.7 ng/g,

95% CIdf=51.4-76.4). However, during a subsequent study conducted 98 days after the

death of the breeding male, we observed no significant difference between the female

(Mdn=148.7 ng/g, 95% CIdf=104.5-189.7) and her male offspring (Mdn=129.6 ng/g, 95%

CIdf=104.4-182.3) in samples collected over a period of 11 days. In addition to sex

differences, we observed diurnal variation of FGM for the pied tamarins, with elevated

levels in the afternoon compared to morning samples for both the female (TF1; AM: n=4,

Mdn=74.5 ng/g, Range=44.2-130.9; PM: n=5, Mdn=399.3 ng/g, Range=303.7-2126.1)

and her male offspring (TM2; AM: n=3, Mdn=106.1 ng/g, Range=55.4-109.2; PM: n=6,

Mdn=520.2 ng/g, Range=312.2-640.0).

Discussion

To our knowledge, this is the first study to provide comparative information on the fecal glucocorticoid response of callitrichine species. All animals assessed in this study (n=13) demonstrated elevated FGM within 24-48 h of a veterinary exam, highlighting the broad applicability of this cortisol EIA to measure the stress response of callitrichine species. In addition, we demonstrated diurnal variation of FGM in pied tamarins. We observed differences between species in the pattern and magnitude of the response to stressors. Specifically, the marmoset group that experienced an exam in response to the death of a social partner and hand rearing of a neonate showed the greatest response and suggests that FGM measures are sensitive to acute and prolonged stressors. Differences in baseline FGM concentrations between males and females were observed in some species, although this may depend on the social status of individuals.

Assessment of fecal markers for use in callitrichine monkeys

As first morning fecal samples were collected from multiple individuals each day,

identification of a reliable fecal marker was necessary. We observed strong differences

across species in their reaction to fecal markers, with glitter being the only marker that was readily consumed by all species. Although all animals within a group reacted similarly, the observed preferences may be group-specific, as other studies have

described different preferences (Fuller et al., 2011).

As callitrichines have demonstrated rapid gut passage times (2.5-4.5 h; Heymann

and Smith, 1999; Power and Oftedal, 1996), fecal markers were fed in the early afternoon

for identification of first morning voids the following day. The pied tamarins appeared to

have a more rapid excretion time than other species, as fecal markers fed in the early

afternoon were identifiable in the late afternoon of the same day. Differences in gut

transit times among callitrichines have been noted and linked to dietary differences, with

shorter retention times for more frugivorous species such as tamarins compared to

marmosets that specialize on gum exudativory (Power and Oftedal, 1996).

Effect of veterinary exams and social changes on FGM concentrations

Veterinary exams produced a consistent FGM increase across most species that

was visible for most individuals within 24 h of the exam, with peak concentrations often

observed within 48 h. Following the initial peak, secondary increases were noted in

several species before FGM concentrations returned to baseline levels. This pattern has

been observed in other species (Steller sea lion, Hunt et al., 2004; greater sage grouse,

Jankowski et al., 2009), including primates (yellow baboon, Wasser et al., 2000), and

these authors have suggested that a secondary increase may be a byproduct of

enterohepatic recirculation of hormones (Roberts et al., 2002) or negative feedback

regulation of HPA activity (Jankowski et al., 2009). Although the presence of

individually unidentified samples post exam prevents a clear understanding of the

absolute magnitude increase of FGM levels for each individual, the callimicos appeared

to exhibit a reduced and variable FGM response compared to the golden lion tamarins

and pied tamarins. Differences in the excretion or metabolism of cortisol may exist

between this species and other callitrichines.

The response of the two male marmosets differed from the overall pattern

observed in the other study species, with a consistent increase in FGM concentrations

observed for 10 days after an exam in response to the death of the breeding female, which

for one individual reached a peak FGM concentration 143 times greater than baseline.

The increase in this male was considerably larger than other individuals in this study and

that reported by other studies, such as the 10.8x increase over baseline Heistermann et al.

(2006) described in two common marmosets after translocation to a new institution. Two

days after the marmoset’s veterinary exam, a neonate that was born 9 days prior to the

death of the breeding female, was returned to the marmoset off-exhibit holding area for hand-rearing in an incubator. The presence of the neonate and its frequent vocalizations may have contributed to this progressive increase in FGM as several studies have found male marmosets are highly motivated by infant vocal cues (e.g. Zahed et al., 2008). The playback of infant distress calls to unrelated male common marmosets increased serum cortisol with no evidence of habituation after repeated exposures (Barbosa and Mota,

2014). As these authors point out, marmosets exhibit a high degree of male parental

54 investment and increased cortisol in response to neonate cues may be an important mechanism to promote care-giving behavior. In addition to the presence of the neonate, increased human activity in the holding area during the hand rearing process may have also negatively influenced the marmosets. Previous studies on laboratory-housed common marmosets have reported increased fecal glucocorticoids (Barbosa and Mota,

2009) and salivary cortisol (Cross et al., 2004) in response to increased human activity.

As monogamous pair-bonded primates, the death of the breeding female marmoset may have contributed to this prolonged stress response. Partner-directed affiliative and protective behaviors have been reported in a wild male common marmoset to a dying female mate (Bezerra et al., 2014). However, it is unlikely that the death of the female contributed directly to the dramatic increase in FGM concentrations observed for two reasons. First, elevated samples were first observed 24-48 h after the exam, which corresponds to 48-72 h after the death of the breeding female and removal of the neonate.

Based on the time lag to elevated FGM concentrations observed in other animals in this study (24-48 h), this initial increase was more likely to be in response to the exam than to the female’s death. Second, this dramatic increase in FGM concentrations that occurred in both the breeding male and adult offspring was not observed after the death of an adult male offspring or after the death of the breeding male. Similarly, the pied tamarins demonstrated a rapid return to baseline FGM levels after a veterinary exam and death of the breeding male, with no prolonged response to the male’s absence. Huck et al. (2005) also reported no increase in FGM in a wild group of mustached tamarins after the death of a breeding female.

Both marmosets in the present study showed a reduction to near baseline levels 27

days after the exam but neither demonstrated a complete return to baseline. During this

subsequent collection period, the neonate had been moved to a temporary cage attached

to the marmoset holding cages that allowed for physical contact between the neonate and

adult marmosets. Human activity from the hand rearing procedure had also decreased by

this point, but was still more frequent than baseline periods. Taken together, it is likely

that a combination of factors related to the nearby hand rearing of a neonate contributed

to the continual FGM response in the marmosets. Although being in audiovisual contact

with conspecifics during the initial hand rearing may benefit the neonate and is a

recommended practice (Ruivo, 2010), the potential adverse effects on adults should be considered and efforts should be made to minimize this disturbance as much as possible.

Demographic and diurnal factors influencing FGM levels

Differences in FGM concentrations were observed in the callimicos and pied

tamarins, with higher concentrations present in females vs. males, suggesting possible sex

differences in these species although the sample size was limited. Higher cortisol

concentrations in females compared to males have also been reported for other

callitrichine species: pied tamarin (Armstrong and Santymire, 2013), common marmoset

(Johnson et al., 1996; Raminelli et al., 2001), and Wied’s black tufted-ear marmoset

(Callithrix kuhlii; Smith and French, 1997). In the pied tamarin group this difference

appeared to depend on the social status of the males. Although the female had higher

baseline FGM levels than both the breeding male and adult offspring during the initial

baseline periods, no significant difference was observed between the female and male

offspring roughly 3 months after the death of the breeding male. While the female’s

FGM level appeared similar during both time periods, the male offspring’s FGM level doubled. This change may reflect the changing social conditions for the male offspring, as Ziegler et al. (1996) reported lower urinary cortisol in experienced breeding males and their male offspring living in the natal group compared to inexperienced males living with or near females. Although FGM differences between sexes may actually be a reflection of possible cross-reactivity of the cortisol EIA antibody with sex-specific hormones as suggested by Goymann (2012), this is unlikely in the present study as previously published cross-reactivity of the cortisol antibody showed strong specificity for cortisol (Young et al., 2004). However, sex differences in the metabolism of cortisol remains possible and requires further assessment.

Diurnal variation of FGM concentrations was evident for the pied tamarins, with increased glucocorticoids present in afternoon samples compared to morning samples. A clear circadian pattern of cortisol production has been described in other callitrichine species, with salivary and urinary cortisol measures increasing from the first morning void to a late morning peak followed by a decrease in the late afternoon (Cross and

Rogers, 2004; McCallister et al., 2004; Smith and French, 1997). In contrast, FGM levels have been shown to be higher in the afternoon (common marmosets: Raminelli et al.,

2001; Sousa and Ziegler, 1998), likely owing to the lag time of excretion through feces.

However, detecting a circadian rhythm of cortisol in feces has not been clearly documented across primates (Huck et al., 2005; Setchell et al., 2008; Weingrill et al.,

2004). Additional research is needed to assess factors such as diet and metabolism that may contribute to species-specific differences in the excretion of cortisol in feces.

This study demonstrated the broad applicability of a fecal cortisol EIA to measure the stress response of callitrichine monkeys through noninvasive methods. We observed differences between species with respect to the magnitude of FGM increase in response to veterinary exams and other potential stressors, although all animals responded to the stress of veterinary handling. Specifically, a progressive increase in FGM levels was observed in two male marmosets during the separation and hand-rearing of a neonate, highlighting the importance of considering the potential impact of hand rearing on group members. Death of conspecifics did not appear to influence FGM levels of the pied tamarins or white-fronted marmosets. Although sample sizes were limited, we did observe some species-specific differences in FGM concentrations between sexes. Similar species-specific sex differences between closely related species have been noted in Ateles

(Rangel‐Negrín et al., 2009; Rimbach et al., 2013) and suggest that HPA activity may not be generalizable across even closely-related species. Future research is needed to explore species-specific differences in HPA activity and to uncover what factors may have contributed to these changes.

Chapter Three: The influence of waterfall sounds and access to off-exhibit areas on the behavior and exhibit use of three species of callitrichine monkeys

Introduction

Noise, or unwanted sound, is a potential source of stress for animals (Morgan and

Tromborg, 2007). With zoos often being located in urban settings and attracting large numbers of visitors, the effects of noise may be particularly concerning for zoo-housed animals. Research has identified potential negative consequences of ambient noise in the zoo setting. For examples, in response to visitor noise, studies have documented increased vigilance behavior (orangutan, Birke, 2002; gorilla, Clark et al., 2012; white- handed gibbon, Cooke and Schillaci, 2007; lion, Farrand, 2007; koala, Larsen et al.,

2014), increased activity (giant panda, Owen et al., 2004), altered maternal (sun bear,

Owen et al., 2014) and infant (orangutan, Birke, 2002) behavior, increased stress-related behaviors (giant panda, Owen et al., 2004), and elevated cortisol levels (giant panda,

Owen et al., 2004). Similar effects have also been described for construction noise (giant panda, Powell et al., 2006; snow leopard, Sulser et al., 2008). Addressing these concerns is challenging and will likely require modifying the sound environment for zoo animals.

One approach that has been suggested is to introduce sounds in order to mask disruptive or distracting noises arising in the environment (e.g. Ogden et al., 1994).

Auditory masking is the process whereby a masking sound interferes with the perception of other sounds resulting from increased hearing thresholds. For people, masking sounds have been successfully introduced into office settings to reduce

59 distracting sounds (Loewen and Suedfeld, 1992) and a recent study identified water sounds as being particularly beneficial (Haapakangas et al., 2011). Similarly, water features have been found to successfully mask road noise and improve the soundscape quality of parks (De Coensel et al., 2011; Galbrun and Ali, 2013; Jeon et al., 2010;

Nilsson et al., 2010). Currently, few studies have investigated the potential of auditory masking for animals, although this has been suggested as a potential explanation for the beneficial effects of auditory enrichment for animals (Patterson-Kane and Farnworth,

2006; Wells, 2009).

However, data on free-ranging wildlife have highlighted potential costs of auditory masking from anthropogenic sources of noise (Barber et al., 2010). One obvious cost is the masking of communication between animals (Slabbekoorn and Ripmeester,

2008). This may even carry direct fitness consequences, as sparrows breeding near a generator showed reduced parental success, an effect the authors attributed to masking of parent-offspring communication (Schroeder et al., 2012). For prey species, auditory masking can impact the ability to detect predators. For example, ground squirrels living near a wind turbine exhibited elevated levels of vigilance and increased likelihood of returning to their burrows in response to the playback of conspecific alarm calls compared to ground squirrels living in a quiet area (Rabin et al., 2006).

The primary aim of the current study was to determine if loud sound from a waterfall feature provided beneficial masking effects for three species of callitrichine monkeys housed nearby: pied tamarin, white-fronted marmoset, and callimico. Also, we investigated whether access to quiet off-exhibit areas was beneficial and could modify the response to the sound environment. Behavior and exhibit use were recorded during

60 experimental modifications of the waterfall feature and access to off-exhibit areas. In addition, we recorded visitor presence and sound levels to describe their potential relationship and effect on behavior. If the waterfall provided benefits through auditory masking of disruptive or distracting sounds, we expected the waterfall sounds to promote decreased vigilance and other behavioral indicators of anxiety (e.g. self-directed behavior and scent marking), and increased use of the exhibit space when animals had access to off-exhibit areas. Overall, we expected increased choices through access to off-exhibit areas would have beneficial effects on behavior.

Materials/Methods

Subjects and housing

Subjects for this study included nine individuals representing three species of callitrichine monkeys: pied tamarin (Saguinus bicolor), white-fronted marmoset

(Callithrix geofroyii), and callimico (Callimico goeldii). Demographic characteristics of the social groups are shown in Table 1. An adult male marmoset died of natural causes during the seventh week of the study and data from this individual were excluded.

Table 1. Demographic background of study subjects. Individual Sex Age (y.o.) Pied tamarin TM1 M 15.3 TF1 F 4.1 TM2 M 1.4 TM3 M 0.9 White-fronted marmoset MM1a M 5.6 MF1 F 3.9 MM2 M 0.8 MM3 M 0.8 Callimico CM1 M 20.2 CM2 F 2.5 a Individual died during study and data were not included in analysis.

Animals were housed in single-species exhibits in the front of the RainForest building at Cleveland Metroparks Zoo. Exhibits were approximately 18 m3 and included

branches and small trees for climbing. Guests were able to view animals through a mesh

barrier in the front of exhibits that was separated from viewing areas by a small planter

that included low bushes. The viewing area in front of the pied tamarin exhibit was

situated in a gift shop area. The gift shop separated this exhibit from an open lobby area

in front of the marmoset and callimico exhibits. A small door at the back of the exhibits

connected to off-exhibit overnight holding cages (4 m3). Exhibits and holding areas of

each species were visually isolated from other species but auditory contact between

species was possible.

A waterfall was located in the front entrance lobby area adjacent to exhibits. The waterfall featured a 30 ft. drop and, in addition to the overhead ventilation system and ground fans near the waterfall, created a loud broadband sound environment within exhibits (Fig. 1). Exhibits were oriented parallel to the waterfall, with the marmoset exhibit being the closest (6 m.) and the callimico and tamarin exhibits at roughly 13 m

from the edge of the waterfall..

Experimental design

This study was conducted during weekdays from February to May 2012.

Modifications to the waterfall feature (on vs. off) and access to off-exhibit areas (access

vs. no access) were systematically manipulated in six two-week experimental phases in an ABACDA design. Baseline conditions (A) with the waterfall on and no access to off-

exhibit areas were alternated with experimental conditions that modified the waterfall or

access features (B, C, D; Table 2). Baseline conditions were followed during weekends.

Table 2. Overview of experimental design. Two-week experimental phase Experimental Modification A B A C D A Waterfall On X X X X Waterfall Off X X Off-exhibit access X X No off-exhibit access X X X X

After the first two-week baseline phase, minor construction in the gift shop took

place that required data collection to be suspended for approximately one week.

Additionally, data from one day of the study were excluded as a result of errors

manipulating the waterfall feature.

Behavioral data collection

Data were recorded during 15 min focal observations (n=505; 55-57 observations per animal) using instantaneous point-sampling at 30 s intervals for state behaviors and exhibit use and all-occurrences sampling for event behaviors (Martin and Bateson, 2007;

Table 3). Additionally, continuous measurement of the time the focal animal was visible was recorded to adjust all-occurrences data. Observations were balanced between morning (AM: 10AM – 1PM, n=253) and afternoon (PM: 1PM – 4PM, n=252) and the daily order of individual observations was randomized.

Table 3. Ethogram of behaviors considered in this study. Behavior Definition

State Behaviors (instantaneous point-sampling) Monitor Visitor Animal is stationary and visually focused on an object (including visitors or observer) outside the exhibit or scanning the visitor area for a minimum of 3 seconds. Locomote Animal travels greater than one body length (not including the tail). Inactive Animal is stationary and either alert and actively monitoring surroundings or resting and not attending to environment, typically with tail curled around body and eyes closed. Social Affiliation Animals are engaged in a non-aggressive positive behavior and includes grooming and play behavior. Event behaviors (all-occurrences sampling) Self-directed Animal manipulates body using hand, feet, or mouth and typically took the form of scratching or self-grooming. Scent Marking Repeated rubbing of scent glands in anogenital, suprapubic, sternal, or facial area across substrate or body part (including tail marking in the callimicos and hand marking in the pied tamarins).

Visitor and sound measurement

To evaluate for the potential effect of visitors, the total number of visitor groups

that stopped at an exhibit to view animals during behavior observations was recorded. A

group was defined as a visitor party of any size that approached the exhibit together and

viewed the exhibit for a minimum of 2 s. This measure was chosen for ease of recording

and based on pilot data that found the majority of groups at this exhibit were small (72%

< 3 visitors). In addition, overall zoo attendance was recorded each day.

Sound levels were measured in each exhibit using a data logging sound level

meter (Model 407760, Extech Instruments, Nashua, NH). The sound level meter was

mounted in a protective enclosure and placed on the floor of the front of the exhibit and

oriented towards the visitor area to minimize the influence of animal vocalizations on

sound level measurements. The sound level meter was capable of measuring sounds

between 20 Hz-20 kHz with an accuracy of ± 2dB. The sound level meter was set to

record sound levels every 0.5s using a ‘Fast’ (125 ms) time-weighting response and ‘A’ frequency weighting curve (dBA). A-weighting adjusts sound levels based on the frequency sensitivity of human hearing and was chosen because most primate species have been shown to have a broadly similar pattern of hearing (Heffner, 2004). As sound pressure levels fluctuated, equivalent continuous sound levels (Leq) were calculated (Eq.

1). The Leq represents the mean energy level during a sampling period expressed on the

decibel scale and has been previously used in studies assessing noise in zoos (Quadros et

al., 2014). Equivalent continuous sound levels were calculated for the entire day (10:00-

16:00; Daily Leq) and during 15 min observations (Observation Leq). Because sound level

meters were not available for each exhibit every day, these data were used to describe the

sound environment and not included as a variable in the behavior analyses.

Eq 1. Formula for calculating the equivalent continuous sound level (Leq) from N equally

spaced sound level measurements (see Raichel, 2006):

1 / Leq = 10log ( 𝑁𝑁 10 ) 𝐿𝐿𝐿𝐿 10 � 𝑁𝑁 𝑖𝑖=1

Statistical analysis

Daily Leq sound levels were compared between days with the waterfall on vs. off

for each exhibit using a Wilcoxon rank sum test with t approximation. In addition,

pairwise comparisons of daily Leq sound levels between the three exhibits when the

waterfall was off vs. on were performed using Wilcoxon rank sum tests. The relationship

between visitors and sound levels was evaluated using a Spearman rank correlation

between Observation Leq sound levels and the number of visitor groups. As we observed

a potential influence of loud animal vocalizations on observation sound levels, we

reassessed this relationship using Leq values that excluded sound levels above 85 dBA,

the minimum sound level of vocalizations we suspected based on exploratory graphs. To

assess differences between exhibits or conditions in the number of visitor groups stopping

during an observation (Groups), a generalized linear mixed model (GLMM; Proc

GLIMMIX; SAS) was constructed. Fixed effects included Exhibit (tamarin, marmoset,

callimico), Waterfall (on or off), Access (access or no access), and overall zoo attendance

as a covariate to account for changes during the study. A random effect of Date was

included with a Variance Components covariance structure to account for the potential

correlation of multiple observations across exhibits on the same day. As the Groups

variable was non-normal, the model was constructed using a negative binomial

distribution with log link function. The negative binomial distribution is commonly used

with overdispersed skewed count data. Parameters were estimated using Laplace

approximation and degrees of freedom was calculated using the between-within method.

Behavioral data were assessed as counts (number of scans for state behaviors and exhibit use or total number of occurrences for event behaviors) and analyzed using

GLMMs. Models were adjusted for the time spent visible using an offset term. The

GLMM model for the time spent in the Front exhibit location was adjusted using an offset term of the scans on exhibit to account for changes in the total available space depending on access to off-exhibit areas. All behaviors except Inactive were modeled using a negative binomial distribution and a log link function to account for overdispersion and a skewed data distribution. As the Inactive behavior model residuals were approximately normal using a log link function, a Gaussian distribution was specified. Parameters were estimated using Laplace approximation and degrees of freedom were calculated using the between-within method. To account for the non- independence of repeated measures data, a random effect of Individual (animal identity) was included in all models using a Variance Components structure.

A full model was first constructed that included fixed effects of Species (tamarin, marmoset, and callimico), Waterfall (on or off), Access (off-exhibit access or no access),

Groups (number of visitor groups present during observation), Waterfall*Access interaction, and Species interactions with Waterfall, Access, and Groups. To account for

variability of behaviors across time of day, a random slope effect of Time (AM or PM)

was assessed with the full model using a likelihood ratio test comparing an intercept-only

model to an intercept-and-slope model (Bolker et al., 2009). The addition of the random

slope effect of Time significantly improved model fit for all behaviors except Inactive

and Off-exhibit. For these behaviors, intercept-and-slope models did not converge and

intercept-only and slope-only models were compared, resulting in a random intercept-

only model for Inactive and random-slope only model for Off-exhibit. As the main

question of the study involved the effects of the waterfall and access on the study species,

these main effects and their species interactions were retained in all models. The

Waterfall*Access, Groups, and Groups*Species effects were removed in a step-wise

fashion from the full model if they were non-significant (p>0.1) and their removal did not

worsen model fit (change in corrected Akaike information criterion <2; Burnham and

Anderson, 2001). The assumption of a normal distribution of random effects was

assessed using Q-Q plots. Post-hoc pairwise comparisons were conducted using least

squares means (LS means) with the Tukey-Kramer adjustment for multiple comparisons.

As the effect of the experimental conditions varied across species, the GLMM results are presented as LS means ± SEM from the species interaction terms. All statistical tests were performed using SAS University Edition (SAS Institute, Cary, NC). Differences below p=0.05 were considered significant and differences approaching significance

(p≤0.1) were identified as trends.

Results

Environmental differences between exhibits and conditions

Daily Leq sound levels were significantly higher in the three exhibits with the

waterfall feature on, increasing by roughly 7-10 dBA (Table 4; Tamarin: W=120, Z=-

5.37, p<0.0001; Marmoset: W=105, Z=-5.18, p<0.0001; Callimico: W=109, Z=-5.03, p<0.0001). Sound level differences between exhibits were observed, with the pied tamarin exhibit located in a gift shop being the quietest of the three exhibits both with the waterfall on (Callimico vs. Tamarin: W=1050, Z=6.3, p<0.0001; Marmoset vs. Tamarin:

W=1161, Z=6.4, p<0.0001) and when it was off (Callimico vs. Tamarin: W=275, Z=2.8, p=0.0088; Marmoset vs. Tamarin: W=308, Z=4.2, p=0.0002). There was no difference in sound levels between the callimico and marmoset exhibits when the waterfall was off.

However, when the waterfall was on, the marmoset exhibit, being the closest to the waterfall feature, was louder than the callimico exhibit (Callimico vs. Marmoset: W=453,

Z=-3.8, p<0.0004). Although not formally measured during this study, subsequent data collection has found off-exhibit sound levels to be approximately 55 dBA, except during brief periods of hosing during cleaning when the sound levels could range from 70-90

dBA.

Table 4. Sound levels (median ± interquartile range) in the study exhibits with the waterfall feature on and off.

Daily Leq Sound Level (dBA) Waterfall On Waterfall Off Pied tamarin 67.7±1.5 60.6±2.0

White-fronted marmoset 75.3±2.0 65.3±4.5

Callimico 73.9±1.2 64.0±5.5

Note. The equivalent continuous sound level (Leq) represents an average sound energy over the day.

Sound levels during observations were positively correlated with the number of visitor groups at the pied tamarin exhibit (WF on: rs=0.46, p<0.0001; WF off: rs=0.27,

p=0.03). At the marmoset exhibit, observation Leq sound levels were negatively

correlated with visitor groups when the waterfall was off (rs=-0.31, p=0.02) and not

correlated when the waterfall was on (p=0.84). We observed no relationship between

observation sound levels and visitor groups at the callimico exhibit (WF on: p=0.38, WF

off: p=0.31). When these relationships were reassessed to account for the potential

influence of animal vocalizations by excluding sound levels above 85 dBA, we observed

no relationship between the number of visitor groups and observation sound levels at the

marmoset exhibit, although results at the pied tamarin exhibit were unchanged.

The number of visitor groups during an observation varied between exhibits

(Generalized linear mixed model: F2,110=40.89, p<0.0001). The highest number of visitor

groups were present at the callimico exhibit (Mdn=4/ observation), with fewer visitors

present at the pied tamarin (Mdn=3/ observation) and white-fronted marmoset exhibits

(Mdn=2/ observation; Callimico vs. Marmoset: t110=9.01, p<0.0001; Callimico vs.

Tamarin: t110=5.36, p<0.0001; Marmoset vs. Tamarin: t110=-4.59, p<0.0001). Although

there was no significant effect of the waterfall feature on visitor groups (p>0.05), fewer

visitors were present during observations when animals had access off-exhibit

(F1,53=9.95, p=0.003; Table 5). The number of visitor groups during observations was

strongly influenced by overall zoo attendance levels (F1,53=40.17, p<0.0001).

Table 5. The median (± IQR) number of visitor groupsa per observation at exhibits across study conditions. No Off-exhibit Access Off-exhibit Access Exhibits Waterfall Waterfall On Waterfall Waterfall On Off Off Pied tamarin 3±2 4±4 2±2 1.5±3 White-fronted marmoset 2±2 2±3 1±2 1±2 Callimico 5±4 4±5 2±1.5 4±3 a A visitor group was defined as a party that approached the exhibit together and paused at the exhibit for a minimum of 2 s. Visitor groups were recorded on an all-occurrence basis during behavior observations.

Effect of waterfall and off-exhibit access on behavior

Modifications to the waterfall feature and off-exhibit access influenced several

behaviors (Fig. 1). The monkeys spent overall less time monitoring the visitor area when

the waterfall was off compared to when it was on (Fig. 1a; F1,6=6.39, p=0.045), although

this effect may have depended on off-exhibit access as a non-significant trend was identified (F1,8=4.24, p=0.074). When the monkeys did not have off-exhibit access, they

monitored the visitor area more often when the waterfall was on vs. off (Fig. 2; t8=-3.54,

p=0.008) but no difference was observed when they had off-exhibit access. The

waterfall feature influenced the activity of the marmosets and tamarins in an opposing

fashion (Fig. 1b; F2,6=11.28, p=0.009), with the marmosets locomoting more with the

waterfall off (t6=3.46, p=0.013), whereas the pied tamarins were less active (t6=-3.17,

p=0.019). Increased affiliative behaviors were also observed in the tamarin group when

the waterfall was turned off (Fig. 1d; t6=2.6, p=0.041).

When access to off-exhibit areas was provided, callimico spent more time

monitoring the visitor area (Fig. 1g; t6=-2.84, p=0.030). There was a general increase in

locomotion across species when off-exhibit access was available (Fig. 1h; F1,6=13.79,

p=0.010). A post-hoc comparison revealed this effect was significant for the marmosets

(t6=-3.65, p=0.011) and a trend to significance for the tamarins (t6=-1.95, p=0.10),

although the Access*Species interaction term was not significant (p=0.34). The

marmosets also displayed increased inactivity with off-exhibit access (Fig. 1i; t6=-2.93,

p=0.026). Animals spent less time engaged in affiliative behaviors when access to off-

exhibit areas was available (Fig. 1j; F1,6=9.66, p=0.021), particularly in the callimico

group (t6=3.05, p=0.022). Differences in all-occurrences behaviors were noted, with

decreased self-directed behavior in the tamarins (Fig. 1k; t6=2.71, p=0.035) and increased scent marking in the marmosets (Fig. 1l; t6=-2.96, p=0.025) when off-exhibit areas were

available.

Figure 1. The least squares means from GLMM behavior models for each species showing the effects of the waterfall (a-f) and access (g-l) conditions. Significant (p<0.05) main effects and post-hoc species comparison are indicated by asterisks and lines, respectively.

Figure 2. The influence of waterfall noise on monitoring the visitor area when monkeys had no access off-exhibit and when they had access off-exhibit. Columns represent LS mean estimates ± standard error of the mean from a GLMM.

Effect of waterfall on exhibit use

Waterfall sound also influenced the use of exhibit and off-exhibit areas (Fig. 3).

When the waterfall was on, the marmosets spent less time in the front half of the exhibit

(t6=4.26, p=0.0053) with a similar trend observed in the callimico group (t6=1.98,

p=0.095). An opposite response was displayed by the tamarins, with a trend of increased

time in the front of the exhibit when the waterfall was on vs. off (t6=-2.21, p=0.069).

When animals had access to off-exhibit areas, they used these spaces significantly more often when the waterfall was on vs. off (F1,6=10.79, p=0.017), particularly in the

callimico group (t6=-3.45, p=0.014).

Figure 3. The least squares means from GLMM exhibit use models for each species showing the effects of the waterfall. Significant (p<0.05) main effects and post-hoc species comparison are indicated by asterisks and lines, respectively.

Effect of visitor groups on behavior and exhibit use

We observed a positive relationship between the number of visitor groups during

observations and the Monitor Visitor behavior (F1,482=3.91, p=0.049). A non-significant

trend of Groups on the Inactive behavior (Groups*Species: F2,480=2.7, p=0.068) and

Front exhibit use (F1,484=2.77, p=0.096) were identified and these terms were retained in

the final model to control for this effect.

Discussion

In this study we sought to determine whether sound from a waterfall feature

provided beneficial auditory masking effects. Our results did not identify benefits of

continuous broadband noise from the waterfall. Instead, the monkeys displayed

increased vigilance and use of quiet off-exhibit areas when the waterfall was on. The waterfall noise did not influence additional behavioral measures of negative welfare, such as self-directed or scent marking behavior, suggesting the animals were able to cope with the impact of the noise. Furthermore, providing access to quiet off-exhibit areas appeared to ameliorate the heightened vigilance when exposed to waterfall noise and provide additional behavioral benefits for the pied tamarin group. However, we did observe fewer visitors at the exhibits when animals had access to off-exhibit areas, which may have influenced results and highlights a potential animal visibility concern when providing out-of-view exhibit choices.

Assessing the behavioral response to the sound environment

Several hypotheses have been suggested for how chronic noise may affect animals. Chan et al. (2010) demonstrated that noise may simply be distracting and exert its negative influence by placing additional demands on attention. Although this cannot be ruled out, the effect of the waterfall noise may have also been a byproduct of auditory masking of important environmental information (Francis and Barber, 2013). For example, the waterfall’s potential auditory masking of sounds from approaching zoo

visitors may have caused the appearance of people to be unexpected by the monkeys.

Humans have been shown to be a threatening stimulus to callitrichines (Cagni et al.,

2009) and monitoring the movement of people may be important for these monkeys.

Callitrichines have been shown to maintain routine vigilance in captive settings and

vigilance has been identified as an important anti-predator behavior for these species

(Barros et al., 2008; Caine, 1984). Indeed, we observed the monkeys to monitor the

visitor area more frequently with increasing visitor groups, as well as when exposed to

waterfall noise. Importantly, providing access to off-exhibit areas, which should not have altered the distracting properties of the waterfall noise, did appear to ameliorate the heightened vigilance of the monkeys from the waterfall. Noise can constrain an animal’s ability to detect potential threats and these results suggest that access to out-of-view areas may reduce the perceived environmental risk. However, it should be noted that access to off-exhibit areas may also modify visitor behavior, as we observed fewer visitors when animals had off-exhibit access. Although it is possible that reduced visitation may have impacted the behavior of the monkeys, visitor levels were consistent within the access conditions (i.e. waterfall off and on periods) and the overall decrease in visitor groups when animals had access was relatively small (1-2 groups less per observation). In addition, animals did appear to avoid the waterfall noise through use of off-exhibit areas, supporting the notion that the sound environment may influence the perception of environmental risk.

Was access to off-exhibit areas inherently beneficial?

Previous studies have highlighted beneficial effects of providing access to off- exhibit areas. For example, when giant pandas were given access to off-exhibit areas,

77 they exhibited lower levels of behaviors indicative of agitation and had lower urinary cortisol concentrations, compared to baseline periods with no access (Owen et al., 2005).

Access to off-exhibit areas also appeared to improve the welfare of a pair of zoo-housed polar bears, reducing pacing and other stereotypic behaviors (Ross, 2006). Similarly, we observed a complete elimination of pacing behavior in a female Malayan sun bear when provided access off exhibit (Rog et al., In review). In the present study, the reaction to having access to off-exhibit areas was mixed. The pied tamarins displayed lower levels of self-directed behavior, an indicator of anxiety in primates (Maestripieri et al., 1992).

However, the behavioral response of the marmosets to off-exhibit access suggested increased arousal, although this finding is confounded by the death of a breeding male during the start of the access experimental conditions. Also, it should be noted that the off-exhibit area for the marmosets was in a kitchen prep area and experienced more frequent traffic by zoo staff than other off-exhibit areas, which may have contributed to this response. Although increased vigilance and decreased affiliation by the two male callimicos in response to off-exhibit access suggest a negative response, these animals also spent the most time off exhibit when exposed to the waterfall noise, suggesting access was a beneficial modification. Overall, access to off-exhibit areas did promote behavioral benefits for some species and provide an opportunity to avoid noise (e.g.

Sulser et al., 2008).

Variation in response to waterfall noise

There appeared to be species differences in the reaction to the waterfall noise, with the pied tamarins displaying a more confident response than the other groups, with increased movement and time spent in the front of the exhibit. The marmoset and

callimico groups, in contrast, spent less time in the front half of the exhibit and the

marmosets decreased movement. Callimico, in particular, appeared to avoid noise

through use of off-exhibit areas. This variation may reflect the differences in sound

levels across exhibits, with the pied tamarin exhibit located in a gift shop area being the

quietest exhibit. Alternatively, these differences may reflect species traits. For example,

pied tamarins have been observed frequently displaying threat behavior towards visitors

and approaching the front of the cage (Wormell et al., 1996), whereas callimico were

found to decrease use of the front of their enclosure when visitor density was high

(Simpson, 2004). Variability in antipredation strategies (Ferrari, 2009) and neophobia

(Addessi et al., 2007; Day et al., 2003; Savastano et al., 2003) have been described within

Callitrichinae. However, given the small sample size of the current study, additional

comparative research in callitrichines is needed to confirm these species differences.

Managing the sound environment of zoos

Zoos are commonly located in urban centers and attract millions of visitors every

year. The effect of visitors on zoo animals is complex and, for some species at least, may

negatively impact welfare (Davey, 2007; Hosey, 2000). Although we were unable to

identify the effect of visitor noise on the callitrchine monkeys in the present study, other

studies have demonstrated a negative impact of visitor noise on animals (Birke, 2002;

Larsen et al., 2014; Owen et al., 2004). In addition, noise from construction activities

may be another source of stress for animals (Powell et al., 2006; Sulser et al., 2008). One

potential method to reduce the negative impact of noise is through the application of

masking sounds (Ogden et al., 1994). Although this has been successful for people in office and urban settings, this technique is focused on reducing the impact of distracting

noises and would likely be counter-productive if applied to sounds perceived as

threatening. Currently, our understanding of how visitor noise and other sounds in the

zoo environment are perceived by animals, either as distractions or threats, is limited and

will likely vary based on the human-animal relationship of an individual, group, or

species and by degrees of habituation to zoo noises (Claxton, 2011; Hosey and Melfi,

2012). For callitrichines, the performance of agonistic threat displays to visitors and staff

suggest masking sounds may not be an appropriate approach for these species. Also,

careful attention to the masking sound is important as it may itself become a source of

stress. As the decibel scale is logarithmic, the 7-10 dBA increase in sound levels from the waterfall feature in the current study represented a doubling of perceived loudness to humans. The location of the masking sounds is also an important consideration, as masking sounds provide greater interference if situated near the listener (Foreman, 2008).

For zoos, this would suggest the placement of water features or speakers for masking sounds should be located within exhibit spaces.

Although wild habitat sounds or waterfall features may be an attractive option from a guest perspective, it may be beneficial for animals to minimize extemporaneous sounds within exhibit spaces. This study and others suggest animals may prefer lower sound levels. With regards to visitor noise, Sherwen et al. (2014) showed signage asking guests to watch animals quietly did successfully reduce noise levels. Increasing vegetation in exhibits could decrease sound levels, provide additional visibility barriers, and promote a naturalistic appearance of the exhibit, potentially influencing guest satisfaction (Davey, 2006; Fàbregas et al., 2012; Melfi et al., 2004). For indoor exhibits, glass barriers provide attenuation of sounds (Tromborg, 1993), although this may also

invite banging and potentially increase the startle to visual stimuli for prey species.

Because these modifications are costly and may not be feasible for some zoo exhibits,

providing access to quiet off-exhibit areas, as done in this study, may be an additional

means to decrease the impact of noise on zoo animals and provide additional behavioral

benefits. However, as access to out-of-view areas may negatively impact animal visibility and unintentionally undermine the education opportunity of zoos, we recommend these changes be made in conjunction with animal visibility surveys and exhibit signage explaining the change (e.g. Kuhar et al., 2010).

We did not find evidence of beneficial auditory masking through sound from a waterfall feature. Instead, we identified a negative response to waterfall noise, with increased vigilance and use of off-exhibit areas. Waterfall noise did not influence rates of self-directed or scent marking behaviors, both common indicators of arousal in callitrichines, and suggests the monkeys were able to cope with the loud sound environment and the overall impact on welfare was minimal. Providing access to off-

exhibit areas did provide an opportunity to avoid noise and behavioral benefits for some

species. Differences between groups in the response to waterfall noise and off-exhibit access were noted and may reflect species differences, although additional research with a larger sample size is needed to confirm this. Increasing the exhibit choices for animals, such as access to off-exhibit areas, is likely to provide welfare benefits and we encourage this practice. Although auditory masking of distracting noises may be beneficial if appropriately designed, additional research is needed to identify how visitor and other noises arising in the zoo environment are perceived by animals, as this may guide appropriate noise management strategies.

Chapter Four: Do zoo animals use off-exhibit areas to avoid noise? A case study

exploring the influence of sound on the behavior, physiology, and exhibit use of two pied

tamarins (Saguinus bicolor)

Introduction

Zoos environments are likely louder than most animals would experience in their

native habitat (Morgan and Tromborg, 2007). Some of these sounds, such as music for

enrichment purposes, may have beneficial effects (Wells and Irwin, 2008). However,

unwanted sounds, or noise, is a concern and a growing body of research in zoos has

highlighted the adverse effects noise may have, including: elevated cortisol levels (Owen et al., 2004; Powell et al., 2006), increased vigilance behavior (Birke, 2002; Cooke and

Schillaci, 2007; Larsen et al., 2014), and increased stress-related behaviors (Owen et al.,

2004; Powell et al., 2006). One potential solution may be to provide animals choices that

allow the opportunity to avoid noise (e.g. Sulser et al., 2008). For some zoo exhibits, this

may be possible by offering access to quiet off-exhibit areas.

Previous research has documented beneficial effects of providing access to off- exhibit areas. In a six-year study of giant pandas housed at the San Diego Zoo, Owen et

al. (2005) found that when pandas were provided daily access to off-exhibit overnight

areas, they spent less time engaging in agitated behaviors (e.g. pacing, scratching, and

door-directed behavior) and exhibited lower urinary cortisol levels compared to days with

no access. It is unclear what aspect of this choice promoted positive welfare, a point

raised by the authors. Possibly having additional choices and consequentially, increased

control, may promote psychological benefits (Hanson et al., 1976). In support of this,

Ross (2006) observed reduced levels of pacing and other stereotypic behaviors in a pair of polar bears when provided access to off-exhibit areas, even though these animals only used these areas for 2% of their time budget. Alternatively, some aspect of the off- exhibit area may be attractive and address a behavioral need, as these areas are typically quiet, visually isolated from visitors, provide a thermal refuge, include sleeping areas, and are closer to animal care staff.

In this study, we assessed the influence of the sound environment on the exhibit use, behavior, and fecal glucocorticoid levels of two pied tamarins (Saguinus bicolor), a

critically endangered, arboreal New World monkey. These monkeys were housed in an

exhibit adjacent to a large waterfall feature that exposed the monkeys to loud, chronic

sound. During the day, the monkeys also had access to quiet off-exhibit areas. The sound environment was modified during two experimental treatment conditions. First, the waterfall feature was deactivated, lowering the sound levels on exhibit (“quiet” condition). Second, the waterfall feature was reactivated and a speaker in the off-exhibit area played white noise at a consistent volume to the waterfall noise on exhibit (“loud” condition). Exhibit use, behavior, and fecal glucocorticoids were evaluated during these experimental periods and a pre- and post-experiment baseline condition with the waterfall feature activated and no sound playback in off-exhibit areas. In addition, we recorded visitor presence and sound levels to describe their relationship and identify potential effects on behavior. We expected decreased use of off-exhibit areas during the quiet and loud treatment conditions, as these areas no longer provided an alternative sound environment to the exhibit space. In addition, we expected behavioral and physiological

indicators of anxiety and arousal to decrease in response to the quiet conditions and

increase during loud conditions.

Methods

Subjects and housing

Subjects included a female pied tamarin (6.2 y.o.) and her adult male offspring

(3.8 y.o.). A breeding male died 3 mo. before this study began. The female pied tamarin

received monthly injections of Depo-Provera (Pfizer Inc., New York, NY, USA) for

contraception.

The pied tamarins were housed in a single-species exhibit near the entrance of the

RainForest building of Cleveland Metroparks Zoo. The exhibit was 18 m3 and included

branches and small trees for climbing. Guests were able to view animals through a mesh

barrier in the front of the exhibit that was separated from a viewing area by a small

planter that included low bushes. The viewing area for this exhibit was situated in a gift

shop. Tamarins had access to off-exhibit holding cages (4 m3) through a small door in the rear of the exhibit. Exhibit and holding areas were visually isolated from other nearby callitrichine species but auditory contact was possible. A large waterfall (30 ft. high) was located in the front entrance lobby area, approximately 13m from the tamarin exhibit. In addition, an overhead ventilation system and ground fans located near the waterfall contributed additional noise.

Experimental conditions

This study was conducted from August to September 2014. Modifications to the sound environment occurred during four two-week phases by manipulating the waterfall

feature and an off-exhibit speaker playing white noise: 1) Baseline 1 – waterfall on and

no off-exhibit noise, 2) Quiet – waterfall off and no off-exhibit noise, 3) Loud – waterfall

on and off-exhibit speaker playing white noise, 4) Baseline 2 – waterfall on and no off-

exhibit noise. White noise was generated using Adobe Audition (Adobe Systems, San

Jose, CA) and played through an iPod (Nano 6th gen., Apple Inc., Cupertino, CA)

attached to an omni-directional speaker (Outcast Jr., Soundcast Systems, Chula Vista,

CA). The speaker was positioned on the floor approximately 1.4m from the holding

cages. During the loud experimental condition, sound levels were calibrated to match the

volume of waterfall sound by recording a 10s average sound reading at the center of the

exhibit and holding cages (iPhone 5s, Apple Inc., Cupertino, CA; SPL Pro app, Studio

Six Digital, Boulder, CO).

Behavioral data collection

Data were recorded during 15 min focal observations (n=104) using 30 s

instantaneous point-sampling for state behaviors and exhibit use and all-occurrences sampling for event behaviors (Martin and Bateson, 2007; Table 1). Additionally, continuous measurement of the time the focal animal was visible was recorded to adjust all-occurrences data. Observations were balanced between morning (10:00-12:00, n=52) and afternoon (14:00-16:00, n=52) during each phase and the order of individual observations was randomized. Data were collected by JW and a research volunteer and they were determined to be greater than 90% reliable.

Table 1. Ethogram of select behaviors considered in this study. Behavior Definition

State Behaviors (instantaneous point-sampling) Monitor Visitor Animal is stationary and visually focused on an object (including visitors or observer) outside the exhibit or scanning the visitor area for a minimum of 3 seconds. Locomote Animal travels greater than one body length (not including the tail). Inactive Animal is stationary and either alert and actively monitoring surroundings or resting and not attending to environment, typically with tail curled around body and eyes closed. Social Affiliation Animals are engaged in a non-aggressive positive behavior and includes grooming and play behavior. Event behaviors (all-occurrences sampling) Self-directed Animal manipulates body using hand, feet, or mouth and typically took the form of scratching or self-grooming. Scent Marking Repeated rubbing of anogenital, suprapubic, sternal, or facial area across substrate. Also included the self-anointing hand marking behavior described by Epple et al. (2002).

Fecal collection

First morning fecal samples were collected in the morning between 7:00-8:00.

Non-toxic glitter was hand-fed in banana pieces during the afternoon (13:00-14:00) to allow individual identification of fecal samples. Fecal samples were stored in Whirl-pak fecal bags at -20°C and feces were lyophilized (FreeZone 4.5 liter freeze dry system,

Model: #7751020, Labconco Corporation, Kansas City, MO, USA), pulverized, and sifted to remove food particles and glitter. Hormonal metabolites were extracted using a method adapted from Brown (2008): 5 ml of methanol were added to 0.2 g dry feces,

briefly hand vortexed, mixed for one hour (large capacity mixer, Model: #099A LC1012,

Glas-Col, Terre Haute, IN, USA), and centrifuged at 2500 RPM for 20 min (Sorvall

Legend RT+). The supernatant was poured off and stored in a -20°C freezer prior to

analysis and assayed within three months.

Hormonal analysis

Fecal glucocorticoid metabolites were analyzed via the enzyme immunoassay (EIA)

protocol of Brown (2008) using polyclonal cortisol antiserum (R4866, 1:300,000

dilution) provided by Coralie Munro (University of California, Davis, CA). The EIA was

validated by demonstrating: 1) parallelism of the binding inhibition curve of pooled fecal

extract dilutions (1:1 to 1:1024) with the cortisol standard (R2=95%), and 2) significant

recovery (89 %) of exogenous cortisol from diluted fecal samples spiked with the quality

control high (200 pg/well) and low (5 pg/well). The mean inter-assay coefficient of variation (CV) of standards and high- and low-value quality controls was 9.9%. Intra-

assay CVs were less than 10%. Microtiter plates were read using a spectrophotometer

(Epoch, Bio-Tek, Winooski, VT, USA) at 450 nm wavelength through the Gen5 software

(v2.03, Bio-Tek, Winooski, VT, USA). This assay was biologically validated in a

previous study by demonstrating a significant increase in fecal glucocorticoids after a

veterinary exam (Chapter 2).

Visitor and sound level measurement

Visitor and sounds level measures were summarized for the observations and

across the day. During observations, the total number of visitor groups that stopped at an

exhibit to view animals was recorded. A group was defined as a visitor party that

approached the exhibit together and viewed the exhibit for a minimum of 2 s. This

measure was chosen for ease of recording and based on previous data that found the

majority of groups at this exhibit were small (78% < 3 visitors; Chapter 3). In addition,

overall daily attendance to the RainForest building was recorded.

Sound levels were measured in each exhibit using a data logging sound level

meter (Model 407760, Extech Instruments, Nashua, NH). The sound level meter was

mounted in a protective enclosure and placed on the floor of the front of the exhibit and

oriented towards the visitor area to minimize the influence of animal vocalizations on

sound level measurements. The sound level meter was capable of measuring sounds

between 20 Hz-20 kHz with an accuracy of ± 2dB. The sound level meter was set to

record sound levels every 0.5s using a ‘Fast’ time-weighting response setting (125 ms) and ‘A’ frequency weighting curve (dBA). A-weighting adjusts sound levels based on the frequency sensitivity of human hearing and was chosen as most primate species have been shown to have a broadly similar pattern of hearing (Heffner, 2004). As sound pressure levels fluctuated, equivalent continuous sound levels (Leq) were calculated (see

Chapter 3 for details). The Leq represents the mean energy sound level during a sampling

period expressed on the decibel scale and has been previously used in studies assessing

noise in zoos (Quadros et al., 2014). In addition, the median (L50) sound level was calculated as this measure more closely reflects the background sound level. Sound level measures were calculated during 15 min observations and for the entire day (10:00-

16:00).

Statistical analysis

Behavior data were summarized as the percent of visible time for scan behaviors

(behavior scans/ scans visible) or the visible rate for event behaviors (number of

occurrences/ minutes visible). Observations in which animals were visible for only one

scan were excluded from behavior analyses to prevent artificially inflated percentages.

Off-exhibit use was summarized as the percent of total time (off-exhibit scans/total scans). Use of the front/back half of the exhibit was adjusted by the number of scans on exhibit (front/back scans/scans on exhibit) to account for changes in exhibit use during the study. Fecal glucocorticoid data were temporally adjusted by -24hrs based on

previous data of the lag time for excretion of glucocorticoids in feces (Wark et al., in

review).

As data were non-normal according to Shapiro-Wilk tests and histograms, we

chose non-parametric tests. Kruskal-Wallis tests were used to compare data across conditions with pairwise comparisons evaluated using Wilcoxon rank sum tests with exact p-values calculated. When significant effects were determined, Pearson’s r was calculated as a measure of effect size (r=Z/√N). In addition, the percent of time spent in off-exhibit areas was compared to the expected percent based on area measurements

(18% of available space) using a Wilcoxon signed rank test on the difference between the actual and expected values. We performed Spearman’s rank correlations to assess the influence of visitors and sound levels on behavioral and hormonal data. Behavioral data were assessed using the number of visitor groups and observation sound levels, whereas fecal glucocorticoid data were assessed using daily building attendance and daily sound levels. Correlations with sound levels were performed separately for days with the

waterfall off and days with the waterfall on. The observation sound level for the female

from one day was excluded as a result of noted vocalizations from the tamarins causing

inaccurate sound level readings. Both the Leq and L50 measures were assessed but as they

yielded identical findings, we present the results for the Leq analysis for simplicity. The

L50 sounds levels are shown for reference purposes.

Data were analyzed separately for each individual with only one observation each

day, thus multiple observations can be considered independent within that individual (e.g.

Owen et al., 2004). This statistical approach was meant to identify significant changes in

these individuals and should not be generalized to a population. Statistical tests were

performed using SAS University Edition (SAS Institute, Cary, NC). Differences below

p=0.05 were considered significant and differences approaching significance (p<0.1)

were identified as trends.

Results

Although the number of visitor groups during an observation was consistent

2 throughout study conditions (χ 3=1.19, p=0.75), overall building attendance was greater

2 during the first baseline period than other conditions (χ 3=21.86, p<0.0001). Exhibit Leq

sound levels were similar during conditions with the waterfall on (Baseline 1, Baseline 2,

2 and Loud conditions; χ 2=2.26, p=0.32). Although off-exhibit sound levels were not

monitored throughout the study, data collected for reference with the off-exhibit speaker on did appear consistent with exhibit sound levels when the waterfall was on (Table 2).

Deactivating the waterfall feature during the Quiet condition significantly lowered sound levels (r=0.72, p<0.0001). The median L50 sound level appeared similar in the quiet exhibit and off-exhibit conditions (waterfall and speaker off) but the off-exhibit area

appeared to have a louder Leq sound level than the exhibit. Sound levels during

observations were positively correlated with the number of visitor groups (Waterfall off:

rs=0.48, p=0.014; waterfall on: rs=0.41, p=0.0003). Similarly, daily Leq sound levels were

positively correlated to building attendance when the waterfall was on (rs=0.42, p=0.011),

but this relationship was not significant when the waterfall was off (p=0.93).

Table 2. The equivalent continuous (Leq) and median (L50) sound level indices (median ± IQR) for the exhibit area when a waterfall feature was on and off and for the off-exhibit area when a speaker broadcast white noise volume matched to the waterfall noise on exhibit.

Day Leq (dBA) Day L50 (dBA) Waterfall Off/ Waterfall On/ Waterfall Off/ Waterfall On/

Speaker Off Speaker On Speaker Off Speaker On

Exhibit 61.6 ± 5.6 66.9 ± 0.7 53.3 ± 1.6 65.9 ± 0.8 Off-exhibita 65.6 67.1 54.7 64.6 a Sound levels in the off-exhibit area were not monitored throughout the study and data from one day with the off-exhibit speaker on and off are provided for reference.

Both individual monkeys displayed a similar pattern of decreased use of off-

exhibit areas during the experimental conditions (Fig. 1). Compared to the baseline

conditions (Mdn=51.7%), the male tamarin spent less time off exhibit during the quiet

(Mdn=10%, r=0.35, p=0.029) and loud (Mdn=13.3%, r=0.34, p=0.034) conditions.

Although the female’s use of off-exhibit areas was not significantly different across

2 conditions (χ 3=4.77, p=0.19), we did detect a trend to decreased time off exhibit during

loud conditions (Mdn=6.7%) compared to baseline (Mdn=30%, r=0.27, p=0.10). For

both individuals, use of exhibit and off-exhibit areas during the quiet and loud

experimental conditions, when sound levels were similar in both areas, was not

significantly different than expected based on area measurements (p>0.10). During

baseline conditions with the waterfall active, the tamarins spent more time off-exhibit than expected (Male: S=111, p=0.0005; Female: S=78, p=0.022). There was no significant difference in use of the front or back half of the exhibit across conditions after

2 accounting for changes in the time spent on exhibit (Male: χ 3=2.58, p=0.46; Female:

χ2(3)=3.11, p=0.37).

Figure 1. The percent of time spent off exhibit during sound conditions involving experimental modifications of a waterfall feature and white noise played from a speaker in the off-exhibit area. Boxes and whiskers represent the 10th, 25th, 75th, and 90th percentiles. The median and mean are indicated using a horizontal line and dot, respectively. The expected percent of use based on space availability (18%) is represented using a dashed line.

There was no significant difference in the female tamarin’s behavior across

conditions (p>0.10 for all behaviors). Behavioral changes were observed in the male

tamarin during the second baseline phase. During the final baseline condition the male spent more time locomoting compared to the Loud condition (Loud vs. Baseline 2: r=0.52, p=0.010) and a similar non-significant trend compared to the first baseline condition (Baseline 1 vs. Baseline 2: r=0.40, p=0.08), decreased inactivity compared to all prior conditions (Baseline 1 vs Baseline 2: r=0.58, p=0.011; Quiet vs. Baseline 2: r=0.44, p=0.029; Loud vs. Baseline 2: r=0.53, p=0.0084), and increased scent marking compared to the Quiet condition (Quiet vs. Baseline 2: r=0.48, p=0.015) and a similar non-significant trend compared to the first baseline condition (Baseline 1 vs. Baseline 2: r=0.39, p=0.090). There was no significant difference in fecal glucocorticoid levels across conditions for either individual (Fig. 2; Male: Baseline 1 Mdn=137.5 ng/g, Quiet

2 Mdn=142.8, Loud Mdn=146.9, Baseline 2 Mdn=126.1, χ 3=0.56, p=0.90; Female:

Baseline 1 Mdn=159.6 ng/g, Quiet Mdn=197.4, Loud Mdn=168.9, Baseline 2

2 Mdn=149.4, χ 3=3.59, p=0.31).

Figure 2. Fecal glucocorticoid metabolite concentrations (ng/g) for a male (grey circles) and female (black Xs) pied tamarin across experimental sound conditions. Fecal samples were collected after first morning voids (7:00-8:00).

We observed no relationship between behavioral or fecal glucocorticoid measures with the number of visitor groups or building attendance, respectively (p>0.05). For the male tamarin, observation Leq sound levels were positively correlated with locomotion when the waterfall was on (rs=0.40, p=0.026) and a trend to significance with self- directed behavior when the waterfall was off (rs=0.52, p=0.07). The female displayed a trend to increased time monitoring the visitor area as observation Leq sound levels

increased when the waterfall was on (rs=0.32, p=0.10). Fecal glucocorticoid levels were not influenced by Daily Leq sound levels when the waterfall was off or on.

Discussion

It has been suggested that beneficial effects of providing zoo animals access to

off-exhibit areas may derive from attractive characteristics of these areas (Owen et al.,

2005), although this has not yet been directly evaluated. In this study, we assessed the

role of the sound environment in influencing use of off-exhibit areas. Both tamarins in

this study altered their use of the exhibit and off-exhibit area in response to changes in the sound environment. During baseline conditions when a nearby waterfall feature that emitted loud, continuous noise was on, individuals spent approximately 25 to 50% their time off exhibit. This was significantly more than expected based on the size of the area

(18% of available space). However, when the waterfall was deactivated, time spent in off-exhibit areas decreased by roughly half and did not differ from expected levels based

on space availability. This exhibit use pattern persisted after the waterfall was reactivated

and an off-exhibit speaker played white noise at the volume of the waterfall noise. This

response suggests these tamarins used off-exhibit areas to avoid noise from the waterfall feature. With the waterfall feature active, background sound levels (L50) on exhibit were

approximately twice as loud (10 dBA increase) as was measured in the off-exhibit area.

However, brief periods of hosing and keeper maintenance of the off-exhibit area, which was restricted during observations, did appear to increase average sound levels (Leq) to a similar level as found on exhibit and suggest attention should be paid to minimize the duration of these activities. Although previous zoo studies have described increased use of off-exhibit areas during noisy conditions (Sulser et al., 2008), we confirmed the

importance of the sound environment of off-exhibit areas through controlled

experimental modifications.

Spatial avoidance of noise has been documented in free-ranging wildlife (Blickley

et al., 2012). For example, black-tufted marmosets living in an urban park in Brazil were observed to avoid noisy areas of the park, even when these areas had high food availability (Duarte et al., 2011). Auditory masking of important cues, such as sounds from predators/prey and intra-specific communication, has been implicated as a primary mechanism for the negative impacts of chronic noise (Barber et al., 2010; Francis and

Barber, 2013). In the present study, the pied tamarin exhibit allowed limited visibility of approaching visitors and it is possible that the waterfall noise masked the activity of zoo visitors, causing a negative reaction to their unexpected presence.

Although many studies have documented a negative impact of visitors on callitrichines (Armstrong and Santymire, 2013; Glaston et al., 1984; Wormell et al.,

1996), we observed no effect of visitors on the behavior or fecal glucocorticoid levels of these pied tamarins. Similarly, observation sound levels, which were positively correlated with the number of visitor groups, also had little influence on behavioral or hormonal measures. This is surprising and possibly reflects the benefits of having access to off-exhibit areas. In a previous study we observed that increased vigilance in response to the waterfall noise was ameliorated when animals had access to off-exhibit areas

(Chapter 3). Although Wormell et al. (1996) reported that pied tamarins housed by

Durrell Wildlife Preservation Trust (DWPT) displayed a negative reaction to visitors,

Holm et al. (2012) more recently found no influence of visitors on the behavior of pied

tamarins at DWPT and suggested that their current exhibit, which was large, naturalistic,

and had access to indoor areas, may have minimized the effect of visitors.

Avoidance of the waterfall noise implies this sound environment may be aversive

and without escape opportunities could negatively impact welfare. It should be noted that

we observed no effect of the sound conditions on individual behaviors or fecal

glucocorticoid levels, suggesting that having an escape may have ameliorated behavioral

indicators of welfare (e.g. Kuhar, 2008). Our current understanding of how animals

respond to noise is limited (Francis and Barber, 2013). It is possible that continuous

noise, such as from the waterfall in the present study, may be less likely to elicit arousal-

related behaviors and influence fecal glucocorticoid levels compared to acute noise although future research is needed to confirm this.

Although the sample size of this study was limited, this is the first study to

experimentally manipulate the sound environment of exhibit and off-exhibit areas. Our results suggest that the sound environment is an important quality to some animals and may be a potential factor in motivating animals to use off-exhibit areas. Sound conditions did not alter other behaviors or fecal glucocorticoids, suggesting the overall impact of the waterfall noise on welfare was minimal. However, it should be noted that animals had access to off-exhibit areas throughout the study and previous research has shown that this may ameliorate negative effects of noise (Chapter 3). Understanding factors associated with the use of off-exhibit areas is important for zoo managers as excessive use of these areas can affect animal visibility for guests and unintentionally undermine the educational opportunity of zoos. Careful evaluations such as these may

97 assist in identifying and integrating potentially negative exhibit sound environments without adversely affecting animal welfare.

Chapter Five: The response of callitrichine monkeys to auditory stimuli and the importance of exhibit choices

Introduction

Increasing environmental complexity through auditory stimulation has been identified as a potential form of enrichment for zoo animals (Wells, 2009). Playing of classical music has been systematically evaluated as a form of auditory enrichment in a variety of species but has had mixed results. Some studies have shown positive influences on behavior (Asian elephant: Wells and Irwin, 2008; domestic dog: Wells et al., 2002; chimpanzee: Videan et al., 2007) but others have failed to identify benefits of classical music (chicken: Campo et al., 2005; cotton-top tamarins: McDermott and

Hauser, 2007; common marmosets: McDermott and Hauser, 2007; Moloch gibbons:

Wallace et al., 2013). It has been suggested that music, which is ultimately designed to impart an emotional response in humans, may not stimulate animals in the same way

(Snowdon and Teie, 2010). Interestingly, playing rainforest sounds to gorillas, which may be more appropriate for this species than music, has also yielded inconsistent results

(Ogden et al., 1994; Robbins and Margulis, 2014; Wells et al., 2006). This mixed response to sounds highlights the need to identify more successful forms of auditory enrichment.

One promising approach is the development of species-specific music (Snowdon and Teie, 2010; Snowdon et al., 2015). This music is designed taking into account characteristics of the species, such as heart rate, hearing range, and aspects of

vocalizations thought to convey emotion. Snowdon and Teie first demonstrated this by

developing music for cotton-top tamarins. Spectral and temporal features of affiliative

and fear or threat vocalizations were used to compose two types of musical pieces and the

final recordings were shifted to higher frequencies to reflect the hearing sensitivity of

these monkeys. After exposure to affiliation-based tamarin music, tamarins reduced movement and spent more time foraging, suggesting a calming response. Playback of fear/threat-based music appeared to agitate the monkeys, with increased movement and arousal-related behaviors compared to the affiliation-based music. Importantly, fewer behavioral differences were observed after playback of human music, a finding consistent with McDermott and Hauser (2007).

In this study, we evaluated traditional forms of auditory enrichment, such as classical music and rainforest sounds, as well as tamarin-specific music for use with three species of callitrichine monkeys: pied tamarin, golden lion tamarin, and callimico.

Sounds were played in the early morning before guests arrived. During the first four weeks, sounds were played in exhibit or off-exhibit areas as a preference test. During the last two weeks sounds were played on exhibit with no off-exhibit access for animals, the standard housing arrangement for these animals at the time. Behavior and exhibit use were compared to a control day with no audio playback. We expected a greater response to the tamarin music than traditional forms of auditory enrichment. In addition, we expected the affiliation-based tamarin music and fear/threat-based tamarin music would elicit a positive and negative response, respectively. As callitrichines have shown a mixed reaction to sounds, the controlled testing of auditory stimuli in this study was critical to identify sounds that could be classified as enrichment for these monkeys.

100

Methods

Subjects and housing

Subjects for this study included three species of callitrichine monkeys: a group of

pied tamarins (Saguinus bicolor) consisting of an adult male (16.0 y.o.) and female (4.7

y.o.) mated pair and two male offspring (2.1 and 1.5 y.o.), a group of golden lion

tamarins (Leontopithecus rosalia) consisting of an adult male (3.3 y.o.) and female (5.7

y.o.) breeding pair with 2 male (2.6 and 0.7 y.o.) and 2 female (1.9 and 0.7 y.o.)

offspring, and a group of callimicos (Callimico goeldii) consisting of an adult male (9.6

y.o.) and female (2.8 y.o.).

Animals were housed in single-species exhibits in the RainForest building at

Cleveland Metroparks Zoo. Exhibits were approximately 18 m3 and included branches for climbing. A small door at the back of the exhibits connected to off-exhibit holding cages (4 m3). Exhibits and holding areas were visually isolated from each other but auditory contact between species was possible. A nearby waterfall feature was deactivated during observations. Animals were fed in the morning and afternoon, with

the first meal at 9:30 occurring after animals transferred onto exhibit and immediately

prior to data collection.

Sound conditions

Animals were exposed to five sound conditions (Table 1). Although some

auditory enrichment studies have presented long, complex stimuli to animals, this

approach makes it challenging to identify the specific characteristics of the sounds being

tested. To address this, we selected two short excerpts (approximately 30 s) of each

101 stimuli to provide limited variability for each piece (see Table 2 and Fig. 1 for a detailed description of each excerpt). These excerpts were repeated in a random order to create 30 min sound files (WAV file format, 44,100 kHz sample rate, 16-bit depth) using Adobe

Audition (Adobe Systems, San Jose, CA). Auditory stimuli were played through an iPod

(Nano 6th gen., Apple Inc., Cupertino, CA) attached to an omni-directional speaker

(Outcast Jr., Soundcast Systems, Chula Vista, CA). The speaker was positioned at a fixed point either in the center of the exhibit or 1.4 m from the off-exhibit holding areas.

Playback sound levels were calibrated in a quiet area to 61-62 dBA by measuring the sound level of the stimuli at a distance of 1 m (iPhone 5s, Apple Inc., Cupertino, CA;

SPL Pro app, Studio Six Digital, Boulder, CO). Although exhibit and off-exhibit sound levels were not recorded during the No Audio condition, previous research has found the background sound level in these areas to be approximately 55 dBA (see Chapter 4).

Table 1. Description of sound conditions. Sound Condition Description No Audio Animals were exposed to sounds naturally arising in the environment, including activities of zoo staff and building ventilation. Mozart Mozart’s Eine Kleine Nachtmusik Movement 2: Romanze Andante from a commercial CD (Mozart for Relaxation, BMG Entertainment). Rainforest Sounds Sounds from a commercial CD (Amazon Rainforest album, Echo Bridge Home Entertainment) that featured bird, frog, and insect sounds with light raining in the background. Affiliation-based “Tamarin serenade” and “Tamarin ballad 2” composed using features of Tamarin Music affiliative tamarin vocalizations and performed by singing and cello (see Snowdon and Teie, 2010 for description). Fear/Threat-based “Tamarin rock” and “Tamarin rock 2” composed using features of Tamarin Music fear/threat vocalizations and performed by singing and cello (Snowdon and Teie, 2010).

102

Table 2. The duration, frequency, and tempo of playback stimuli. Playback Stimuli Duration (s) Frequency Range (Hz) Notes / minutea Mozart Excerpt 1 29 172 – 514 75 Excerpt 2 28 93 – 505 181 Rainforest Sounds Excerpt 1 29 855 – 8790 163 Excerpt 2 30 4629 – 8440 72 Affiliation-based Tamarin Music Excerpt 1 36 1856 – 3721 53 Excerpt 2 32 1835 – 4180 60 Fear/threat-based Tamarin Music Excerpt 1 27 1518 – 7684 578 Excerpt 2 30 938 – 5265 482 a For rainforest sounds, the number of bird and frog vocalizations were counted as notes.

103

Figure 1. Spectrograms of playback stimuli used in this study (A,B: Mozart; C,D: Rainforest Sounds; E,F: Affiliation-based Tamarin Music; G,H: Fear/Threat-based Tamarin Music). Two 30 s excerpts of each stimulus type are shown. The color intensity in the spectrograms represents the amplitude of the sound.

104

Experimental procedure

This six-week study was conducted during weekdays in the morning before the building opened to the public (9:30-10) from November to December, 2012. Sound

conditions were randomly assigned each day and all conditions were tested each week

(see Fig. 2). This study involved two phases. During the first four weeks (Phase 1), animals had access to off-exhibit areas during testing and sounds were played either in the exhibit or off-exhibit area. To balance exposure to the audio-location combinations

during Phase 1, the location of the playback was pseudo-randomized such that animals

were exposed to every stimulus once in the exhibit and once in off-exhibit areas during

the first 2 weeks and again during the second two weeks of Phase 1. In addition,

playback in an area occurred no more than three times in a given week. During the last

two weeks of the study (Phase 2), animals did not have access to off-exhibit areas and all

sounds were played on exhibit. All species experienced the same testing conditions each

day.

105

Figure 2. Diagram of experimental playback procedure.

Behavioral data collection

Animals were video recorded during the 30 min experiments using a camcorder

and tripod stationed in front of the exhibits (15 h per species). Reliable identification of individuals on video was not possible and group data were scored. During Phase 1, the number of animals on and off exhibit were continuously recorded (Martin and Bateson,

2007). The duration of bouts in each area was multiplied by the number of animals in the

area to generate a group duration. For example, two animals off exhibit for 30 s would

equal 1 min of group time spent off exhibit. All-occurrences of self-directed and scent marking behavior were scored as indicators of arousal. Feeding/foraging behavior was scored as an indicator of relaxed behavior. A minimum of 3 s needed to elapse after a bout for a new bout to be scored.

106

Statistical analysis

Exhibit use data during Phase 1 were summarized for each day as the percent of

group time by dividing the total group duration in the exhibit or off-exhibit area by the

group observation time (observation duration multiplied by the number of animals in a

group). As the audio speaker was moved between the exhibit and off-exhibit areas, the percent of group time in the speaker location was used for analysis (i.e. percent of group time on exhibit when the speaker was on exhibit and percent of group time off exhibit when the speaker was off exhibit). Behavior was adjusted by dividing the number of occurrences of a behavior by the group duration of time animals were visible on exhibit.

Data are presented as group means and differences were evaluated using 95% confidence

intervals. As a guide, confidence intervals that overlap by less than half would

correspond to a significant difference using null hypothesis statistical tests (p<0.05;

Cumming and Fitch, 2005). It should be noted that this analysis was chosen to identify

changes in these groups and, as sample size was limited, we urge caution in generalizing

these results to callitrichines housed at other facilities. All data were assessed using SAS

University Edition (SAS Institute, Cary, NC).

Results

Influence of auditory stimuli on exhibit use

During Phase 1 when the speaker was alternated between exhibit and off-exhibit

areas, there was no difference in time spent in the speaker location across auditory

conditions (Fig. 3).

107

Figure 3. The mean percent of time three callitrichine groups (pied tamarin, golden lion tamarin, and callimico) spent in the location a speaker was placed during auditory conditions. The speaker was randomly placed in either the exhibit or off-exhibit area each day and time in the respective area with the speaker was calculated. Error bars represent 95% confidence intervals around the mean.

Influence of auditory conditions and off-exhibit access on behavior

The behavioral response to auditory conditions was similar when animals had access off exhibit during Phase 1 and when they did not have access during Phase 2.

Overall, the pied tamarins performed self-directed behavior more frequently after exposure to Mozart (M=1.6, CI95%[1.2, 2.1]; Fig. 4A). No behavior differences across

108

auditory conditions were observed in the golden lion tamarin or callimico groups (Fig 4B

and 4C). During Phase 2 when animals did not have off-exhibit access, pied tamarins

scent marked more frequently (Fig. 5A) and the callimicos performed self-directed

behavior more often (Fig. 5C) compared to Phase 1 with off-exhibit access. No differences were observed in the golden lion tamarin group in response to off-exhibit access.

109

Figure 4. The mean group rate (occurrences/min visible) of behavior for three callitrichine species (A: pied tamarin; B: golden lion tamarin; C: callimico) during auditory conditions. Animals were exposed to each condition once per week for 6 weeks (Phases 1 and 2). Error bars represent 95% confidence intervals around the mean.

110

Figure 5. The mean group rate (occurrences/min visible) of behavior for three callitrichine species (A: pied tamarin; B: golden lion tamarin; C: callimico) during periods with access to off-exhibit areas (Phase 1) and no access (Phase 2). Error bars represent 95% confidence intervals around the mean.

111

Discussion

In this study we exposed three species of callitrichine monkeys to playback of

acoustic stimuli during the morning prior to zoo opening. We compared the behavioral

response to classical music and rainforest habitat sounds, two traditional types of auditory

enrichment, with music composed for cotton-top tamarins (Snowdon and Teie, 2010).

Overall, we found few differences between stimuli and control conditions with no audio

playback. During Phase 1 when the speaker was alternated daily between exhibit and

off-exhibit areas, we saw no spatial preference for the speaker location. With the

exception of increased self-directed behavior by the pied tamarins when exposed to

Mozart, we observed no differences between auditory conditions for the behaviors

assessed in this study – self-directed behavior, scent marking, and feeding/foraging. In

addition, the behavioral response to auditory conditions was similar between Phase 1

with access and during Phase 2 with no off-exhibit access, suggesting that exhibit choices did not modify the response to sound. Both the pied tamarin and callimico groups exhibited increased behavioral indicators of anxiety when they did not have off-exhibit access (Phase 2) compared to the access period (Phase 1).

The indifference to traditional forms of auditory enrichment is consistent with previous studies on callitrichines (McDermott and Hauser, 2007; Snowdon and Teie,

2010). However, the lack of response to the affiliation-based and fear-threat based tamarin music was unexpected and contrasts with the findings of Snowdon and Teie

(2010). Several methodological differences between their work and the present study may help explain this discrepancy. First, they exposed animals to a single brief exposure to stimuli (30 s) and assessed behavior for 5 min before and after playback. In the

112

present study, two short excerpts of each stimulus type were combined to form a 30 min

piece and it is possible that animals may have habituated to the repeated and prolonged

exposure of the stimuli. Second, Snowdon and Teie assessed behavior during calm

baseline conditions. We performed playback experiments in the morning when animals

were provided access to exhibit areas and fed their morning meal. During this time

animals appeared focused on exploration and feeding and may have been less attentive to

sounds. In addition to these potential issues, the tamarin music was composed using

features of cotton-top tamarin vocalizations and it is possible this may not have been

appropriate for the related callitrichine species in this study. Differences between cotton- top tamarin vocalizations and other callitrichines have been noted, with long distance contact calls by cotton-top tamarins being lower frequency than has been recorded in other species (Snowdon, 1993).

Providing access to off-exhibit areas during Phase 1 did appear to benefit the pied tamarins and callimicos. Other zoo studies have also reported beneficial effects of off- exhibit access on behavior (Owen et al., 2005; Ross, 2006). In addition to the potential benefits of increased choice and complexity, access to off-exhibit areas may provide animals additional retreat space. Callitrichines are a small-bodied prey species and have been shown to react negatively to humans (Armstrong and Santymire, 2013; Glaston et al., 1984; Wormell et al., 1996). Interestingly, we observed this effect in the morning before the zoo was opened to the public, although zoo staff were active in front of exhibits during this time.

Research on the utility of music and other sounds as auditory enrichment have often yielded conflicting results. For callitrichine species, we found no benefits of

113 auditory stimulation, although additional work with longer, more complex pieces of tamarin music is needed. As studies of noise in the zoo setting have highlighted individual differences, it is possible that the response to music and other sounds may be primarily individual-based, an issue this study was not able to address. If the use of auditory enrichment is desired, these sounds should be used with caution in animal facilities and providing animals the ability to avoid these sounds is encouraged.

114

Chapter Six: A pilot investigation on the use of startle reactivity as a measure of affect in

callitrichine monkeys

Introduction

Concern for animal welfare is based on the assumption that animals are sentient beings capable of experiencing conscious, subjective states such as pain and suffering

(Mendl et al., 2010). Assessing these subjective experiences, often characterized as affective states or emotional processes, has proven challenging. Recent research into cognitive biases that evaluate “optimistic-like” and “pessimistic-like” responses to

ambiguous cues have yielded promising results (Mendl et al., 2009). However these

methodologies are often time-consuming, requiring operant conditioning of

discrimination tasks, and may not be practical in a zoo setting. Alternatively, assessing

the startle reflex in animals, an unconditioned behavior response, has been highlighted as

a potential unexplored measure of animal welfare (Paul et al., 2005).

In both people and animals, emotional processes have been shown to be involved

in the startle response, either sensitizing or buffering the reaction to the startle event. For

example, it is well documented that fear can increase the startle response of people and

animals (“fear-potentiated startle effect”, Lang et al., 2000). Environmental stimuli, such

as music and other sounds, may also be able to modulate the affective state and thus the

startle responsiveness of people and animals. Nature sounds and music have been shown

to modify the startle response of people, with sounds rated as unpleasant causing an

increased startle response to a loud startle sound compared to pleasant sounds (Bradley

115

and Lang, 2000; Roy et al., 2009). In exploring the startle responses of rats, Hoffman and Fleshler (1963) found that introducing a white noise background during their experiments caused a heightened startle response by the animals, a phenomenon that has been attributed to increased arousal from the white noise leading to a sensitization of the startle event.

The startle reflex is often very brief (<1 s) and consists of an eyeblink and contraction of facial, neck, and skeletal muscles (Koch, 1999). Research on the startle response of animals often focuses on whole body startle, measuring body movement in a specialized device (Cassella and Davis, 1986). In addition to this immediate autonomic response, startle reactions can also involve a sequence of behavioral changes. In callitrichine monkeys for example, animals typically react to a startle event with rapid flight accompanied with alarm calls followed by freezing and visual scanning (Stevenson and Poole, 1976). Although measuring the immediate startle reflex may not be feasible in a zoo setting, analyzing these startle-related behaviors may be a potential means to assess the startle response and underlying affective state of zoo-housed animals.

Assessing startle-related behaviors has been previously done in studies of predator recognition and anti-predator behavior. For example, Searcy and Caine (2003) found that white-fronted marmosets were more startled by the playback of hawk calls compared to the playback of raven calls and the sound of a power drill. Specifically, they found animals increased freezing times, moved higher in their cage, and were more likely to alarm call after exposure to the hawk call.

In this study, we evaluated the potential of various sounds to influence behavior and modify the startle response of two species of callitrichine monkeys: pied tamarin

116

(Saguinus bicolor) and white-fronted marmoset (Callithrix geoffroyi). Monkeys were

exposed to sounds (5 min) that were immediately followed by an acoustic startle event

(90 dB white noise). We evaluated typical sounds zoo animals may be exposed to, such

as visitor noise and traditional forms of auditory enrichment (classical music and

rainforest sounds), as well as sounds designed for tamarins (Affiliation- and Fear/threat-

based tamarin music, Snowdon and Teie, 2010). In addition, we assessed a no audio

condition as a negative control and playback of loud (80 dB) white noise, as a positive

control. Data collected during the seven auditory conditions and after the startle event

were compared to baseline periods at the start of experiments with no sound playback.

As a validation of the startle methodology, we expected an exaggerated startle response

after the startle event following 80 dB white noise compared to the no audio control

condition. In general, the playback of sounds that promoted calm behaviors were

expected to buffer the response to the startle event, whereas sounds that increased

agitation should sensitize animals to the startle event. Because exposure to startle events

may negatively affect welfare, fecal glucocorticoid metabolites were monitored before

and after experiments. This research is expanding our knowledge of the sound

environment preferences of zoo-housed animals and investigating startle modification as

an unexplored potential indicator of welfare.

Methods

Subjects and housing

Subjects for this study included members of a family group of pied tamarins and

white-fronted marmosets. The pied tamarin group consisted of an adult male (17 y.o.)

and female (6 y.o.) breeding pair, and an adult male offspring (3 y.o.). The marmoset

117

group consisted of an adult male (8 y.o.) and female (6 y.o.) breeding pair and two adult

male twin offspring (3 y.o.). One of the male offspring marmosets died after the third

week of the study from surgery complications. The breeding male pied tamarin was

euthanized after the fifth week of the study as a result of an age-related health decline.

Animals were housed in single-species exhibits in the RainForest building at

Cleveland Metroparks Zoo. Exhibits were approximately 18 m3 and included branches for climbing. A nearby waterfall feature was deactivated during experiments. Feeding of the morning meal (typically 9:30) was delayed until after the startle experiments (10:00) to prevent the potential confound of food presence on behavior and attention to sounds.

Sound conditions

Animals were exposed to seven sound conditions (Table 1, see Chapter 5 for a detailed description of Mozart, Rainforest Sounds, and Tamarin Music conditions).

Although some auditory enrichment studies have presented long, complex stimuli to animals, this approach makes it challenging to identify the specific characteristics of the sounds being tested. To address this, we selected two short excerpts (approximately 30 s) of each stimuli (excluding 80 dB White Noise) to provide limited variability of each piece. These excerpts were repeated in a random order to create 5 min sound files (WAV file format, 44,100 kHz sample rate, 16-bit depth) using Adobe Audition (Adobe

Systems, San Jose, CA). The exact length of stimuli varied to allow for resolution of excerpts and ranged from 4.73 – 5.17 min. A 500 ms burst of white noise was included approximately 1 s after the sound stimuli ended to create an acoustic startle event. All sound files were adjusted using Adobe Audition’s Amplify tool to ensure the stimuli and startle sound played at their respective amplitudes.

118

Table 1. Description of sound conditions. Sound Condition Description No Audio Animals were exposed to sounds naturally arising in the environment, including activities of zoo staff and building ventilation. 80 dB White Noise Broadband white noise generated using Adobe Audition and played at 80 dB (other stimuli played at 64 dB). Mozart Mozart’s Eine Kleine Nachtmusik Movement 2: Romanze Andante from a commercial CD (Mozart for Relaxation, BMG Entertainment). Rainforest Sounds Sounds from a commercial CD (Amazon Rainforest album, Echo Bridge Home Entertainment) that featured bird and insect sounds with light raining in the background. Affiliation-based “Tamarin serenade” and “Tamarin ballad 2” composed using features of Tamarin Music affiliative tamarin vocalizations and performed by singing and cello (see Snowdon and Teie, 2010 for description). Fear/Threat-based “Tamarin rock” and “Tamarin rock 2” composed using features of Tamarin Music fear/threat vocalizations and performed by singing and cello (Snowdon and Teie, 2010). Visitor Noise Audio recording of visitor sounds during a crowded zoo event.

Auditory stimuli were played through an iPod (Nano 6th gen., Apple Inc.,

Cupertino, CA) attached to an omni-directional speaker (Outcast Jr., Soundcast Systems,

Chula Vista, CA) placed in the center of the exhibit. The speaker was also present during the No Audio control condition but no sounds were played. Sound levels were calibrated each morning in a quiet area by measuring the sound level of the stimuli at a distance of 1 m (iPhone 5s, Apple Inc., Cupertino, CA; SPL Pro app, Studio Six Digital, Boulder, CO).

All stimuli were played at a sound level of 64 dBA, with the exception of the 80 dB white noise. The white noise startle sound was 90 dBA.

Experimental procedure

This seven-week study was conducted during weekdays in the morning before the building opened to the public (9:30-10:00) from March-May, 2014. Startle experiments

119 consisted of an initial 5 min baseline period with no sound exposure, followed by one of seven sound conditions (see Table 1). This playback period lasted approximately 5 min and was immediately (<2 s) followed by an acoustic startle stimulus (90 dB white noise).

Observations continued until 10:00 (approximately 20 min). Startle experiments were conducted only once per week for each species, with tamarins and marmosets being tested on separate days. Animals were exposed to each sound condition only once and the presentation order of sound conditions was randomized. Both species were exposed to the same sound condition each week.

Behavior data collection

Animals were video recorded during the 30 min startle experiments. Two camcorders

(Panasonic HCV700K and Samsung HMX-H200N) were stationed on tripods in front of the exhibits to capture the entire exhibit space. General behavior during the observation was scored from videos using a continuous sampling methodology (Martin and Bateson,

2007; Table 2). In addition, specific startle-related behaviors were assessed following the startle event. This included the duration of time freezing (stationary period that occurred either immediately or after rapid locomotion following the startle event that consisted of visually scanning or vocalizing before the animal reinitiated movement) and the latency and duration of alarm calling (rapid, loud high-frequency chirp and trill vocalizations).

Also, the number of animals that moved immediately following the startle event (<1 s) and whether they moved to out-of-view areas in the exhibit (corners or behind overhanging portion of ceiling) was recorded.

120

Table 2. Ethogram of general behaviors considered in this study. Behavior Definition

Scent Marking Repeated rubbing of anogenital, suprapubic, sternal, or facial area across substrate. Also included the self-anointing hand marking behavior described by Epple et al. (2002). Self-directed Animal manipulates body using hand, feet, or mouth and typically took the form of scratching or self-grooming. Feed / Foragea Visual scanning or fixating on an area associated with food and/or physically manipulating food or objects containing food, including eating and drinking. Locomote Animal travels greater than one body length (not including the tail). Object-directeda Animal is inspecting or manipulating non-edible object or substrate. Included tree gouging behavior in the marmosets. Inactive Animal is stationary and either alert and actively monitoring surroundings or resting and not attending to environment, typically with tail curled around body and eyes closed. Other Solitarya Animal is engaged in a solitary behavior not defined above. Groomingb Animal is oro-manually manipulating a social partner’s hair or skin. Other Social Animals are engaged in a non-aggressive positive behavior Affiliationb including play and sexual behaviors. Social Agonisticb Animal is engaged in an aggressive interaction with a social partner, including contact and non-contact aggression and food stealing. Other Socialb Animal is engaged in a social behavior not defined above. Behavior Not Visible Animal is not clearly visible and behavior cannot be observed. Animal Not Visible Animal’s body is out-of-sight. a Behaviors occurred at low frequencies and were combined for analysis. b Social behaviors did not occur frequently and were not assessed.

121

Fecal collection and fecal glucocorticoid analysis

First morning fecal samples were collected between 7:00-8:00. Fecal samples

were individually identified using non-toxic glitter that was hand-fed to animals in banana pieces during the afternoon (13:00-14:00). Samples were stored in Whirl-pak fecal bags at -20 °C and lyophilized (FreeZone 4.5 liter freeze dry system, Model:

#7751020, Labconco Corporation, Kansas City, MO, USA) within 1 month. Samples were processed and assayed according to previously described methodology (see Chapter

2). The mean inter-assay coefficient of variation (CV) of standards and high- and low- value quality controls was 14.1%. Intra-assay CVs were less than 10%. Microtiter plates were read using a spectrophotometer (Epoch, Bio-Tek, Winooski, VT, USA) at 450 nm wavelength through the Gen5 software (v2.03, Bio-Tek, Winooski, VT, USA).

Statistical analysis

Behavior data were analyzed as the percent of visible time during 5 min periods

(baseline, sound playback, 0-5, 5-10, 10-15, and 15-20 min post-startle) by dividing the duration of behavior during the 5 min period by the time visible. Behavior was evaluated for each sound condition by comparing the percent of visible time animals were engaged in a behavior during the playback period and post-startle periods to their baseline frequency using two-tailed Wilcoxon signed-ranks tests (S test statistic represents the sum of positive ranks minus the sum expected under the null hypothesis: n(n+1)/4). Data analysis of the post-startle periods first compared the response during the 0-5 min period and if no difference was observed at this time, subsequent periods were not analyzed

(Searcy and Caine, 2003). Freeze times were compared across sound conditions to control conditions (No Audio and 80 dB White Noise) using Wilcoxon signed-rank tests.

122

The Wilcoxon signed-rank test is a non-parametric alternative to a paired-samples t-test

and was chosen because of the non-normal distribution of data arising from the small

sample size in this study. Because two individuals died during the study, the number of

paired comparisons ranged from seven to five. Individuals from both species were

included in these paired comparisons to identify broad effects of the sounds and startle

responses. Group-specific responses could not be assessed statistically given the small

sample size and are presented graphically.

Fecal glucocorticoid levels were compared across sampling days (0 - 48 h post startle experiment) using a mixed-effects model. Prior to analysis, daily mean values were first calculated for individuals when multiple daily samples were collected, and daily fecal glucocorticoid levels were then log-transformed. Fixed effects of Species and

Day were included in the model, along with a random intercept effect of Individual to account for repeated measures. Degrees of freedom were calculated using the

Satterthwaite adjustment.

All statistical tests were performed using SAS University Edition (SAS Institute,

Cary, NC). Differences below p=0.05 were considered significant and differences approaching significance (p<0.01) were identified as trends.

Results

Behavioral response to sound conditions

No overall behavior changes were observed across species during the No Audio or Fear/Threat-based Tamarin Music conditions, compared to the baseline period at the start of observations. For other sound conditions, animals typically responded to

123

playback with either a trend toward increased inactivity (Table 3; 80 dB White Noise:

S=-9.5, p=0.063; Visitor Noise: S=-7.5, p=0.063) and/ or decreased movement

(Rainforest Sounds: S=14, p=0.016; Affiliation-based Tamarin Music: S=13, p=0.031; 80 dB White Noise: S=9.5, p=0.062 non-significant trend). This effect was particularly

apparent in the marmoset group and was observed to varying degrees after all sound

conditions except the No Audio control condition (Fig 1). Decreased time spent in other

active behavior was observed during playback of Mozart (S=10.5, p=0.031).

124

Table 3. Behavior of tamarins and marmosets before, during, and after exposure to sound conditions and acoustic startle event. Rainforest Sounds Affiliation-based 80 dB White Noise Visitor Noise Mozart Tamarin Music Inactive Baseline 72.2 (14.8) 73.5 (8.3) 75.2 (22.5) 74.5 (15.0) 70.5 (22.3) Playback 86.3 (19.0) 77.4 (16.8) 95.9 (2.1) 93.7 (8.9) 67.3 (30.1) 0-5 min Post-startle 87.9 (9.2) 77.8 (13.1) 75.4 (16.0) 87.3 (9.9) 67.8 (27.7) 5-10 min Post-startle 83.8 (8.0) 10-15 min Post-startle 78.3 (9.0) Locomotion Baseline 15.5 (5.7) 19.6 (7.0) 10.2 (6.8) 4.5 (5.8) 9.0 (3.7) Playback 7.2 (6.4) 12.4 (6.9) 3.3 (1.7) 3.8 (5.6) 6.6 (5.6) 0-5 min Post-startle 8.3 (6.2) 15.0 (7.1) 9.4 (6.0) 5.6 (4.3) 6.9 (3.5) 5-10 min Post-startle 9.8 (6.4) 16.6 (9.4) 10-15 min Post-startle 9.1 (6.2) 15-20 min Post-startle 7.8 (9.1) Self-directed Baseline 3.8 (5.4) 4.0 (1.4) 3.7 (3.9) 6.2 (6.7) 14.5 (20.5) Playback 0.9 (1.6) 8.7 (10.9) 0 1.9 (2.7) 24.2 (26.8) 0-5 min Post-startle 3.7 (4.0) 4.6 (4.9) 11.2 (13.1) 5.6 (4.3) 15.1 (20.3) Other Baseline 7.2 (10.4) 1.9 (2.2) 10.4 (18.6) 1.3 (2.8) 5.5 (3.7) Playback 5.4 (14.2) 1.0 (0.8) 0.8 (1.9) 0.5 (0.7) 1.0 (1.6) 0-5 min Post-startle 0 2.1 (3.0) 2.7 (4.9) 1.6 (1.5) 8.7 (18.0) Notes. Values represent the mean (SD) percent of visible time for each time block. Data for tamarins and marmosets were combined and compared to Baseline periods with no audio exposure using Wilcoxon signed-rank tests. Significant differences (p<0.05) are shown in bold and trends are shown in italics. No differences occurred after No Audio and Fear/Threat-based Tamarin Music conditions.

125

Figure 1. The percent of visible time marmosets spent inactive and locomoting during baseline and playback periods for each of the seven sound conditions. The sound environment was the same during baseline and playback periods for the No Audio condition. Each line represents an individual’s data (n=3-4). Note the differences in the y-axis scale for locomote behavior.

126

Behavioral response after acoustic startle event

Many of the behavior changes that occurred during sound playback had recovered

to baseline levels by 5 min (Table 3; 80 dB White Noise, Visitor Noise, Mozart).

Prolonged differences were noted following the first startle trial, Rainforest Sounds, with

elevated inactivity not returning to baseline levels until 15 min (0-5 min Post-startle: S=-

14, p=0.02; 5-10 min Post-startle: S=-11, p=0.08) and no return to baseline levels of locomotion at 20 min (0-5 min Post-startle: S=14, p=0.02; 5-10 min Post-startle: S=13, p=0.03; 10-15 min Post-startle: S=14, p=0.02; 15-20 min Post-startle: S=13, p0.03).

Although not significant across species, the pied tamarins did appear to increase self- directed behavior following the 80 dB White Noise and acoustic startle event, with no apparent return to baseline levels (Fig. 2).

127

Figure 2. The percent of visible time pied tamarins spent engaged in self-directed behavior during the 80 dB White Noise startle trial. After a 5 min baseline period, animals were exposed to 80 dB white noise for 5 min that was immediately followed by an acoustic startle stimulus (90 dB white noise for 500 ms). No sounds were played during the baseline or post-startle periods.

Startle response

No startle response was observed at the start of sound conditions, including the 80

White Noise condition. The 90 dB burst of white noise following sound conditions elicited a consistent startle response in all individuals with one exception: no physical startle was observed after 80 dB White Noise conditions in the marmoset group. In addition, the marmoset group also displayed a prolonged delay before moving following

128

the 80 dB White Noise condition (Table 4). Overall, the startle response appeared most

severe during the initial startle trials compared to later weeks. A prolonged freezing

response was observed in several individuals during week 1 (Rainforest Sounds). Also,

an adult female marmoset and her male offspring consistently retreated to out-of-view areas in the exhibit after the startle event during the first 2 weeks, a behavior that was only observed once more in this group during the study. After the startle event, the pied tamarins frequently began alarm calling and this behavior was observed after all sound conditions except the last, Fear/Threat-based Tamarin Music. The tamarins displayed the most alarm calling following the Rainforest Sounds and Affiliation-based Tamarin

Music, the first and second treatments. No alarm calls were observed in the marmoset group during the study.

129

Table 4. Summary of startle response measures after an acoustic startle event following sound conditions. Freeze time (s) Movement (number) Alarm calls (s)c Sound Conditiona Mean (Range) Total moved Moved out-of-viewb Latency to call Duration White-fronted marmoset Rainforest Sounds 40.0 (9-120)d 3/4 2/3 NA NA Tamarin Affiliation Music 7.5 (6-10 3/4 2/3 NA NA No Audio 10.5 (8-17) 3/4 0/3 NA NA 80 dB White Noise 39.0 (34-43) 0/3 0/3 NA NA Mozart 28.0 (8-67) 3/3 0/3 NA NA Visitor Noise 15.0 (11-19) 2/3 1/2 NA NA Tamarin Fear/Threat Music 8.3 (7-11) 3/3 0/3 NA NA Pied tamarin Rainforest Sounds 20.0 (3-41) 2/3 0/2 3 22 Tamarin Affiliation Music 3.0 (1-4) 2/3 0/2 4 16 No Audio 3.3 (2-5) 2/3 0/2 2 7 80 dB White Noise 3.3 (2-5) 2/3 1/2 9 7 Mozart 10.0 (2-20) 1/3 0/1 3 11 Visitor Noise 7.0 (6-8) 0/2 NA 2 4 Tamarin Fear/Threat Music 9.0 (7-11) 1/2 0/1 NA NA a The presentation of sound conditions was randomized and is shown in chronological order. b This is the proportion of animals that moved following the startle event. c It was not possible to identify the individual vocalizing and data represent the overall group time. d One male marmoset did not move after the startle sound for 13 min and was given a max trial time of 120 s. The group mean without this individual was 13 s.

130

Effect of startle experiments on fecal glucocorticoids

Startle experiments did not affect fecal glucocorticoid levels – there was no

difference in fecal glucocorticoid levels between samples collected on the days following

startle experiments and baseline samples collected the morning of the experiment

(F2,99=0.76, p=0.47).

Discussion

In this study, we assessed whether the playback of sounds could influence

behavior and modify the startle response, providing a potential indicator of affective

state. Behavior changes were observed during sound playback, compared to a baseline

period at the start of observations, with increased inactivity and/or decreased movement

occurring across species during four of the six sound conditions. This effect was

particularly apparent in the marmoset group and occurred to varying degrees after all

sound conditions except the No Audio control condition. Playback of 80 dB white noise,

a positive control condition expected to adversely influence the animals, caused a near

cessation of behavior in both groups, suggesting this sound condition may have

successfully modulated their affective state. We expected the 80 dB white noise to

sensitize animals and elicit an exaggerated response to the startle event. After the loud

white noise, prolonged freezing did occur in the marmoset group post startle but no

physical startle response was observed and was the only trial a physical startle did not

occur. We observed no apparent difference in the startle response of the tamarins

following 80 dB white noise but these monkeys did appear to increase self-directed behavior post-startle, a behavioral indicator of arousal. Most of the changes that occurred during playback had recovered to baseline levels by 5 min post-startle. In general, there

131

appeared to be a decreased response to the startle event over time, suggesting potential

habituation to the startle event.

Behavioral response to sound conditions

Although reduced activity may indicate calmer behavior (e.g. Snowdon and Teie,

2010), the restrained response by the marmosets during sound exposure is likely to have

indicated a defensive reaction, as it occurred consistently across sound conditions, including those sound conditions expected to be aversive – 80 dB White Noise,

Fear/Threat-based Tamarin Music, and Visitor Noise. Searcy and Caine (2003) also observed decreased movement by white-fronted marmosets in response to the playback of a hawk calls, although this was not observed after the playback of other sounds. After the startle event, animals generally displayed a rapid recovery to baseline levels of behavior, often within 5 min. Marmosets have been observed to exhibit a rapid return to baseline levels of behavior following predator confrontations (Cagni et al., 2011; Caine,

1998), highlighting the adaptability of callitrichines to quickly assess threats and reduce costs of antipredator behavior. Similar to previous studies (see Chapter 5), we observed no behavioral benefits of auditory stimulation and urge caution in applying sounds as a potential form of enrichment for callitrichine species.

Potential of startle reactivity as a measure of affect

Startle experiments did not influence fecal glucocorticoid measures, suggesting these brief aversive events did not have long-term consequences and thus, opening the possibility of these methods as an indicator of welfare of zoo animals. We did identify a modified response to the acoustic startle following 80 dB white noise suggestive of

132 increased arousal and providing partial evidence of an affective modulation of the startle response. However, this response took different forms in the two groups, with the tamarins showing an unaltered startle response but increased self-directed behavior post- startle whereas the marmosets exhibited prolonged freezing. In addition, the marmoset group failed to display a physical startle response after the 80 dB white noise. This may reflect prepulse inhibition, a phenomenon whereby a pulse of white noise of a lower amplitude preceding a startle sound decreases the magnitude of the startle response

(Davis et al., 2008). Attention may also modulate the startle response (Braff et al., 2001), but, as both species were vigilantly scanning prior to the startle event, attentional mechanisms may not explain this discrepancy. Differences in the startle response between the two groups makes quantifiable assessments challenging and question the practicality of assessing startle-related behaviors, compared to the detailed measurement of the startle reflex in laboratory studies (Davis et al., 2008). Future studies should explore the use of wearable accelerometers to provide detailed startle information.

More problematic is the potential habituation of startle-related behaviors we observed during the study. Although a physical startle was observed throughout the study in response to the startle event, hiding behavior of the marmosets and alarm calling by the tamarins appeared to wane after the initial trials. This was surprising as startle experiments occurred at least a week apart. Searcy and Caine (2003) saw no evidence of habituation by white-fronted marmosets across 8 presentations of hawk calls. However, rapid habituation of marmosets to predator models have been described (Barros et al.,

2004; Dacier et al., 2006). In the present study, the location of the speaker in the center of the exhibit may have promoted habituation as animals could quickly identify the

133 source of the sounds, unlike in Searcy and Caine’s study where the speaker was hidden in bushes near the marmoset enclosures. Although attempts were made to hide the speaker prior to this study, it was not feasible for these exhibits.

This study explored whether sounds experienced in the zoo environment, including music, habitat sounds, and visitor noise, could modify the startle response, a novel indicator of animal welfare. As expected, 80 dB white noise did appear to sensitize animals to the acoustic startle event. However, we observed no clear evidence of other sound conditions to modify the startle response. Although this may indicate these sounds did not influence the affective state of the monkeys, we did observe a defensive response to sounds by the marmosets, with increased inactivity and decreased movement. As it is likely this behavioral response was indicative of a change in affective state, this suggests the startle response, as measured in this study, may not have been sensitive enough to detect these changes. Habituation and group-specific differences in the startle response were observed, making interpretation of these results challenging and question the utility of assessing startle-related behaviors as a reliable indicator of affective state. In general, we did not identify benefits of exposure to sounds, indicating auditory stimulation may not be a suitable form of enrichment for callitrichine species.

134

Chapter Seven: General discussion and future outlook

This project explored the impact of the sound environment on several species of callitrichine monkeys housed at Cleveland Metroparks Zoo’s RainForest building. One study focus was to evaluate the effect of loud, broadband sound arising from a nearby waterfall feature. It has been suggested that broadband sound, such as from the waterfall, may serve to provide auditory masking of arousing noises that arise in the zoo environment. We observed no evidence of these benefits for callitrichine monkeys through controlled experimental manipulations of the waterfall feature. In contrast, monkeys appeared to avoid the waterfall noise through increased use of quiet off-exhibit areas when available. In a follow-up study, we confirmed the role of the sound environment in modifying space use by additionally manipulating the off-exhibit sound environment. When background sound levels were consistent across the exhibit and off- exhibit areas, either equally loud or quiet, pied tamarins used these areas as expected based on space availability. Providing the exhibit choices for animals to retreat from noise appeared beneficial and may have ameliorated some effects of waterfall noise. For example, the monkeys were observed to be more vigilant when exposed to the waterfall noise but only when they did not have off-exhibit access. It should be noted that we observed no effect of the waterfall noise on behavioral or hormonal indicators of welfare, suggesting animals were able to cope with the negative effects of this noise.

In addition to evaluating the impact of waterfall noise, a secondary focus was to evaluate sounds that may promote calming effects and represent potential forms of auditory enrichment. Thus, we investigated the effect of a classical music piece

135

(Mozart’s Eine Kleine Nachtmusik) and rainforest habitat sounds, both traditional forms

of auditory stimulation that have been previously applied as enrichment, as well as music

of their vocalizations. We observed no beneficial effects of auditory stimulation in

controlled early morning playback experiments. This was not surprising with regards to

the classical music and rainforest sounds, which have previously shown mixed effects

across species, but the lack of a response to tamarin music was unexpected. Although a

complete explanation of this discrepancy is not possible, methodological and species

differences may have contributed to the lack of response.

Mechanism of response to noise

Several hypotheses have been suggested for how noise can affect animals,

including as a perceived threat (Frid and Dill, 2002); through auditory masking of

important information (Barber et al., 2010); or as a distraction (Chan et al., 2010).

Francis and Barber (2013) suggested that the characteristics of the noise may dictate the response, with unpredictable sudden sounds likely causing a disturbance response whereas chronic noise would cause interference through auditory masking.

In this study, the increased vigilance when exposed to the chronic waterfall noise would suggest a masking response, similar to what has been observed in free-ranging species exposed to chronic noise (Rabin et al., 2006). Callitrichines have been noted to perform high levels of vigilance, even in captive settings with no risk of predation (Caine,

1984). For these species, monitoring the activity of people may be important but, as multiple studies have documented a negative effect of visitors on these species, may also be stressful. Providing access to off-exhibit areas may have decreased the perception of

136

risk by allowing escape opportunities. In contrast to the waterfall noise, the effect of

music and habitat sounds appeared generally minor, possibly a reflection of the lower

sound levels during playback experiments. However, the decreased activity by the

marmosets during playback exposure in the startle modification experiment was similar

to their response to waterfall noise and suggest noise, even at this lower sound level (64

dBA), may have altered their assessment of environmental risk. As expected, the

response to the acoustic startle stimulus (90 dBA) was typically an immediate startle

and—in the case of the pied tamarins—alarm calling and threat displays. This response

was distinct from other sounds (including the onset of 80 dB white noise during those

experiments) and indicate that the acoustic startle sound was perceived as threatening.

Interestingly, the response to the startle event did appear to wane during the study,

suggesting possible habituation.

Do animals habituate to noise in the zoo setting?

There is a general belief that, through frequent exposure, animals will habituate to

disturbances in the zoo setting. Formally, habituation is defined as the persistent waning

of a response as a result of repeated stimulation without reinforcement (Bejder et al.,

2009). Commonly, habituation is evoked to argue that a decreased response over time to

a stressor indicated that the stimulus was no longer perceived as threatening (Bejder et al., 2009). However, although this may be the case, animals may stop responding behaviorally but still exhibit a physiological reaction (Coe et al., 1983). Animals may also be constrained from persistent responding. For example, the risk allocation hypothesis (Lima and Bednekoff, 1999) suggests that when a perceived risk is brief, animals will perform high levels of antipredator behavior. However, when the period of

137

risk is protracted or frequent, animals may reduce antipredator behaviors to avoid the

costs to foraging and other biologically important activities. Brown et al. (2012) found

pronghorn and elk to be less vigilant during periods of increased human activity and

anthropogenic noise. These authors suggested that although this response may indicate

habituation, alternatively it may reflect that these animals could not afford to maintain

high levels of responsiveness to human activity.

Sometimes a lack of response is mistakenly taken to indicate habituation. A lack

of a response may alternatively indicate that an animal had sufficient ability to cope with

the stimulus. For example, Hanson et al. (1976) showed that providing control over 100

dB noise decreased plasma cortisol levels in rhesus macaques. Given the amplitude of

the noise (100 dB), which is similar to the sound level from a jackhammer and can cause

hearing damage if sustained, it is highly unlikely that the noise itself was less aversive

and instead suggests the benefits derived from a change in the perception of control and

not necessarily how the stimulus was perceived.

In this study, animals avoided the waterfall noise even though all individuals had

been either born into these exhibits or experienced this noise for at least two years prior

to the start of this research. This would suggest animals did not habituate to chronic loud

noise. Providing the monkeys with access to off-exhibit areas appeared to ameliorate

some of the negative behavioral effects of the waterfall noise even though animals still

showed a preference for the quieter off-exhibit areas. Thus, the waterfall noise may not

necessarily have been less aversive with off-exhibit access but instead, animals may have derived benefits from having opportunities to cope with the noise. Surprisingly we saw

little overall influence of visitors on these monkeys but, as previously noted, this should

138

not be taken as evidence that these animals had habituated to human visitation.

Throughout much of the research animals had access to off-exhibit areas which may have

influenced the response to visitors. Anecdotally, brief threat displays did routinely occur

after the approach of visitors or the observer, indicating these animals did still perceive

humans as a potential threat. The marmosets and tamarins did appear to behaviorally

habituate to the acoustic startle event over successive weeks, with decreased alarm

calling and hiding after the initial trials. It is unclear if the animals also exhibited a

physiological habituation, as the aggregate fecal glucocorticoid measure assessed in this

study was not time-sensitive enough to detect the short-term response to the startle sound.

Differences between groups in response to sounds

Several differences in the response to sounds were noted between the species

groups in this study. When exposed to waterfall noise, the pied tamarins appeared to show a bold response (e.g. increased locomotion and use of the front of the exhibit) compared to the more defensive reaction of the marmosets and callimicos (e.g decreased

locomotion, increased use of the back of the exhibit). The marmosets also responded

with decreased activity when presented with sounds during the startle modification

experiments. Differences were also noted in response to acoustic startle events, with the

pied tamarins emitting alarm calls and performing threat displays, behaviors never

observed from the marmoset group.

Anecdotally, differences between the groups were noted when animals were being

hand fed pieces of banana with fecal markers. An initial attempt was made to hand feed

the pied tamarins on exhibit. However, these monkeys responded aggressively—

performing head shaking threat displays, piloerection, and attempted to steal the food

139

bowl—and all subsequent hand feeding was performed in off-exhibit areas through caging for protection. In contrast, hand feeding of the marmosets occurred on exhibit and was relatively uneventful (with the exception of several incidents of aggression from the female marmoset). The callimicos appeared more wary of accepting food by hand than the other species. These species differences are consistent with the observations of

Savastano et al. (2003) during their evaluation of a training program at the Bronx Zoo.

Also, the breeding pair of callimicos and a breeding male marmoset were introduced to these exhibits during the study and displayed a high degree of cautiousness. The callimicos were reluctant to enter the exhibit space after moving to these areas, even after lights in off-exhibit areas were turned off and food was only available in the exhibit, and did not begin reliably entering the exhibit until their second week in the building. The male marmoset was noted to spend a considerable amount of time in an out-of-view crevice of the exhibit and this behavior persisted for months.

It is possible that the behavior differences between these groups may reflect species differences in their respective natural histories. For example, the pelage of callimicos is all black, contrasting sharply with the conspicuous colorful coats observed in other callitrichines. Leila Porter was the first researcher to perform long-term studies on this species and described callimicos as “extremely shy animals and take a very long time to become habituated to people” (Hanson et al., 2006). In addition, differences in feeding ecology between these monkeys may also play a role. For example, pied tamarins have been noted to spend more time hunting for insects than white-fronted marmosets (Egler, 1992; Passamani and Rylands, 2000), possibly conferring a more ballistic response pattern. Previous comparative research on callitrichines have noted

140

differences in neophobia (Day et al., 2003) and performance on memory tasks (Platt et

al., 1996), which the authors have attributed to differences in the feeding ecology

between species. Given the small sample size in the present study, firm conclusions

regarding the differences between these species are not possible. The results do suggest

that closely related callitrichine species may have species-specific coping strategies in response to noise but additional comparative work is warranted.

Sound environment recommendations for zoos

Noise has been identified as a potential source of stress for animals and zoos should consider monitoring sound levels in animal areas. This can be easily accomplished through traditional sound level meters as well as apps available for smartphones (Kardous and Shaw, 2014). However, careful attention should be paid to how the instrument weights sound frequencies (see Pater et al., 2009 for noise assessment recommendations). The A-weighting curve that preferentially weights sounds between 1 and 5 kHz based on human hearing would be the most appropriate choice for monitoring sound levels for most primate species (Heffner, 2004). However, for Strepsirrhine primates and other animal species that have notably higher frequency hearing than humans (see Heffner, 2004), typical sound level meters (frequency range 20 Hz- 20 kHz) may be inadequate and monitoring of ultrasound (sounds >20 kHz) is recommended. It is also possible to develop a species-specific frequency weighting for evaluating the sound environment if the hearing range and sensitivity of the species is known (rat, Björk et al.,

1999; spotted owl; Delaney et al., 1999).

The response of the monkeys in this study would suggest ‘silence is golden’.

From an exhibit design standpoint, the use of glass exhibit barriers, as opposed to the

141

mesh barriers of the exhibits in this project, would provide attenuation of noise arising in

the visitor area. However, it may be valuable for these monkeys to monitor the sound

environment and glass barriers may not be necessary in the absence of other sources of

noise (e.g. waterfall features, building ventilation). Exhibit placement may also be

important and some authors have recommended for callitrichine exhibits to be situated in

lower trafficked areas of the zoo (Glaston et al., 1984; Wormell et al., 1996). Foliage in

the exhibit may also provide some degree of attenuation of noise. The results of this

study do not support the use introducing sounds for auditory masking purposes and this is

not recommended for callitrichine monkeys. Alternatively, providing an opportunity to

avoid noise through access to off-exhibit areas did confer benefits to animals in this project and is recommended as a low-cost solution when noise is a concern. As this change may compromise animal visibility for the guests, access to quiet out-of-view areas should be provided in conjunction with animal visibility surveys (Kuhar et al.,

2010) and exhibit signage explaining the housing changes.

Future outlook

This project shed light on the sound environment for the monkeys in this study.

Incorporating these findings from the zoo environment with the broader framework of noise effects on free-ranging wildlife was valuable and identified similar responses to chronic noise. However, this project also highlighted a distinction for zoo species—the ability to avoid noise, or inability as may be the case in some exhibits. Thus, research in zoos should consider the avoidance opportunities a particular exhibit allows when evaluating noises. In addition, a greater understanding is needed of the individual or species characteristics that influence the response to noise. It is likely that the

142 predator/prey dynamic, human-animal relationship, and individual or species temperament will likely play a role.

When evaluating the sound environment, the central question that should be addressed is how are the sounds perceived? Our relationship to the sound environment is heavily influenced by our experience, including our hearing range, knowledge of sounds, ability to explore the environment, and safety from threats, to name a few. Thus, an umwelt of the animal’s world may be a valuable starting point.

Additional measures may help evaluate how animals perceive sounds. First, with increasingly popular touchscreen cognition work, it may now be possible to easily implement the direct control over sounds. Second, as the response to sounds may wax and wane over time, future studies should seek to incorporate immediate physiological responses after exposure to sounds, such as salivary cortisol, oxytocin, and alpha- amylase. Third, modifications to the startle methodology of this project may improve its utility as an indicator of welfare. Most obviously, hiding the speaker in out-of-view areas is strongly recommended. In addition, the use of a wearable actigraph may provide detailed information on the startle reflex. When assessing sounds, the use of a visual startle may be valuable comparison across modalities and reduce the potential for auditory inhibition.

Beyond animal welfare, research of the sound environment of zoos may have important implications for conservation. The effect of anthropogenic noise and other types of human disturbance extend across land, sea, and air. Research on noise in the zoo setting may provide an understanding of tolerance limits for a species and help develop predictions for the impact of noise.

143

References

Addessi E, Chiarotti F, Visalberghi E, Anzenberger G. 2007. Response to novel food and

the role of social influences in common marmosets (Callithrix jacchus) and

Goeldi's monkeys (Callimico goeldii). American Journal of Primatology

69(11):1210-1222.

Akiyama K, Sutoo D. 2011. Effect of different frequencies of music on blood pressure

regulation in spontaneously hypertensive rats. Neuroscience Letters 487(1):58-60.

Angelucci F, Ricci E, Padua L, Sabino A, Tonali PA. 2007. Music exposure differentially

alters the levels of brain-derived neurotrophic factor and nerve growth factor in

the mouse hypothalamus. Neuroscience Letters 429(2-3):152-155.

Anzenberger G, Falk B. 2012. Monogamy and family life in callitrichid monkeys:

deviations, social dynamics and captive management. International Zoo Yearbook

46(1):109-122.

Armstrong DM, Santymire RM. 2013. Hormonal and behavioral variation in pied

tamarins housed in different management conditions. Zoo Biology 32(3):299-306.

Association of Zoos and Aquariums Animal Welfare Committee. 2015.

https://www.aza.org/membership/detail.aspx?id=378.

Badihi I. 2006. The effects of complexity, choice and control on the behaviour and the

welfare of captive common marmosets (Callithrix jacchus). Stirling Online

Research Repository. (http://hdl.handle.net/1893/120).

144

Bales KL, French JA, Hostetler CM, Dietz JM. 2005. Social and reproductive factors

affecting cortisol levels in wild female golden lion tamarins (Leontopithecus

rosalia). American Journal of Primatology 67(1):25-35.

Bales KL, French JA, McWilliams J, Lake RA, Dietz JM. 2006. Effects of social status,

age, and season on androgen and cortisol levels in wild male golden lion tamarins

(Leontopithecus rosalia). Hormones and Behavior 49(1):88-95.

Barber JR, Crooks KR, Fristrup KM. 2010. The costs of chronic noise exposure for

terrestrial organisms. Trends in Ecology & Evolution 25(3):180-189.

Barbosa MN, Mota MTS. 2009. Behavioral and hormonal response of common

marmosets, Callithrix jacchus, to two environmental conditions. Primates

50(3):253-260.

Barbosa MN, Mota MTS. 2014. Do newborn vocalizations affect the behavioral and

hormonal responses of nonreproductive male common marmosets (Callithrix

jacchus)? Primates 55(2):293-302.

Barros M, Alencar C, de Souza Silva M, Tomaz C. 2008. Changes in experimental

conditions alter anti-predator vigilance and sequence predictability in captive

marmosets. Behavioural Processes 77(3):351-356.

Barros M, de Souza Silva MA, Huston JP, Tomaz C. 2004. Multibehavioral analysis of

fear and anxiety before, during, and after experimentally induced predatory stress

in Callithrix penicillata. Pharmacology Biochemistry and Behavior 78(2):357-

367.

Bee MA, Swanson EM. 2007. Auditory masking of anuran advertisement calls by road

traffic noise. Animal Behaviour 74(6):1765-1776.

145

Bejder L, Samuels A, Whitehead H, Finn H, Allen S. 2009. Impact assessment research:

use and misuse of habituation, sensitisation and tolerance in describing wildlife

responses to anthropogenic stimuli. Marine Ecology Progress Series 395:177-185.

Bezerra BM, Keasey MP, Schiel N, Souto AS. 2014. Responses towards a dying adult

group member in a wild New World monkey. Primates 55(2):185-188.

Bigand E, Filipic S, Lalitte P. 2005. The time course of emotional responses to music.

Annals of the New York Academy of Sciences 1060(1):429-437.

Birke L. 2002. Effects of browse, human visitors and noise on the behaviour of captive

orang utans. Animal Welfare 11(2):189-202.

Björk E, Nevalainen T, Hakumäki M, Voipio H-M. 2000. R-weighting provides better

estimation for rat hearing sensitivity. Laboratory Animals 34(2):136-144.

Blickley JL, Blackwood D, Patricelli GL. 2012. Experimental evidence for the effects of

chronic anthropogenic noise on abundance of greater sage‐grouse at leks.

Conservation Biology 26(3):461-471.

Blickley JL, Patricelli GL. 2012. Potential acoustic masking of Greater Sage-Grouse

(Centrocercus urophasianus) display components by chronic industrial noise.

Ornithological Monographs 2012(74):23-35.

Boinski S, Gross TS, Davis JK. 1999. Terrestrial predator alarm vocalizations are a valid

monitor of stress in captive brown capuchins (Cebus apella). Zoo Biology

18(4):295-312.

Boissy A, Manteuffel G, Jensen MB, Moe RO, Spruijt B, Keeling LJ, Winckler C,

Forkman B, Dimitrov I, Langbein J. 2007. Assessment of positive emotions in

animals to improve their welfare. Physiology & Behavior 92(3):375-397.

146

Braccini SN, Caine NG. 2009. Hand preference predicts reactions to novel foods and

predators in marmosets (Callithrix geoffroyi). Journal of Comparative Psychology

123(1):18.

Bradley MM, Lang PJ. 2000. Affective reactions to acoustic stimuli. Psychophysiology

37(02):204-215.

Braff DL, Geyer MA, Swerdlow NR. 2001. Human studies of prepulse inhibition of

startle: normal subjects, patient groups, and pharmacological studies.

Psychopharmacology 156(2-3):234-258.

Brent L, Weaver O. 1996. The physiological and behavioral effects of radio music on

singly housed baboons. Journal of medical primatology 25(5):370-374.

Brown CL, Hardy AR, Barber JR, Fristrup KM, Crooks KR, Angeloni LM. 2012. The

effect of human activities and their associated noise on ungulate behavior. PloS

one 7(7):e40505.

Brown JL, editor. 2008. Wildlife Endocrinology Manual. Front Royal, VA: National

Zoological Park Conservation and Research Center. 75 p.

Brown JL, Schmitt DL, Bellem A, Graham LH, Lehnhardt J. 1999. Hormone secretion in

the Asian elephant (Elephas maximus): Characterization of ovulatory and

anovulatory luteinizing hormone surges. Biology of Reproduction 61(5):1294-

1299.

Burman OH, Ilyat A, Jones G, Mendl M. 2007. Ultrasonic vocalizations as indicators of

welfare for laboratory rats (Rattus norvegicus). Applied Animal Behaviour

Science 104(1):116-129.

147

Burnham KP, Anderson DR. 2001. Kullback-Leibler information as a basis for strong

inference in ecological studies. Wildlife Research 28(2):111-119.

Cagni P, Gonçalves I, Ziller F, Emile N, Barros M. 2009. Humans and natural predators

induce different fear/anxiety reactions and response pattern to diazepam in

marmoset monkeys. Pharmacology Biochemistry and Behavior 93(2):134-140.

Cagni P, Sampaio AC, Ribeiro NB, Barros M. 2011. Immediate, but no delayed,

behavioral response to a snake model by captive black tufted-ear marmosets.

Behavioural Processes 87(3):241-245.

Caine NG. 1984. Visual scanning by tamarins. Folia Primatologica 43(1):59-67.

Caine NG. 1993. Flexibility and co-operation as unifying themes in Saguinus social

organization and behaviour: The role of predation pressures. In: Rylands AB,

editor. Marmosets and Tamarins: Systematics, Behaviour, and Ecology. p 200-

219.

Caine NG. 1998. Cutting costs in response to predatory threat by Geoffroy's marmosets

(Callithrix geoffroyi). American Journal of Primatology 46(3):187-196.

Campo J, Gil M, Davila S. 2005. Effects of specific noise and music stimuli on stress and

fear levels of laying hens of several breeds. Applied Animal Behaviour Science

91(1):75-84.

Carlstead K, Shepherdson D. 2000. Alleviating stress in zoo animals with environmental

enrichment. In: Moberg GP, Mench JA, editors. The Biology of Animal Stress:

Basic Principles and Implications for Animal Welfare. New York, NY: CABI. p

337-354.

148

Cassella JV, Davis M. 1986. The design and calibration of a startle measurement system.

Physiology & Behavior 36(2):377-383.

Chan AAY-H, Giraldo-Perez P, Smith S, Blumstein DT. 2010. Anthropogenic noise

affects risk assessment and attention: the distracted prey hypothesis. Biology

Letters 6(4):458-461.

Clark FE, Fitzpatrick M, Hartley A, King AJ, Lee T, Routh A, Walker SL, George K.

2012. Relationship between behavior, adrenal activity, and environment in zoo‐

housed western lowland gorillas (Gorilla gorilla gorilla). Zoo Biology 31(3):306-

321.

Clark FE, Melfi VA. 2012. Environmental enrichment for a mixed‐species nocturnal

mammal exhibit. Zoo Biology 31(4):397-413.

Claxton AM. 2011. The potential of the human–animal relationship as an environmental

enrichment for the welfare of zoo-housed animals. Applied Animal Behaviour

Science 133(1):1-10.

Coe CL, Glass JC, Wiener SG, Levine S. 1983. Behavioral, but not physiological,

adaptation to repeated separation in mother and infant primates.

Psychoneuroendocrinology 8(4):401-409.

Colborn D, Thompson Jr D, Roth T, Capehart J, White K. 1991. Responses of cortisol

and prolactin to sexual excitement and stress in stallions and. Journal of Animal

Science 69:2556-2562.

Cooke CM, Schillaci MA. 2007. Behavioral responses to the zoo environment by white

handed gibbons. Applied Animal Behaviour Science 106(1):125-133.

149

Costall B, Domeney AM, Farre AJ, Kelly ME, Martinez L, Naylor RJ. 1992. Profile of

action of a novel 5-hydroxytryptamine1A receptor ligand E-4424 to inhibit

aversive behavior in the mouse, rat and marmoset. Journal of Pharmacology and

Experimental Therapeutics 262(1):90-98.

Cross N, Pines MK, Rogers LJ. 2004. Saliva sampling to assess cortisol levels in

unrestrained common marmosets and the effect of behavioral stress. American

Journal of Primatology 62(2):107-114.

Cross N, Rogers LJ. 2004. Diurnal cycle in salivary cortisol levels in common

marmosets. Developmental Psychobiology 45(3):134-139.

Cumming G, Finch S. 2005. Inference by eye: Confidence intervals and how to read

pictures of data. American Psychologist 60(2):170.

Dacier A, Maia R, Agustinho D, Barros M. 2006. Rapid habituation of scan behavior in

captive marmosets following brief predator encounters. Behavioural Processes

71(1):66-69.

Davey G. 2006. Relationships between exhibit naturalism, animal visibility and visitor

interest in a Chinese Zoo. Applied Animal Behaviour Science 96(1):93-102.

Davey G. 2007. Visitors' effects on the welfare of animals in the zoo: A review. Journal

of Applied Animal Welfare Science 10(2):169-183.

Davis M, Antoniadis EA, Amaral DG, Winslow JT. 2008. Acoustic startle reflex in

rhesus monkeys: a review. Reviews in the Neurosciences 19(2-3):171-186.

Dawkins M. 2004. Using behaviour to assess animal welfare. Animal Welfare 13:S3-S7.

150

Day RL, Coe RL, Kendal JR, Laland KN. 2003. Neophilia, innovation and social

learning: a study of intergeneric differences in callitrichid monkeys. Animal

Behaviour 65(3):559-571.

De Coensel B, Vanwetswinkel S, Botteldooren D. 2011. Effects of natural sounds on the

perception of road traffic noise. The Journal of the Acoustical Society of America

129(4):EL148-EL153.

Delaney DK, Grubb TG, Beier P, Pater LL, Reiser MH. 1999. Effects of helicopter noise

on Mexican spotted owls. The Journal of Wildlife Management:60-76.

Diette GB, Lechtzin N, Haponik E, Devrotes A, Rubin HR. 2003. Distraction therapy

with nature sights and sounds reduces pain during flexible bronchoscopy: A

complementary approach to routine analgesia. Chest Journal 123(3):941-948.

Duarte MH, Vecci MA, Hirsch A, Young RJ. 2011. Noisy human neighbours affect

where urban monkeys live. Biology Letters 7(6):840-842.

Egler S. 1992. Feeding ecology of Saguinus bicolor bicolor (Callitrichidae: Primates) in

a relict forest in Manaus, Brazilian Amazonia. Folia Primatologica 59(2):61-76.

Ekman P, Friesen WV. 1971. Constants across cultures in the face and emotion. Journal

of personality and social psychology 17(2):124.

Epple G, Epple A, Baker A. 2002. Scent Marking Patterns in a Group of Pied Tamarins

(Saguinus bicolor bicolor, Callitrichinea). Primate Report:55-62.

Evans D. 2002. The effectiveness of music as an intervention for hospital patients: A

systematic review. Journal of Advanced Nursing 37(1):8-18.

Fàbregas MC, Guillén‐Salazar F, Garcés‐Narro C. 2012. Do naturalistic enclosures

provide suitable environments for zoo animals? Zoo Biology 31(3):362-373.

151

Farrand A. 2007. The effect of zoo visitors on the behaviour and welfare of zoo mammals

Stirling Online Research Repository. (http://hdl.handle.net/1893/120).

Fernandez-Duque E, Di Fiore A, Huck M. 2012. The behavior, ecology, and social

evolution of New World monkeys. In: Mitani JC, Call J, Kappeler PM, Palombit

RA, Silk JB, editors. The Evolution of Primate Societies. Chicago, IL: The

University of Chicago Press. p 43-64.

Ferrari SF. 2009. Predation risk and antipredator strategies. In: Garber PA, Estrada A,

Bicca-Marques JC, Heymann EW, Strier KB, editors. South American Primates:

Comparative Perspectives in the Study of Behavior, Ecology, and Conservation.

New York, NY: Springer. p 251-277.

Fitch WT. 2006. The biology and evolution of music: A comparative perspective.

Cognition 100(1):173-215.

Fontani S, Vaglio S, Beghelli V, Mattioli M, Bacci S, Accorsi PA. 2014. Fecal

concentrations of cortisol, testosterone, and progesterone in cotton-top tamarins

housed in different zoological parks: Relationships among physiological data,

environmental conditions, and behavioral patterns. Journal of Applied Animal

Welfare Science 17(3):228-252.

Foreman C. 2008. Sound System Design. In: Ballou G, editor. Handbook for Sound

Engineers. 4th ed. Burlington, MA: Focal Press.

Francis CD, Barber JR. 2013. A framework for understanding noise impacts on wildlife:

An urgent conservation priority. Frontiers in Ecology and the Environment

11(6):305-313.

152

Fraser D. 2009. Animal behaviour, animal welfare and the scientific study of affect.

Applied Animal Behaviour Science 118(3):108-117.

Friant SC, Campbell MW, Snowdon CT. 2008. Captive‐born cotton‐top tamarins

(Saguinus oedipus) respond similarly to vocalizations of predators and sympatric

nonpredators. American Journal of Primatology 70(7):707-710.

Frid A, Dill LM. 2002. Human-caused disturbance stimuli as a form of predation risk.

Conservation Ecology 6(1):11.

Fuller G, Margulis SW, Santymire R. 2011. The effectiveness of indigestible markers for

identifying individual animal feces and their prevalence of use in North American

zoos. Zoo Biology 30(4):379-398.

Galbrun L, Ali TT. 2013. Acoustical and perceptual assessment of water sounds and their

use over road traffic noise. The Journal of the Acoustical Society of America

133(1):227-237.

Garber PA. 1992. Vertical clinging, small body size, and the evolution of feeding

adaptations in the Callitrichinae. American Journal of Physical Anthropology

88(4):469-482.

Gerrards‐Hesse A, Spies K, Hesse FW. 1994. Experimental inductions of emotional

states and their effectiveness: A review. British Journal of Psychology 85(1):55-

78.

Glaston AR, Geilvoet-Soeteman E, Hora-Pecek E, van Hooff JARAM. 1984. The

influence of the zoo environment on social behavior of groups of cotton-topped

tamarins, Saguinus oedipus oedipus. Zoo Biology 3:241-253.

153

Gomez P, Danuser B. 2004. Affective and physiological responses to environmental

noises and music. International Journal of psychophysiology 53(2):91-103.

Gomez P, Danuser B. 2007. Relationships between musical structure and

psychophysiological measures of emotion. Emotion 7(2):377.

Goymann W. 2012. On the use of non-invasive hormone research in uncontrolled, natural

environments: The problem with sex, diet, metabolic rate and the individual.

Methods in Ecology and Evolution 3(4):757-765.

Haapakangas A, Kankkunen E, Hongisto V, Virjonen P, Oliva D, Keskinen E. 2011.

Effects of five speech masking sounds on performance and acoustic satisfaction.

Implications for open-plan offices. Acta Acustica united with Acustica 97(4):641-

655.

Hanson AM, Hall MB, Porter LM, Lintzenich B. 2006. Composition and nutritional

characteristics of fungi consumed by Callimico goeldii in Pando, Bolivia. Int J

Primatol 27(1):323-346.

Hanson JD, Larson ME, Snowdon CT. 1976. The effects of control over high intensity

noise on plasma cortisol levels in rhesus monkeys. Behavioral Biology 16(3):333-

340.

Heffner RS. 2004. Primate hearing from a mammalian perspective. The Anatomical

Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology

281(1):1111-1122.

Heistermann M, Palme R, Ganswindt A. 2006. Comparison of different

enzymeimmunoassays for assessment of adrenocortical activity in primates based

on fecal analysis. American Journal of Primatology 68(3):257-273.

154

Heymann EW, Smith AC. 1999. When to feed on gums: Temporal patterns of

gummivory in wild tamarins, Saguinus mystax and Saguinus fuscicollis

(Callitrichinae). Zoo Biology 18(6):459-471.

Hoffman HS, Fleshler M. 1963. Startle reaction: Modification by background acoustic

stimulation. Science 141(3584):928-930.

Holm CM, Priston NE, Price EC, Wormell D. 2012. An investigation into the

environmental factors affecting behavioural stress in captive pied tamarins,

Saguinus bicolor. Canopy 12(2):14-16.

Hosey G, Melfi V. 2012. Human–animal bonds between zoo professionals and the

animals in their care. Zoo Biology 31(1):13-26.

Hosey GR. 2000. Zoo animals and their human audiences: What is the visitor effect?

Animal Welfare 9(4):343-357.

Howell S, Schwandt M, Fritz J, Roeder E, Nelson C. 2003. A stereo music system as

environmental enrichment for captive chimpanzees. Lab Animal 32(10):31-36.

Hu Y, Cardoso GC. 2010. Which birds adjust the frequency of vocalizations in urban

noise? Animal Behaviour 79(4):863-867.

Huck M, Löttker P, Heymann E, Heistermann M. 2005. Characterization and social

correlates of fecal testosterone and cortisol excretion in wild male Saguinus

mystax. Int J Primatol 26(1):159-179.

Hunt KE, Trites AW, Wasser SK. 2004. Validation of a fecal glucocorticoid assay for

Steller sea lions (Eumetopias jubatus). Physiology & Behavior 80(5):595-601.

155

Ison JR, Hammond GR. 1971. Modification of the startle reflex in the rat by changes in

the auditory and visual environments. Journal of Comparative and Physiological

Psychology 75(3):435.

Jankowski M, Wittwer D, Heisey D, Franson J, Hofmeister E. 2009. The adrenocortical

response of Greater Sage Grouse (Centrocercus urophasianus) to capture, ACTH

injection, and confinement, as measured in fecal samples. Physiological and

Biochemical Zoology 82(2):190.

Jeon JY, Lee PJ, You J, Kang J. 2010. Perceptual assessment of quality of urban

soundscapes with combined noise sources and water sounds. The Journal of the

Acoustical Society of America 127(3):1357-1366.

Johnson EO, Kamilaris TC, Carter CS, Calogero AE, Gold PW, Chrousos GP. 1996. The

biobehavioral consequences of psychogenic stress in a small, social primate

(Callithrix jacchus jacchus). Biological Psychiatry 40(5):317-337.

Juslin PN, Laukka P. 2003. Communication of emotions in vocal expression and music

performance: Different channels, same code? Psychological Bulletin 129(5):770.

Juslin PN, Västfjäll D. 2008. Emotional responses to music: The need to consider

underlying mechanisms. Behavioral and Brain Sciences 31(05):559-575.

Kardous CA, Shaw PB. 2014. Evaluation of smartphone sound measurement

applicationsa). The Journal of the Acoustical Society of America 135(4):EL186-

EL192.

Keay JM, Singh J, Gaunt MC, Kaur T. 2006. Fecal glucocorticoids and their metabolites

as indicators of stress in various mammalian species: A literature review. Journal

of Zoo and Wildlife Medicine 37(3):234-244.

156

Kemper KJ, Danhauer SC. 2005. Music as therapy. Southern Medical Journal 98(3):282-

288.

Khalfa S, Bella S, Roy M, Peretz I, Lupien S. 2003. Effects of relaxing music on salivary

cortisol level after psychological stress. Annals of the New York Academy of

Sciences 999:374.

Kirkden RD, Pajor EA. 2006. Using preference, motivation and aversion tests to ask

scientific questions about animals’ feelings. Applied Animal Behaviour Science

100(1):29-47.

Knight WE, Rickard NS. 2001. Relaxing music prevents stress-induced increases in

subjective anxiety, systolic blood pressure, and heart rate in healthy males and

females. Journal of Music Therapy 38(4):254-272.

Koch M. 1999. The neurobiology of startle. Progress in Neurobiology 59(2):107-128.

Koger SM, Chapin K, Brotons M. 1999. Is music therapy an effective intervention for

dementia? A meta-analytic review of literature. Journal of Music Therapy

36(1):2-15.

Krohn TC, Salling B, Hansen AK. 2011. How do rats respond to playing radio in the

animal facility? Laboratory Animals 45(3):141-144.

Kuhar C, Miller L, Lehnhardt J, Christman J, Mellen J, Bettinger T. 2010. A system for

monitoring and improving animal visibility and its implications for zoological

parks. Zoo Biology 29(1):68-79.

Kuhar CW. 2008. Group differences in captive gorillas’ reaction to large crowds. Applied

Animal Behaviour Science 110(3):377-385.

157

Kweon B-S, Ulrich RS, Walker VD, Tassinary LG. 2008. Anger and stress: The role of

landscape posters in an office setting. Environment and Behavior 40(3):355-381.

Lang PJ, Bradley MM, Cuthbert BN. 1990. Emotion, attention, and the startle reflex.

Psychological Review 97(3):377.

Lang PJ, Davis M, Öhman A. 2000. Fear and anxiety: Animal models and human

cognitive psychophysiology. Journal of Affective Disorders 61(3):137-159.

Larsen MJ, Sherwen SL, Rault J-L. 2014. Number of nearby visitors and noise level

affect vigilance in captive koalas. Applied Animal Behaviour Science 154:76-82.

Lazaro-Perea C, Castro CSS, Harrison R, Araujo A, Arruda MF, Snowdon CT. 2000.

Behavioral and demographic changes following the loss of the breeding female in

cooperatively breeding marmosets. Behav Ecol Sociobiol 48(2):137-146.

LeDoux J. 2000. Emotion circuits in the brain. Annual review of neuroscience 23:155.

Lemmer B. 2008. Effects of music composed by Mozart and Ligeti on blood pressure and

heart rate circadian rhythms in normotensive and hypertensive rats.

Chronobiology International 25(6):971-986.

Lenti Boero D, Bottoni L. 2008. Why we experience musical emotions: Intrinsic

musicality in an evolutionary perspective. Behavioral and Brain Sciences

31(05):585-586.

Li WJ, Yu H, Yang JM, Gao J, Jiang H, Feng M, Zhao YX, Chen ZY. 2010. Anxiolytic

effect of music exposure on BDNFMet/Met transgenic mice. Brain Research

1347:71-79.

158

Lima SL, Bednekoff PA. 1999. Temporal variation in danger drives antipredator

behavior: The predation risk allocation hypothesis. The American Naturalist

153(6):649-659.

Loewen LJ, Suedfeld P. 1992. Cognitive and arousal effects of masking office noise.

Environment and Behavior 24(3):381-395.

Lutz C, Well A, Novak M. 2003. Stereotypic and self‐injurious behavior in rhesus

macaques: A survey and retrospective analysis of environment and early

experience. American Journal of Primatology 60(1):1-15.

Lutz CK, Novak MA. 2005. Environmental enrichment for nonhuman primates: Theory

and application. ILAR Journal 46(2):178-191.

Maestripieri D, Schino G, Aureli F, Troisi A. 1992. A modest proposal: Displacement

activities as an indicator of emotions in primates. Animal Behaviour 44(5):967-

979.

Markowitz H, Aday C, Gavazzi A. 1995. Effectiveness of acoustic “prey”:

Environmental enrichment for a captive African leopard (Panthera pardus). Zoo

Biology 14(4):371-379.

Markowitz H, Line S. 1989. Primate research models and environmental enrichment. In:

Segal E, editor. Housing, Care and Psychological Wellbeing of Captive and

Laboratory Primates. New Jersey, US: Noyes Publications.

Martin PR, Bateson PPG. 2007. Measuring Behaviour: An Introductory Guide.

Cambridge, UK: Cambridge University Press.

Mason G, Mendl M. 1993. Why is there no simple way of measuring animal welfare?

Animal Welfare 2(4):301-319.

159

McCallister JM, Smith TE, Elwood RW. 2004. Validation of urinary cortisol as an

indicator of hypothalamic-pituitary-adrenal function in the bearded emperor

tamarin (Saguinus imperator subgrisescens). American Journal of Primatology

63(1):17-23.

McDermott J, Hauser M. 2004. Are consonant intervals music to their ears? Spontaneous

acoustic preferences in a nonhuman primate. Cognition 94(2):B11-B21.

McDermott J, Hauser M. 2005. Probing the evolutionary origins of music perception.

Annals of the New York Academy of Sciences 1060:6.

McDermott J, Hauser MD. 2007. Nonhuman primates prefer slow tempos but dislike

music overall. Cognition 104(3):654-668.

Melfi VA, McCormick W, Gibbs A. 2004. A preliminary assessment of how zoo visitors

evaluate animal welfare according to enclosure style and the expression of

behavior. Anthrozoos: A Multidisciplinary Journal of The Interactions of People

& Animals 17(2):98-108.

Mendl M, Burman OH, Parker RM, Paul ES. 2009. Cognitive bias as an indicator of

animal emotion and welfare: Emerging evidence and underlying mechanisms.

Applied Animal Behaviour Science 118(3):161-181.

Mendl M, Burman OH, Paul ES. 2010. An integrative and functional framework for the

study of animal emotion and mood. Proceedings of the Royal Society B:

Biological Sciences 277(1696):2895-2904.

Mizoguchi K, Ishige A, Aburada M, Tabira T. 2003. Chronic stress attenuates

glucocorticoid negative feedback: involvement of the prefrontal cortex and

hippocampus. Neuroscience 119(3):887-897.

160

Moberg GP, Mench JA. 2000. The biology of animal stress: Basic principles and

implications for animal welfare. New York, NY: CABI.

Morgan KN, Tromborg CT. 2007. Sources of stress in captivity. Applied Animal

Behaviour Science 102(3):262-302.

Mormède P, Andanson S, Aupérin B, Beerda B, Guémené D, Malmkvist J, Manteca X,

Manteuffel G, Prunet P, van Reenen CG. 2007. Exploration of the hypothalamic–

pituitary–adrenal function as a tool to evaluate animal welfare. Physiology &

behavior 92(3):317-339.

Möstl E, Palme R. 2002. Hormones as indicators of stress. Domestic Animal

Endocrinology 23(1–2):67-74.

Murray CM, Heintz MR, Lonsdorf EV, Parr LA, Santymire RM. 2013. Validation of a

field technique and characterization of fecal glucocorticoid metabolite analysis in

wild chimpanzees (Pan troglodytes). American Journal of Primatology 75(1):57-

64.

Nagel T. 1974. What is it like to be a bat? The Philosophical Review:435-450.

Newberry RC. 1995. Environmental enrichment: Increasing the biological relevance of

captive environments. Applied Animal Behaviour Science 44(2):229-243.

Nilsson ME, Alvarsson J, Rådsten-Ekman M, Bolin K. 2010. Auditory masking of

wanted and unwanted sounds in a city park. Noise Control Engineering Journal

58(5):524-531.

Novak MA, Drewsen KH. 1989. Enriching the lives of captive primates: Issues and

problems. In: Segal E, editor. Housing, Care and Psychological Wellbeing of

Captive and Laboratory Primates. New Jersey, US: Noyes Publications.

161

O'Neill P. 1989. A room with a view for captive primates: Issues, goals, related research

and strategies. In: Segal E, editor. Housing, Care, and Psychological Well-being

of Captive and Laboratory Primates. New Jersey: Noyes Publications.

Ogden JJ, Lindburg DG, Maple TL. 1994. A preliminary study of the effects of

ecologically relevant sounds on the behaviour of captive lowland gorillas.

Applied Animal Behaviour Science 39(2):163-176.

Owen MA, Hall S, Bryant L, Swaisgood RR. 2014. The influence of ambient noise on

maternal behavior in a Bornean sun bear (Helarctos malayanus euryspilus). Zoo

Biology 33(1):49-53.

Owen MA, Swaisgood RR, Czekala NM, Lindburg DG. 2005. Enclosure choice and

well‐being in giant pandas: Is it all about control? Zoo Biology 24(5):475-481.

Owen MA, Swaisgood RR, Czekala NM, Steinman K, Lindburg DG. 2004. Monitoring

stress in captive giant pandas (Ailuropoda melanoleuca): Behavioral and

hormonal responses to ambient noise. Zoo Biology 23(2):147-164.

Palakanis KC, DeNobile JW, Sweeney WB, Blankenship CL. 1994. Effect of music

therapy on state anxiety in patients undergoing flexible sigmoidoscopy. Diseases

of the Colon and Rectum 37(5):478-481.

Papoutsoglou S, Karakatsouli N, Batzina A, Papoutsoglou E, Tsopelakos A. 2008. Effect

of music stimulus on gilthead seabream Sparus aurata physiology under different

light intensity in a re‐circulating water system. Journal of Fish Biology 73(4):980-

1004.

Papoutsoglou S, Karakatsouli N, Louizos E, Chadio S, Kalogiannis D, Dalla C, Polissidis

A, Papadopoulou-Daifoti Z. 2007. Effect of Mozart's music (Romanze-Andante

162

of “Eine Kleine Nacht Musik”, sol major, K525) stimulus on common carp

(Cyprinus carpio L.) physiology under different light conditions. Aquacultural

Engineering 36(1):61-72.

Papoutsoglou SE, Karakatsouli N, Papoutsoglou ES, Vasilikos G. 2010. Common carp

(Cyprinus carpio) response to two pieces of music (“Eine Kleine Nachtmusik”

and “Romanza”) combined with light intensity, using recirculating water system.

Fish physiology and biochemistry 36(3):539-554.

Passamani M, Rylands AB. 2000. Feeding behavior of Geoffroy's marmoset (Callithrix

geoffroyi) in an Atlantic forest fragment of south-eastern Brazil. Primates

41(1):27-38.

Pater LL, Grubb TG, Delaney DK. 2009. Recommendations for improved assessment of

noise impacts on wildlife. The Journal of Wildlife Management 73(5):788-795.

Patricelli GL, Blickley JL. 2006. Avian communication in urban noise: Causes and

consequences of vocal adjustment. The Auk 123(3):639-649.

Patterson-Kane EG, Farnworth MJ. 2006. Noise exposure, music, and animals in the

laboratory: A commentary based on laboratory animal refinement and enrichment

forum (LAREF) discussions. Journal of Applied Animal Welfare Science

9(4):327-332.

Paul ES, Harding EJ, Mendl M. 2005. Measuring emotional processes in animals: The

utility of a cognitive approach. Neuroscience and Biobehavioral Reviews

29(3):469-491.

163

Peretz I. 2001. Listen to the brain: A biological perspective on musical emotions. In:

Juslin PN, Sloboda JA, editors. Music and Emotion: Theory and Research. New

York, NY: Oxford University Press. p 105-134.

Pines M, Kaplan G, Rogers L. 2004. Stressors of common marmosets (Callithrix jacchus)

in the captive environment: Effects on behaviour and cortisol levels. Folia

Primatologica 75(Suppl. 1):317-318.

Platt ML, Brannon EM, Briese TL, French JA. 1996. Differences in feeding ecology

predict differences in performance between golden lion tamarins (Leontopithecus

rosalia) and Wied’s marmosets (Callithrix kuhli) on spatial and visual memory

tasks. Animal Learning & Behavior 24(4):384-393.

Porsolt RD, Le Pichon M, Jalfre M. 1977. Depression: A new animal model sensitive to

antidepressant treatments. Nature 266:730-732.

Powell DM, Carlstead K, Tarou LR, Brown JL, Monfort SL. 2006. Effects of

construction noise on behavior and cortisol levels in a pair of captive giant pandas

(Ailuropoda melanoleuca). Zoo Biology 25(5):391-408.

Power ML, Oftedal OT. 1996. Differences among captive callitrichids in the digestive

responses to dietary gum. American Journal of Primatology 40(2):131-144.

Quadros S, Goulart VD, Passos L, Vecci MA, Young RJ. 2014. Zoo visitor effect on

mammal behaviour: Does noise matter? Applied Animal Behaviour Science

156:78-84.

Quinn JL, Whittingham MJ, Butler SJ, Cresswell W. 2006. Noise, predation risk

compensation and vigilance in the chaffinch Fringilla coelebs. Journal of Avian

biology 37(6):601-608.

164

Rabin LA, Coss RG, Owings DH. 2006. The effects of wind turbines on antipredator

behavior in California ground squirrels (Spermophilus beecheyi). Biological

Conservation 131(3):410-420.

Raichel DR. 2006. The Science and Applications of Acoustics. New York, NY: Springer.

Raminelli JLF, Sousa MBC, Cunha MS, Barbosa MFV. 2001. Morning and afternoon

patterns of fecal cortisol excretion among reproductive and non-reproductive male

and female common marmosets, Callithrix jacchus. Biological Rhythm Research

32(2):159-167.

Rangel‐Negrín A, Alfaro J, Valdez R, Romano M, Serio‐Silva J. 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.

Rimbach R, Heymann EW, Link A, Heistermann M. 2013. Validation of an enzyme

immunoassay for assessing adrenocortical activity and evaluation of factors that

affect levels of fecal glucocorticoid metabolites in two New World primates.

General and Comparative Endocrinology 191(0):13-23.

Robbins L, Margulis SW. 2014. The effects of auditory enrichment on gorillas. Zoo

Biology 33(3):197-203.

Roberts M, Magnusson B, Burczynski F, Weiss M. 2002. Enterohepatic circulation:

Physiological, pharmacokinetic, and clinical implications. Clin Pharmacokinet

41(10):751-790.

Rog JE, Lukas KE, Wark JD. In review. Social and environmental influences on pacing

in a female Malayan sun bear (Helarctos malayanus).

165

Ross SR. 2006. Issues of choice and control in the behaviour of a pair of captive polar

bears (Ursus maritimus). Behavioural Processes 73(1):117-120.

Roy M, Mailhot J-P, Gosselin N, Paquette S, Peretz I. 2009. Modulation of the startle

reflex by pleasant and unpleasant music. International Journal of

Psychophysiology 71(1):37-42.

Ruivo EB, editor. 2010. EAZA husbandry guidelines for the Callitrichidae. Beauval,

France: ZooParc de Beauval.

Rukstalis M, French JA. 2005. Vocal buffering of the stress response: exposure to

conspecific vocalizations moderates urinary cortisol excretion in isolated

marmosets. Hormones and Behavior 47(1):1-7.

Russell JA. 1980. A circumplex model of affect. Journal of Personality and Social

Psychology 39(6):1161.

Savastano G, Hanson A, McCann C. 2003. The development of an operant conditioning

training program for New World primates at the Bronx Zoo. Journal of Applied

Animal Welfare Science 6(3):247-261.

Schapiro SJ, Bloomsmith MA. 1995. Behavioral effects of enrichment on singly‐housed,

yearling rhesus monkeys: An analysis including three enrichment conditions and a

control group. American Journal of Primatology 35(2):89-101.

Schneider H, Sampaio I. 2015. The systematics and evolution of New World primates–A

review. Molecular Phylogenetics and Evolution 82:348-357.

Schroeder J, Nakagawa S, Cleasby IR, Burke T. 2012. Passerine birds breeding under

chronic noise experience reduced fitness. PLoS One 7(7):e39200.

166

Schwarzenberger F. 2007. The many uses of non-invasive faecal steroid monitoring in

zoo and wildlife species. International Zoo Yearbook 41(1):52-74.

Searcy YM, Caine NG. 2003. Hawk calls elicit alarm and defensive reactions in captive

Geoffroy's marmosets (Callithrix geoffroyi). Folia Primatologica 74(3):115-125.

Setchell JM, Smith T, Wickings EJ, Knapp LA. 2008. Factors affecting fecal

glucocorticoid levels in semi‐free‐ranging female mandrills (Mandrillus sphinx).

American Journal of Primatology 70(11):1023-1032.

Shepherdson D, Bemmet N, Carman M, Reynolds S. 1989. Auditory enrichment for Lar

gibbons Hylobates lar at London Zoo. International Zoo Yearbook 28(1):256-

260.

Sherwen SL, Magrath MJ, Butler KL, Phillips CJ, Hemsworth PH. 2014. A multi-

enclosure study investigating the behavioural response of meerkats to zoo visitors.

Applied Animal Behaviour Science 156:70-77.

Siemers BM, Schaub A. 2011. Hunting at the highway: Traffic noise reduces foraging

efficiency in acoustic predators. Proceedings of the Royal Society B: Biological

Sciences 278:1646-1652.

Simpson L. 2004. The effect of visitors on captive non-human primates. Zoo Federation

Research Newsletter 5(3):6.

Slabbekoorn H, Ripmeester EA. 2008. Birdsong and anthropogenic noise: Implications

and applications for conservation. Molecular Ecology 17(1):72-83.

Smith AS, Birnie AK, French JA. 2011. Social isolation affects partner-directed social

behavior and cortisol during pair formation in marmosets, Callithrix geoffroyi.

Physiology & Behavior 104(5):955-961.

167

Smith TE, French JA. 1997. Psychosocial stress and urinary cortisol excretion in

marmoset monkeys. Physiology & Behavior 62(2):225-232.

Snowdon CT. 1993. A vocal taxonomy of the callitrichids. In: Rylands AB, editor.

Marmosets and Tamarins: Systematics, Behaviour and Ecology. Oxford, UK:

Oxford University Press. p 78-93.

Snowdon CT, Teie D. 2010. Affective responses in tamarins elicited by species-specific

music. Biology Letters 6(1):30-32.

Snowdon CT, Teie D. 2013. Emotional communication in monkeys: Music to their ears?

In: Altenmuller E, Schmidt S, Zimmermann E, editors. The Evolution of

Emotional Communication: From Sounds in Nonhuman Mammals to Speech and

Music in Man. Oxford, UK: Oxford University Press. p 133-151.

Snowdon CT, Teie D, Savage M. 2015. Cats prefer species-appropriate music. Applied

Animal Behaviour Science 166:106-111.

Sousa MBC, Ziegler TE. 1998. Diurnal variation on the excretion patterns of fecal

steroids in common marmoset (Callithrix jacchus) females. American Journal of

Primatology 46(2):105-117.

Stevenson MF, Poole TB. 1976. An ethogram of the common marmoset (Calithrix

jacchus jacchus): General behavioural repertoire. Animal Behaviour 24(2):428-

451.

Suied C, Susini P, McAdams S. 2008. Evaluating warning sound urgency with reaction

times. Journal of Experimental Psychology: Applied 14(3):201-212.

168

Sulser C, Steck B, Baur B. 2008. Effects of construction noise on behaviour of and

exhibit use by Snow leopards Uncia uncia at Basel zoo. International Zoo

Yearbook 42(1):199-205.

Swaisgood RR, White AM, Zhou X, Zhang H, Zhang G, Wei R, Hare VJ, Tepper EM,

Lindburg DG. 2001. A quantitative assessment of the efficacy of an

environmental enrichment programme for giant pandas. Animal Behaviour

61(2):447-457.

Touma C, Palme R. 2005. Measuring fecal glucocorticoid metabolites in mammals and

birds: The importance of validation. Annals of the New York Academy of

Sciences 1046(1):54-74.

Trehub SE, Hannon EE. 2006. Infant music perception: Domain-general or domain-

specific mechanisms? Cognition 100(1):73-99.

Tromborg CT. 1993. Behavioral effects of exposing captive cotton-top tamarins to

controlled auditory stimuli (Unpublished master's thesis). San Francisco, CA: San

Francisco State University.

Tromborg CT. 1999. Figure and Ground Squirrels: Acoustic environments and their

influence on the behavior of California ground squirrels (Unpublished doctoral

dissertation): University of California, Davis.

Tromborg CT, Coss RC. Denizens, decibels, and dens; 1995; Seattle, WA. p 521-528.

Uetake K, Hurnik J, Johnson L. 1997. Effect of music on voluntary approach of dairy

cows to an automatic milking system. Applied Animal Behaviour Science

53(3):175-182.

169

Ulrich RS, Simons RF, Losito BD, Fiorito E, Miles MA, Zelson M. 1991. Stress recovery

during exposure to natural and urban environments. Journal of Environmental

Psychology 11(3):201-230.

Updike PA, Charles DM. 1987. Music Rx: Physiological and emotional responses to

taped music programs of preoperative patients awaiting plastic surgery. Annals of

Plastic Surgery 19(1):29-33.

Videan EN, Fritz J, Howell S, Murphy J. 2007. Effects of two types and two genre of

music on social behavior in captive chimpanzees (Pan troglodytes). Journal of the

American Association for Laboratory Animal Science 46(1):66-70.

Vrana SR, Spence EL, Lang PJ. 1988. The startle probe response: A new measure of

emotion? Journal of abnormal psychology 97(4):487.

Wallace EK, Kingston‐Jones M, Ford M, Semple S. 2013. An investigation into the use

of music as potential auditory enrichment for moloch gibbons (Hylobates

moloch). Zoo Biology 32(4):423-426.

Waser PM, Brown CH. 1986. Habitat acoustics and primate communication. American

Journal of Primatology 10(2):135-154.

Wasser SK, Hunt KE, Brown JL, Cooper K, Crockett CM, Bechert U, Millspaugh JJ,

Larson S, Monfort SL. 2000. A generalized fecal glucocorticoid assay for use in a

diverse array of nondomestic mammalian and avian species. General and

Comparative Endocrinology 120(3):260-275.

Watanabe S, Nemoto M. 1998. Reinforcing property of music in Java sparrows (Padda

oryzivora). Behavioural Processes 43(2):211-218.

170

Weingrill T, Gray DA, Barrett L, Henzi SP. 2004. Fecal cortisol levels in free-ranging

female chacma baboons: Relationship to dominance, reproductive state and

environmental factors. Hormones and Behavior 45(4):259-269.

Wells D, Graham L, Hepper P. 2002. The influence of auditory stimulation on the

behaviour of dogs housed in a rescue shelter. Animal Welfare 11(4):385-393.

Wells DL. 2009. Sensory stimulation as environmental enrichment for captive animals: A

review. Applied Animal Behaviour Science 118(1):1-11.

Wells DL, Coleman D, Challis MG. 2006. A note on the effect of auditory stimulation on

the behaviour and welfare of zoo-housed gorillas. Applied Animal Behaviour

Science 100(3):327-332.

Wells DL, Hepper PG, Coleman D, Challis MG. 2007. A note on the effect of olfactory

stimulation on the behaviour and welfare of zoo-housed gorillas. Applied Animal

Behaviour Science 106(1):155-160.

Wells DL, Irwin RM. 2008. Auditory stimulation as enrichment for zoo-housed Asian

elephants (Elephas maximus). Animal Welfare.

Westermann R, Spies K, Stahl G, Hesse FW. 1996. Relative effectiveness and validity of

mood induction procedures: A meta-analysis. European Journal of Social

Psychology 26:557-580.

Wormell D, Brayshaw M, Price E, Herron S. 1996. Pied tamarins Saguinus bicolor

bicolor at the Jersey Wildlife Preservation Trust: Management, behaviour and

reproduction. Dodo-Journal of the Wildlife Preservation Trusts 32:76-97.

171

Wright AA, Rivera JJ, Hulse SH, Shyan M, Neiworth JJ. 2000. Music perception and

octave generalization in rhesus monkeys. Journal of Experimental Psychology:

General 129(3):291.

Wright AJ, Soto NA, Baldwin AL, Bateson M, Beale CM, Clark C, Deak T, Edwards EF,

Fernández A, Godinho A. 2007. Anthropogenic noise as a stressor in animals: a

multidisciplinary perspective. International Journal of Comparative Psychology

20(2).

Yeates J, Main D. 2008. Assessment of positive welfare: A review. The Veterinary

Journal 175(3):293-300.

Young KM, Walker SL, Lanthier C, Waddell WT, Monfort SL, Brown JL. 2004.

Noninvasive monitoring of adrenocortical activity in carnivores by fecal

glucocorticoid analyses. General and Comparative Endocrinology 137(2):148-

165.

Zahed SR, Prudom SL, Snowdon CT, Ziegler TE. 2008. Male parenting and response to

infant stimuli in the common marmoset (Callithrix jacchus). American Journal of

Primatology 70(1):84-92.

Ziegler TE, Scheffler G, Snowdon CT. 1995. The relationship of cortisol levels to social

environment and reproductive functioning in female cotton-top tamarins,

Saguinus oedipus. Hormones and Behavior 29(3):407-424.

Ziegler TE, Sousa MBC. 2002. Parent–daughter relationships and social controls on

fertility in female common marmosets, Callithrix jacchus. Hormones and

Behavior 42(3):356-367.

172

Ziegler TE, Wegner FH, Snowdon CT. 1996. Hormonal responses to parental and

nonparental conditions in male cotton-top tamarins, Saguinus oedipus, a New

World primate. Hormones and Behavior 30(3):287-297.