Lighting and Nocturnal Strepsirrhine Care in Zoos

Home , Angwantibo, Cathemerality, Colugo, Diurnality, Loris, Lorisidae

THE NIGHT SHIFT: LIGHTING AND NOCTURNAL STREPSIRRHINE CARE IN ZOOS

GRACE FULLER

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

January 2014 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of Grace Fuller, candidate for the Doctor of Philosophy degree*.

Signed:

______Kristen E. Lukas, Ph.D. (chair of the committee)

______Mark Willis, Ph.D.

______Mary Ann Raghanti, Ph.D.

______Patricia M. Dennis, D.V.M., Ph.D.

______Christopher W. Kuhar, Ph.D.

Date: 10 October 2013

* We also certify that written approval has been obtained for any proprietary material contained within.

Dedicated to:

Rene Culler, my mother and my role model; my father Charles Fuller, who would have been so proud;

and all the big-eyed friends I made along the way.

TABLE OF CONTENTS

Acknowledgments vii Abstract xii Chapter 1. Introduction: Light, Activity Patterns, and the Exhibition of 1 Nocturnal Primates in Zoos Chapter 2. A Survey of Husbandry Practices for Lorisid Primates in 30 North American Zoos and Related Facilities Chapter 3. A Retrospective Review of Mortality in Lorises and Pottos 57 in North American Zoos, 1980-2010 Chapter 4. Validating Actigraphy for Circadian Monitoring of Behavior 90 in the Pygmy Loris (Nycticebus pygmaeus) and Potto (Perodicticus potto) Chapter 5. Methods for Measuring Salivary Melatonin in the Potto, 113 Perodicticus potto, and Pygmy Loris, Nycticebus pygmaeus Chapter 6. A Case Study Comparing Hormonal and Behavioral 135 Responses to Red and Blue Exhibit Lighting in the Aye-Aye, Daubentonia madagascariensis Chapter 7. A Comparison of Nocturnal Strepsirrhine Behavior in 148 Exhibits Illuminated with Red and Blue Light Chapter 8. Endocrine Responses to Exhibit Lighting in the Potto, 175 Perodicticus potto Chapter 9. General Discussion 195 Appendix I. Questions for Multi-Institutional Husbandry Survey 200 Appendix II. Detailed Tables for Cause of Death in Lorisid Primates by 202 Species and Age Group References 208

LIST OF TABLES

Chapter Two

Table 1. Group compositions of lorisid primates in North American facilities. 37

Table 2. Enclosure design features of lorisid primates in North American 40

facilities.

Table 3. Lighting design of the primary enclosure. 43

Table 4. Animal care practices. 45

Table 5. Estimated reproductive success of lorisid primates. 49

Chapter Three

Table 1. Recorded type of death for lorises and pottos in North American 63

facilities 1980-2010.

Table 2. Distribution of loris and potto deaths by age class in North American 64

facilities 1980-2010.

Table 3. Primary cause of death or reason for euthanasia in lorises and pottos 67

housed in North American facilities 1980-2010.

Table 4. Percent of lorises and pottos with pathology diagnosed by organ 69

system upon postmortem examination 1980-2010.

Table 5. Neoplasia reported for lorises and pottos in North American facilities 74

1980-2010.

Table 6. Circumstances surrounding traumas related to death in lorises and 80

pottos in North American zoos 1980-2010.

Chapter Four

Table 1. Ethogram for behavioral data collection for actigraph study. 99

Chapter Six

Table 1. Ethogram for behavioral data collection on the aye-aye. 138

Chapter Seven

Table 1. Nocturnal strepsirrhine subjects and housing conditions for the 154

multi-zoo study.

Table 2. Ethogram for behavioral data collection. 158

Chapter Eight

Table 1. Nocturnal strepsirrhine subjects and housing conditions for the 181

multi-zoo study.

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LIST OF FIGURES

Chapter Two

Figure 1. Age pyramids for lorisid primates in North American institutions. 38

Figure 2. Primary enclosure size for lorisid primates in North American 41

facilities.

Chapter Four

Figure 1 a-b. a) Attaching the actigraph harness to the pygmy loris subject; b) 95

the potto wears the actigraph harness on exhibit.

Figure 2. General activity budget for the potto subject with and without the 101

actigraph harness in place.

Figure 3. General activity budget for the pygmy loris subject with and without 103

the actigraph harness in place.

Figure 4. Actigraph data for the potto subject. 105

Figure 5. Actigraph data for the pygmy loris subject. 106

Figure 6. Mean activity counts associated with behaviors observed in a potto 107

and a pygmy loris.

Chapter Five

Figure 1. Collection of saliva from the female potto. 118

Figures 2 a-c. Melatonin concentrations measured following nocturnal 127

exposure to test lights in a potto (a), and pygmy lorises PSL1 (b) and

PSL2 (c).

Figure 3. 24-hour patterns of salivary melatonin expression in a potto and 130

three pygmy lorises (PSL).

Chapter Six

Figure 1. Daily time of emergence from the nest box by the aye-aye subject 140

based on keeper reports.

Figure 2. Time spent performing active behaviors (move, feed, self-directed, or 141

object examination) by the aye-aye subject during the baseline red

and experimental blue lighting conditions.

Figure 3. Dark phase activity budget (1000-2200 hrs) for the aye-aye subject 142

during the baseline red and experimental blue lighting conditions.

Figure 4. Dark phase salivary melatonin concentrations in the aye-aye during 144

the baseline red and experimental blue lighting conditions.

Figure 5. Salivary cortisol rhythms in the aye-aye during the baseline red and 146

experimental blue conditions.

Chapter Seven

Figure 1 a-b. Percent of time spent performing active behaviors during the dark 162

phase by animals at Cleveland Metroparks Zoo (a) and Cincinnati Zoo

and Botanical Garden (b).

Figure 2 a-d. Changes in behavior across the three study conditions (C1-3) 164

comparing red and blue light for pottos (a) and pygmy slow lorises

(b) at Cleveland Metroparks Zoo (CMZ) and pottos (c) and a bamboo

lemur (d) at Cincinnati Zoo and Botanical Garden (CZBG).

Figure 3 a-c. 24-hour activity level data for potto PP2 (a) and pygmy slow 167

lorises NP1 (b) and NP2 (c) (see Table 1 for subject IDs) at Cleveland

Metroparks Zoo.

Chapter Eight

Figure 1 a-c. Melatonin concentrations measured in potto saliva six hours after 185

dark phase onset in red and blue light.

Figure 2. Scatterplot of light intensity compared to salivary melatonin 186

concentrations measured in pottos at six hours after dark phase

onset.

Figure 3. Salivary cortisol concentrations measured in pottos living in blue and 188

red dark phase lighting conditions.

Acknowledgments

It is humbling to look back over these many years and contemplate how many people contributed to my development as a scientist as well as to the body of work I am offering here. First and foremost, I would like to thank my dissertation advisor, Dr. Kristen Lukas. Meeting Kristen inspired me to pursue a path of study examining the care of animals in captivity. Over the years she has been a mentor and a friend, and I am a better person and scientist for having known her.

All of my committee members have had a profound influence on how I see the world and have also been a source of great strength and support throughout the process of completing my dissertation research. I am grateful to Dr. Mary Ann

Raghanti for her years of friendship and encouragement, for believing in me, and always reminding me to think like an anthropologist. I am grateful to Dr. Pam

Dennis for pushing me to ask hard questions and for encouraging me to believe that

I could acquire the skills to answer them, and for always reminding me to stand up for the little guys. I am grateful to Dr. Chris Kuhar for pushing me to strive for both scientific rigor and practicality in my research endeavors. Not only did Dr. Mark

Willis teach me to think like a scientist, he also demonstrated what it means to be a great teacher both in and outside the classroom. Finally, Dr. Mandi Vick selflessly gave me so much guidance, personally and professionally, that I will always consider her an honorary part of my committee. I look forward to many years of friendship and collaboration with you all.

I am also grateful to my academic peers and friends for their support and encouragement over these many years. Dr. Elena Less was a source of inspiration to

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me for both her work ethic and her kindness, and I am so grateful for all the experiences we had together as students. Jason Wark made me laugh every day, and pushed me to think critically and have fun doing it. Christine Cassella was also a great source of friendship and support, and an inspiration for how to live a better life. I am incredibly grateful to Austin Leeds for his assistance in collecting saliva samples for this research, as I truly could not have done it without his help. Bonnie

Baird is going to do great things and I will remember that I knew her when. I look forward to many years of friendship and collaboration with you all as well, and I cannot wait to see all the amazing contributions that you make to our field.

Working in the Conservation and Science area at Cleveland Metroparks Zoo was amazing because literally everyone I worked with was wonderful. Laura

Amendolagine is a top-notch laboratory manager and therapist and has the biggest heart of anyone I know. I will always remember fondly our time working in the lab together. I am also grateful to Kym Gopp for her dedication to loris conservation and for offering me opportunities to contribute, in some small way, to this goal. Finally, I am grateful that I met Sonia DiFiore there too.

Of course there are many other staff members at the zoo that I would like to thank for their assistance with my research, especially Becky Johnson, Dawn Stone, and Andrew Smyser, who put in many, many hours of work to assist in my research efforts all while providing exceptional care to the lorises and many other animals. I am also grateful to Terri Rhyner, Heather Mock Strawn, and the other animal keepers in PCA for their dedication and hard work. At Cleveland Metroparks Zoo, I would also like to thank Linda DeHoff, Sharon Gehri, Andi Kornak, Pam Krentz, Dr.

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Albert Lewandowski, Tad Schoffner, and Dr. Mike Selig. Apologies to any one I have omitted—what really makes Cleveland Metroparks Zoo great is that all the staff are dedicated, good people.

I had a wonderful experience collecting data at Cincinnati Zoo and Botanical

Garden, in large part because the staff members were so welcoming and helpful. I am extremely grateful to Michael Guilfoyle for his support of my research project and for sharing with me his many years of knowledge about caring for pottos and other nocturnal mammals. I would also like to thank Matt Miller for his assistance with lighting changes and for creating a little lab for me in Jungle Trails. Finally, I would also like to thank Patrick Callahan, Mike Dulaney, Ron Evans, Valerie Haft,

Janet Hutson, Michael Land, Mike Maciariello, Kate MacKinnon, Dr. Terri Roth,

Stephanie Schuler, Vicki Ulrich, Amanda Weisel, and any other staff that assisted with this project.

I would like to thank Nicole Smith and Jeannine Jackle for coordinating sample collection from animals at Franklin Park Zoo. I had a great experience visiting the zoo and really enjoyed working with you both. Many thanks are also due to the other Tropical Forest staff members who assisted with this project.

There are many others who have contributed to my professional development and I would like to thank them here: Dr. Colleen McCann, Helena Fitch-

Snyder, Dr. Dean Gibson, Dr. Anna Nekaris, and Dr. Tara Stoinski. All of these brilliant women have been a great source of inspiration to me. Also a great supporter and inspiration was my master’s thesis advisor, Dr. Richard Feinberg.

I would like to express my gratitude to the following institutions for completing the husbandry survey: Akron Zoological Park, Albuquerque Biological

Park, Aquarium & Rainforest at Moody Gardens, The Calgary Zoo, Botanical Garden

& Prehistoric Park, Capron Park Zoo, Chicago Zoological Society- Brookfield Zoo,

Cincinnati Zoo & Botanical Garden, Cleveland Metroparks Zoo, Duke Lemur Center,

El Paso Zoo, Franklin Park Zoo, Houston Zoo, Inc., Lee Richardson Zoo, Lincoln Park

Zoo, Little Rock Zoo, Los Angeles Zoo and Botanical Gardens, Louisiana Purchase

Gardens & Zoo, Memphis Zoo, Mesker Park Zoo & Botanic Garden, Minnesota

Zoological Garden, Omaha’s Henry Doorly Zoo, The Philadelphia Zoo, Prospect Park

Zoo, Pueblo Zoo, San Diego Zoo, Trevor Zoo, Wildlife Conservation Society- Bronx

Zoo, Woodland Park Zoo, and Zoo de Granby.

I wish also to thank the following facilities for contributing medical records to this study: Albuquerque Biological Park, Aquarium & Rainforest at Moody

Gardens, The Calgary Zoo, Botanical Garden & Prehistoric Park, Chicago Zoological

Society- Brookfield Zoo, Cincinnati Zoo & Botanical Garden, Cleveland Metroparks

Zoo, Columbus Zoo and Aquarium, Denver Zoo, Detroit Zoo, Duke Lemur Center, El

Paso Zoo, Franklin Park Zoo, Houston Zoo, Inc., Lake Superior Zoological Gardens,

Lee Richardson Zoo, Lincoln Park Zoo, Los Angeles Zoo and Botanical Gardens, The

Maryland Zoo in Baltimore, Memphis Zoo, Mesker Park Zoo & Botanic Garden,

Minnesota Zoological Garden, Oglebay’s Good Zoo, Omaha’s Henry Doorly Zoo, The

Philadelphia Zoo, San Antonio Zoo, San Diego Zoo and San Diego Wild Animal Park,

San Francisco Zoo, Santa Ana Zoo, Trevor Zoo, Virginia Zoological Park, Wildlife

Conservation Society- Bronx Zoo, and Woodland Park Zoo. Several individuals went

above and beyond to accommodate this request, including Julie Parks Taylor, Jenn

Harrison, Jeannine Jackle, Andrea Katz, and Mark Campbell. I am also grateful to the many volunteers who assisted with entering medical data, including Gail Simpson,

Jessica Taylor, Alyssa Mills, and Lauren Starkey.

I am grateful to all the lorises and pottos, the big-eyed friends we met and sometimes lost along the way. I will never forget you: Vijay, Sweetie Pie, Fozzy, Moe,

Nox, Harry, Hermione, Sing, Tai Sanlo, Philbert, Nicole, Ringo, Jahzira, Caliban, Mojo,

Chachi, Rendille, Lil Bit, Jabari, Tiombe, Lucy, Jabari, Amare, and Gabriel. You touched more lives than you could ever know.

Finally, to friends and family, both those lost and those whose company I still enjoy, I offer my love and gratitude. Peace.

The Night Shift: Lighting and Nocturnal Strepsirrhine Care in Zoos

GRACE FULLER

Abstract

Over billions of years of evolution, light from the sun, moon, and stars has provided organisms with reliable information about the passage of time. Photic cues entrain the circadian system, allowing animals to perform behaviors critical for survival and reproduction at optimal times. Modern artificial lighting has drastically altered environmental light cues. Evidence is accumulating that exposure to light at night

(particularly blue wavelengths) from computer screens, urban light pollution, or as an occupational hazard of night-shift work has major implications for human health.

Nocturnal animals are the shift workers of zoos; they are generally housed on reversed light cycles so that daytime visitors can observe their active behaviors. As a result, they are exposed to artificial light throughout their subjective night. The goal of this investigation was to examine critically the care of nocturnal strepsirrhine primates in North American zoos, focusing on lorises (Loris and Nycticebus spp.) and pottos (Perodicticus potto). The general hypothesis was that exhibit lighting design affects activity patterns and circadian physiology in nocturnal strepsirrhines. The first specific aim was to assess the status of these populations. A multi-institutional husbandry survey revealed little consensus among zoos in lighting design, with both red and blue light commonly used for nocturnal illumination. A review of medical

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records also revealed high rates of neonate mortality. The second aim was to develop methods for measuring the effects of exhibit lighting on behavior and health. The use of actigraphy for automated activity monitoring was explored.

Methods were also developed for measuring salivary melatonin and cortisol as indicators of circadian disruption. Finally, a multi-institutional study was conducted comparing behavioral and endocrine responses to red and blue dark phase lighting.

These results showed greater activity levels in strepsirrhines housed under red light than blue. Salivary melatonin concentrations in pottos suggested that blue light suppressed nocturnal melatonin production at higher intensities, but evidence for circadian disruption was equivocal. These results add to the growing body of evidence on the detrimental effects of blue light at night and are a step towards empirical recommendations for nocturnal lighting design in zoos.

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Chapter One

Introduction: Light, Activity Patterns, and the Exhibition of Nocturnal Primates in Zoos

Nocturnal species make up more than half of the world’s land-dwelling vertebrates but often are cryptic and difficult to observe in the wild (Conway, 1969).

Zoos provide an opportunity for close encounters with nocturnal animals, but their care in captivity poses unique challenges. Nocturnal species are generally housed on reversed light cycles in zoos. Artificial lighting is designed to simulate nighttime conditions in exhibits during the day so that nocturnal animals are active when zoo visitors and staff can observe them. Lighting in nocturnal exhibits must be carefully designed to entrain the animal’s circadian system to the reversed light cycle, providing enough light for zoo visitors to view nocturnal species without overwhelming the animals’ sensitive visual systems or suppressing their activity

(Erkert, 1989). This perceptual tug-of-war may have negative effects on the behavior, health, and reproduction of nocturnal species in zoo environments.

Prior to the advent of artificial lighting, light from the environment provided reliable information about the time of day or year to physiological systems regulating animal behavior, allowing animals to maximize reproductive success by timing behaviors like foraging and sleeping to appropriate environmental conditions (Ashby, 1972; Halle and Stenseth, 2000b). It is now becoming clear that artificial lighting can disrupt internal timekeeping systems, adversely affecting health, reproduction, and behavior in humans and other animals (Navara and

Nelson, 2007; Rea et al., 2008). These effects are dramatic enough that the World

Health Organization categorizes night-shift work as a possible carcinogen (Straif et al., 2007). Nocturnal animals are the shift workers of zoos, and the means and effects of altering their activity patterns in the captive setting are in need of systematic study.

The goal of these studies was to examine current husbandry practices for nocturnal strepsirrhines in zoos, focusing on members of the primate family

Lorisidae (lorises and pottos), and the behavioral and physiological impacts of exhibit lighting. The general hypothesis was that lighting designs for nocturnal exhibits vary among North American zoos, and these differences have implications for the behavior, circadian rhythms, and health of captive nocturnal strepsirrhines.

NOCTURNALITY IN THE PRIMATE ORDER

The majority of primates are diurnal, with only one nocturnal haplorhine genus (Aotus) and a preponderance of nocturnal forms among the strepsirrhines

(Ankel-Simons and Rasmussen, 2008). However, this simple dichotomy between nocturnal and diurnal forms based along taxonomic lines has been repeatedly challenged by field observations of primates engaged in active behaviors outside their presumed temporal niche (Ankel-Simons and Rasmussen, 2008; Curtis and

Rasmussen, 2006). Many lemur species may be instead termed ‘cathemeral’, meaning that they are active opportunistically during both day and night (Curtis and

Rasmussen, 2006). Many now believe that primate activity patterns and their associated visual adaptations are evolutionarily flexible, allowing species to exploit different temporal niches depending on local conditions (Ankel-Simons and

Rasmussen, 2008; Pariente, 1979). This new understanding of primate activity patterns casts doubt on the assumption based on fossil evidence that the earliest primates were nocturnal (Ankel-Simons and Rasmussen, 2008).

Activity in darkness exposes animals to predators and other dangers

(Bearder et al., 2002) and there are several hypotheses to explain the evolutionary benefits of a nocturnal activity pattern (Curtis and Rasmussen, 2006). Nocturnality allows animals to partition environments temporally, minimizing competition for resources. Charles-Dominique (1975) suggests that in tropical environments, nocturnal primates benefit by reduced feeding competition from birds at fruiting trees. Nocturnal species may also benefit by using the cover of night to surreptitiously hunt prey, or as a means of avoiding predation (Bearder et al., 2002).

Restricting activity to nighttime can also aid in thermoregulation by avoiding hotter times of the day (Curtis and Rasmussen, 2006; Fernandez-Duque, 2003).

Decreased visual information available in darkness has led to a suite of perceptual adaptations that allow nocturnal primates to gather information from the environment using other sensory systems, leading to a great emphasis on chemical (Delbarco-Trillo et al., 2011) and auditory signals (Charles-Dominique,

1975). Visual adaptations that make use of the little light available at night are also common.

Nocturnal Primate Visual Systems

Given the degree of flexibility in primate activity patterns, it is perhaps not surprising that primate visual systems are highly variable as well and do not

separate clearly into diurnal and nocturnal groups. Many nocturnal primates possess morphological features that confer improved vision at night, such as large eyes (Ross and Kirk, 2007). Nocturnal species often have greater numbers of rods

(sensitive to brightness) relative to cones (sensitive to color) in the retina relative to diurnal species (Silveira, 2004). For example, humans have an estimated 5 or 6 cones for every 100 rods in the retina, while in the potto this ratio is 1 to 300

(Goffart et al., 1976). Many species such as the potto (Goffart et al., 1976) also possess a tapetum lucidum, a reflective layer of cells behind the retina that serves to amplify ambient light. However, a more nuanced examination reveals that variation is the norm for these features. Eye size does not correlate reliably with orbital aperture and among the nocturnal primates, only predatory species (such as Loris and Tarsius) have exceptionally large eyes compared to diurnal species of the same body size (Kirk, 2006). Even the presence of a tapetum lucidum does not correlate reliably with activity pattern among primates (Ankel-Simons and Rasmussen,

2008). Thus, one must take care in making assumptions about species activity patterns based on morphological evidence alone.

Color vision is also highly variable in the primate lineage, as Ankel-Simons and Rasumussen (2008) explain. Photosensitive cells in the retina are divided into two classes: rods, which are specialized for vision under dark or ‘scotopic’ conditions; and cones which are active under bright or ‘photopic’ conditions and are specialized for visual acuity and color perception. Rod cells contain a single (RH1) opsin (protein that absorbs light), meaning that vision under scotopic conditions is essentially monochromatic and color blind. The extent to which members of a given

species perceive color depends on which genes that code for cone opsins are present. Most mammals are functionally dichromatic, possessing the short wavelength sensitive type one (SWS1) cone opsin and the middle to long wavelength (M/LWS) cone opsin. Additionally, many haplorhine primates have evolved trichromatic vision by diversification of the M/LWS gene (Kawamura and

Kubotera, 2004). Each type of cone responds maximally to different wavelengths of light, and the peak sensitivity of a given opsin varies among different species as well

(Melin et al., 2012).

The extent and nature of color vision varies among strepsirrhine primates.

Many nocturnal lemurs, including mouse lemurs (Microcebus spp.) and aye-ayes

(Daubentonia madagascariensis) retain the SWS1 pigment and are functionally dichromatic. Melin et al. (2012) propose that this locus is under active selection in the aye-aye, which is often active during the blue-hued twilight and for whom the color blue may play an important role in foraging and social communication. The cathemeral brown lemur (Eulemur fulvus) retains the SWS1 gene as well

(Kawamura and Kubotera, 2004) and trichomatic color vision has evolved among several diurnal lemurs (Tan and Li, 1999). Lorisiform primates express only a single

M/LWS opsin and are therefore monochromatic (Deegan and Jacobs, 1996). It appears that the common ancestor of lorises and galagos lost functionality of the

SWS1 opsin gene and as a result these species are truly color-blind (Kawamura and

Kubotera, 2004).

So how do nocturnal primates see the world? All primates have high visual acuity, even nocturnal species that lack a fovea, the central cluster of photoreceptors

where visual acuity is greatest (Pariente, 1979). Vision also plays an important role in nocturnal primate orientation and communication behaviors (Bearder et al.,

2006; Pariente, 1979). Nocturnal primates are able to navigate complex forest environments easily in what humans perceive as almost complete darkness. Yet, many nocturnal species have the visual flexibility to engage in daytime activity, and a surprising number of nocturnal species appear to be capable of at least some degree of color vision (Melin et al., 2012). This high level of flexibility and variability among species has important implications for the care of nocturnal primates in zoos.

Lighting designs in enclosures are likely to be perceived very differently based on the visual capabilities of the species in question, and some species may adapt more readily to artificial changes in the light environment than others.

Natural History of Lorisid Primates

The infraorder Lorisiformes includes two families, the Lorisidae and the

Galagonidae (Groves, 2001). The Galagonidae consists of bushbabies or galago species, nocturnal primates which may be found living sympatrically with African lorisines but who exhibit a vertical clinging and leaping locomotor style that contrasts sharply with the slow, quadrupedal climbing of the Lorisidae (Bearder,

1999). The taxonomic diversity of galagos is relatively well described in part due to the conspicuous, species-specific vocalization patterns they exhibit - unlike the quiet, cryptic lorises (Grubb et al., 2003). Groves (2001) further divides the

Lorisidae into two subfamilies: the Asiatic Lorisinae (slow and slender lorises) and the African Perodicticinae (pottos and angwantibos).

The taxonomic diversity of lorisines has not yet been fully described. With

Nycticebus menagensis, N. bancanus, and N. borneanus, the newly named N. kayan makes four slow loris species recognized in Borneo alone (Munds et al., 2012). In addition to the pygmy loris, N. pygmaeus, three additional slow loris species are recognized: N. coucang, N. bengalensis, and N. javanicus (Nekaris et al., 2008). The pygmy loris’s range extends into southern China, and the other slow lorises are found across continental Asia. Slender lorises are found only in India and Sri Lanka and include at least two species, the red (Loris tardigradus) and grey (L. lydekkerianus) slender lorises (Brandon-Jones et al., 2004).

The taxonomy of the perodicticines is even less developed than that of lorises. As Grubb et al. (2003) explain, only two genera are recognized currently:

Perodicticus, the potto, and the golden potto or angwantibo, genus Arctocebus. Two species of angwantibo are recognized, the Calabar (A. calabarensis) and the golden anwangtibo (A. aureus). Although only a single potto species (P. potto) is currently recognized, the true diversity of this group has yet to be described. Pottos are found from the west coast of Africa east into Kenya, and the three known subspecies are divided geographically into the western (P. p. potto), central (P. p. edwarsi) and eastern (P. p. ibeanus) pottos. In discussing the natural history of lorises and pottos, it is important to keep in mind that their true behavioral variation, like their true taxonomic breadth, has yet to be fully delineated.

Lorises and pottos are found in a variety of forested habitats ranging from primary rainforest, to evergreen and bamboo forests, and agroforests (Streicher,

2004). All lorisines are nocturnal foragers that traverse their environments by

climbing along tree branches and lianas (Bearder, 1987). Their muscular limbs and strong grip allow for careful exploration of the environment, and their slow, deliberate movements may be an adaptation to avoid predator detection (Charles-

Dominique, 1977). However, it should be noted that rapid arboreal locomotion has been observed in pygmy (Duckworth, 1994) and slender lorises (Nekaris and

Stevens, 2007).

Charles-Dominique (1977) likens the niche of the potto in Africa to the slow loris in Asia, and that of the more faunivorous angwantibo to the slender loris.

Pottos are known to consume exudates, fruit, and insects (Oates, 1984). Fruit appears to play a larger role in the potto diet than that of slow lorises

(Gonzalezkirchner, 1995), which are dedicated exudativores that actively gouge trees to elicit gums and saps. In Cambodia, the pygmy loris primarily consumes exudates, followed by fruits and arthropods (Starr and Nekaris, 2013). The Bengal slow loris (N. bengalensis) feeds almost exclusively on gums during the winter, suggesting that exudates may be a particularly important resources for lorises occupying highly seasonal environments (Swapna et al., 2010). The diet of the slender loris is more faunivorous, and the Mysore slender loris (L. lydekkerianus lydekkerianus) has been observed consuming animal prey at 96% of feeding events

(Nekaris and Rasmussen, 2003).

When pottos do consume insects, they generally concentrate on slow-moving or noxious species, much like the slow loris (Charles-Dominique, 1977; Wiens et al.,

2006). Slow lorises may additionally benefit from this diet by sequestering secondary compounds from arthropod prey for incorporation into venom (Wiens et

al., 2006). The venomous bite of the slow loris is unique among primates and results from mixing saliva with secretions from a brachial gland (Krane et al., 2003). Toxic bites have led to anaphylactic shock in human caretakers (Kalimullah et al., 2008) as well as necrotic wounds in sanctuary-housed conspecifics (Streicher, 2004), and venom likely plays a role in both interspecific and intraspecific defense. The most striking defensive adaptation of the potto is a series of spines on the cervical vertebrae, which are thrust at attackers when pottos are threatened (Charles-

Dominique, 1977).

Lorisids are often found foraging alone, but they are far from asocial and their social structure may be better described as dispersed rather than solitary

(Pimley et al., 2005). Pottos forage alone but share “delayed” communication via olfactory signals (Charles-Dominique, 1977). Male and female home ranges overlap and pairs seem to associate with exclusivity (Pimley et al., 2005). A spacing system wherein male home ranges overlap with the ranges of several females is also typical of slender (Nekaris, 2003b) and slow lorises (Wiens and Zitzmann, 2003).

Individuals tend to be more social at dawn or dusk. Female slender lorises (L.t. lydekkerianus) sleep in groups with their offspring or other relatives, and adult males are often present in sleeping groups as well (Nekaris, 2003b; Radhakrishna and Singh, 2002). Thus, social networks are maintained through overlapping home ranges, sleeping contact, olfactory and auditory signals, and interactions surrounding mating and infant care.

Pottos are relatively common throughout their range, and their populations are thought to be stable (Oates et al., 2008). However, it is clear that all lorises are

declining in the wild (Nekaris & Jayewardene 2004, Nekaris & Nijman 2007, Iseborn et al. 2011). The greatest threats to Asiatic lorisines are deforestation (for palm oil and other cash crops); and human exploitation for use in traditional Asian medicine, the tourist photo prop trade, and the illegal pet trade (Nekaris et al., 2010). In recent years the pet trade has had an increasingly devastating impact on wild slow loris populations, in part due to the nouveau celebrity of lorises in “cute” web videos

(Nekaris et al., 2013). Many animals rescued from this illegal trade have suffered physical injuries that make reintroduction to the wild impossible, and the need for sanctuary housing grows along with the popularity of lorises as pets (Nekaris & Jaffe

2007). Information gleaned from decades of caring for lorises and pottos in zoos may take on new relevance to in situ conservation efforts as sanctuaries play a more critical role in managing wild populations.

STATUS AND CARE OF NOCTURNAL STREPSIRRHINES IN NORTH AMERICAN ZOOS

North American zoos and related facilities accredited by the Association of

Zoos and Aquariums (AZA) currently house nine genera of nocturnal strepsirrhines according to the most recent population analysis and breeding/transfer plan (Kuhar et al., 2011). This group includes five species from continental Africa: the potto, (P. potto), two lesser bushbabies (G. senegalensis and G. moholi), and two species of greater bushbaby (Otolemur garnettii and O. crassicaudatus). Malagasy strepsirrhines housed in North American zoos include the aye-aye (D. madagascariensis), gray mouse lemur (Microcebus murinus), giant mouse lemur

(Mirza zaza), and the fat-tailed dwarf lemur (Cheirogaleus medius). Asian lorisines in zoos include representatives of both the genera Nycticebus and Loris.

With one exception, these populations all are classified as “red” Species

Survival Plans® under AZA’s current sustainability framework, meaning that their populations contain fewer than 50 individuals and do not meet minimum standards for genetic diversity (Kuhar et al., 2011). The pygmy loris population, which contains 71 animals at 22 North American facilities, is classified as a yellow SSP®

(Gibson et al., 2013) and likely shows the greatest potential for future exhibition of nocturnal strepsirrhines in zoos.

Caring for Lorisid Primates in Zoos

Lorisid primates in AZA zoos include the potto, pygmy loris, red slender loris

(L. tardigradus tardigradus) and a hybridized population of N. coucang and N. bengalensis managed under the coucang moniker. Although AZA zoos historically housed an additional subspecies of red slender loris, L.t. nordicus, this population is no longer represented in North America. According to Fitch-Snyder and Schulze

(2001), the history of slow and slender lorises in North America dates back to late

19th/early 20th century exhibits at the Philadelphia and Bronx zoos, while a breeding population of pygmy slow lorises was established in North America in 1987. Current lorisid populations are mostly in decline. At the most recent published population analysis, there were fifteen pottos, thirteen slow lorises, and twelve slender lorises remaining in AZA accredited facilities (Kuhar et al., 2011).

High infant mortality and traumatic death appear to be major impediments to population growth in lorises and pottos. Debyser (1995) analyzed mortality trends for strepsirrhine juveniles in zoos and primate centers and found that more offspring died prior to weaning in N. coucang, L.t. tardigradus, and P. potto than the thirteen lemur and galago species that were also surveyed. Sutherland-Smith and

Stalis (2001) reviewed mortality in lorises from the San Diego Zoo (1982-1995) compared to Duke University Lemur Center (1980-1994). These data show that in addition to trauma, significant contributors to captive loris morbidity and mortality include dental, renal, and respiratory diseases.

A comprehensive review of current knowledge of Asian loris captive care is provided in Fitch-Snyder and Schulze’s (2001) husbandry manual and is also available at www.loris-conservation.org. Guidelines for habitat design are briefly summarized from the manual as follows. The minimum cage size should be no less than 15.6 m3. Relative humidity should be maintained between 40-60% and the temperature between 65.5-85.5 °F. Frequent cage cleaning is not recommended as lorises may be stressed by the procedure, but accumulated urine marks should be regularly removed. Finally, an enriched environment should contain nest boxes, leafy branches that provide cover for the animals, other hiding places, and floor substrates that can be used for sleeping and olfactory stimulation.

Social and reproductive requirements are also outlined in detail in the husbandry manual (Fitch-Snyder and Schulze, 2001). A challenging aspect of lorisid care in captivity is balancing their naturally dispersed social system with the need for social opportunities in a setting where space is restricted. Lorises should be

housed as breeding or mother-offspring pairs. Even more ideal would be separate cages that share a common area where lorises can interact; this design mimics the most common natural ranging system in which one male’s territory overlaps with several distinct female territories. Aggression among individuals should be monitored and is especially common surrounding breeding activity and parturition.

Mixed-species housing may also provide a source of enrichment, and has the additional benefits of utilizing exhibit space more efficiently and creating public education opportunities (Ferrie et al., 2011; Fitch-Snyder and Schulze, 2001).

Despite the fact that the spectral composition of moonlight is equivalent to that of sunlight (Erkert, 1989), nocturnal primates are often housed under light shifted toward either red or blue wavelengths (Davis, 1961; Frederick and

Fernandes, 1994). The first successful zoo exhibits of nocturnal animals, such as the

Bronx Zoo’s “World of Darkness,” were illuminated by dim red light (Conway, 1969).

Because the spectral sensitivity of rods is shifted toward the blue end of the spectrum, electrical activity in the potto retina appears similar in response to blue and white light but is attenuated in response to red light (Goffart et al., 1976). This difference between the sensitivity of differing retinal cell types is the rationale for exhibiting nocturnal species under red light, which would arguably be perceived as less bright by them (Davis, 1961).

Current guidelines for illumination of nocturnal primate exhibits are limited.

For the aye-aye, the Duke Lemur Center’s (DLC) housing guidelines call for red light of less than one lux for dark phase illumination (Williams et al., 2013). The extent to which these husbandry guidelines are followed in practice is unknown, but anecdotal evidence suggests that zoos are using a variety of approaches to create night lighting in the absence of more concrete standards. Given the pivotal role of light in regulating the circadian system, and the potential consequences for behavior and health when this signal is disrupted, lighting design for nocturnal animals in the captive environment should be empirically examined.

LIGHT AND THE REGULATION OF PRIMATE ACTIVITY PATTERNS

‘Chronoecology’ is the study of factors that shape the distribution of animal activities over time (Halle and Stenseth, 2000a). Animals have evolved internal time keeping systems, known as biological clocks, that allow them to predict and respond behaviorally and physiologically to regular patterns of environmental change that occur on a daily or seasonal basis (Rietveld et al., 1993). Located in the suprachiasmatic nucleus (SCN) of the hypothalamus, the internal timekeeping system calibrates rhythmic outputs of behavior, such as sleep-wake cycles, to match environmental conditions (Challet, 2007).

Light is the primary signal that entrains the mammalian circadian system.

The mammalian retina contains intrinsically photosensitive retinal ganglion cells

(IpRGCs) that project to the SCN, as well as neural areas involved in mood and cognition (Bailes and Lucas, 2010). IpRGCs contain a photosensitive opsin called melanopsin that responds strongly to short wavelengths but little to red light (Bailes

and Lucas, 2010). Consequently, blue light has a greater effect on circadian entrainment than other wavelengths, an effect that has been demonstrated in hamsters (Boulos, 1995), nocturnal mouse lemurs (Perret et al., 2010), and humans

(Brainard et al., 2008).

Internal clocks also have an inherent flexibility so that they can be synchronized with environmental inputs by entraining agents known as ‘zeitgebers’

(timekeepers). Clocks are also subject to direct effects of environmental inputs that acutely alter the outputs of internal oscillators, known as ‘masking’ effects (Rietveld et al., 1993). Masking agents serve the purpose of allowing short-term adaptation to environmental change (Erkert, 2008; Rietveld et al., 1993). The interactions between internal clocks, zeitgebers, and masking agents produce rhythmic outputs of behavior in the form of activity patterns (Fernandez-Duque, 2003).

Primatologists are only beginning to examine activity patterns, and there is a great need for research on the chronoecology of primate behavior (Curtis and Rasmussen,

2006).

Environmental conditions, including illuminance levels, interact with other factors such as temperature and humidity, behavioral patterns of predator species, morphological characteristics, and patterns of locomotion and food acquisition to structure primate activity patterns in a complex, species-specific manner (Bearder et al., 2002; Fernandez-Duque, 2003). In the captive environment, these variables are under anthropogenic control and represent both artificial zeitgebers and masking agents that interact to shape activity patterns in zoos (Richter, 2006).

Proximate Factors Shaping Activity Patterns in Lorisids

Light is the major proximate factor that entrains activity patterns in nocturnal mammals (Erkert and Cramer, 2006). When housed in constant darkness, the slow loris (N. coucang) has an internal pacemaker that creates an endogenous activity rhythm with a period close to 24 hours (Kavanau and Peters, 1974; Redman,

1979). When housed in artificial lighting, the lorises’ active period becomes entrained to the dark phase of the light-dark (LD) cycle (Ehrlich, 1968; Redman,

1979; Tenaza et al., 1969). An outdoor colony of slow lorises at a university reliably timed the onset of their behavior to the twilight period despite seasonal changes in day length, leading Kavanau (1976) to argue that lorises have a strong endogenous activity rhythm that is primarily entrained by light levels at twilight. He suggests that twilight may be an important zeitgeber because becoming active as soon as lighting conditions are favorable confers a survival advantage (Kavanau, 1976).

Surprisingly, the majority of nocturnal primate species that have been studied are more active under the brighter light of the full moon than on darker nights (Ankel-Simons and Rasmussen, 2008; Fernandez-Duque, 2003; Gursky,

2003). Galagos are known to travel and vocalize more on bright moonlit nights, perhaps because light aids in finding food or navigating the forest (Nash, 1986).

However, lorisids may be the exception to this trend. The pottos and angwantibos in

Charles-Dominique’s (1977) study site limited their active period to that of total darkness. Both species also emerged from their sleeping sites later and returned to them earlier than sympatric galagos, a pattern which Charles-Dominique (1977) attributed to the emphasis on cryptic behavior in the perodicticinae.

Although some studies have found that Asian lorisines do not alter resting or travel patterns in relation to moonlight levels (Bearder et al., 2002; Bearder et al.,

2006), in a seasonal environment the pygmy loris is less likely to forage on cold and bright moonlit nights (Starr et al., 2012). These findings highlight the complexity of activity patterns among species, which are shaped not only by a multitude of variables but also by interactions between them.

Husbandry practices in captivity can shape the intensity and patterning of loris and potto activities as well. Daschbach et al. (1982/83) found that slow lorises were more active in larger, more enriched cages. Similarly, Frederick and Fernandes

(1996) found that naturalizing an exhibit for two pottos, by adding elements such as leaves and a cricket dispenser, led to an increase in activity and an expansion of the pottos’ behavioral repertoire to include more exploratory and sexual behaviors.

However, the causal relationship between the emergence of sexual behavior and other activity changes is unclear. Furthermore, some of these behaviors declined after an initial peak, suggesting the increase in activity may have been at least partially a result of novelty (Frederick and Fernandes, 1996). Other factors documented to affect captive slow loris activity include zoo visitor presence

(Oswald and Kuyk, 1978) and extreme temperatures (Kavanau, 1976).

A collection of small-scale studies supports the claim that loris activity is inhibited under high illuminance levels. A reduction in activity with increasing light intensity has been documented in captive galagos (Randolph, 1971), as well as three slow lorises at the Woodland Park Zoo (Trent et al., 1977). Frederick and Fernandes

(1994) simultaneously increased the intensity of simulated day lighting, decreased

the intensity of night lighting, and changed night lighting from blue to full-spectrum lighting for two pottos at Franklin Park Zoo. They found increased activity and behavioral diversity following these changes, but the causal relationship between these findings and the increase in reproductive behavior they also observed is unclear (Frederick and Fernandes, 1994). Also, because so many changes were simultaneously made to the lighting conditions in this study, it is difficult to draw any firm conclusions about their efficacy.

Other elements of nocturnal primate lighting design have received less scientific scrutiny than intensity. Frederick et al. (1995) examined the effect of adding simulated dawn and dusk periods to the lighting regimen for two captive pottos, but it is difficult to draw any conclusions from their data because they simultaneously changed the length of the photoperiod in the exhibit as well. Despite the prevalence of differed colored night lighting in zoos, at present there are no systematic studies examining the effects of light wavelength on nocturnal primate behavior.

The effects of light wavelength on locomotor rhythms have been examined in

Drosophila (Subramanian et al., 2009) and migratory birds (Malik et al., 2004) but have been little studied in mammals. Boulos (1995) demonstrated that the effectiveness of light pulses for altering rhythms of wheel-running in Syrian hamsters (Misocricetus auratus) was dependent on wavelength, with blue light exerting a strong effect compared to green and red. Tests with humans have not shown differential effects of light color on nighttime alertness. Shifts workers chronically exposed to bright light filtered to block short wavelengths reported few

differences in fatigue and concentration compared to an unfiltered light condition

(Schobersberger et al., 2007). Exposure to both red and blue light at night reportedly arouses alertness in human subjects experimentally asked to stay awake and perform work at night, as measured by heart rate and electroencephalogram

(EEG) activity (Figueiro et al., 2009).

As these studies show, the complexities of circadian regulation in relation to the light environment are far from being understood. This complexity, along with the natural diversity in activity patterns and visual systems among nocturnal primates, complicates the task of providing recommendations for nocturnal primate lighting design. To this aim, Erkert (1989) proposed that artificial lighting regimens for nocturnal primates are effective when animals show evidence of stable circadian rhythms without any apparent masking of activity during the dark phase. However, these criteria may be difficult to judge in lorisid primates for the same reason that it is hard to make inferences about welfare states in nocturnal strepsirrhines that have naturally inactive behavioral profiles (Wright et al., 1989). Examining the effects of lighting on endocrine functioning and health may provide further insight for determining optimal lighting conditions.

HEALTH IMPLICATIONS OF EXPOSURE TO LIGHT AT NIGHT

Lighting levels that render lorises visible to the zoo-going public may essentially amount to continuous light exposure, which human epidemiological studies and laboratory animal research suggests can have wide-ranging health effects. Constant exposure to light can be a source of stress for mice (Van der Meer

et al., 2004), and disrupts circadian activity patterns (Albers et al., 1981) and sleep- wake cycles (Ikeda et al., 2000) in laboratory rats. The deleterious effects of light at night appear to be largely endocrine-mediated.

Melatonin

The principal hormone responsible for conveying information about light to the internal timekeeping system is melatonin. Information about lighting conditions is transmitted to the SCN, the neural seat of the “master” biological clock, via the retino-hypothalamic track, and ultimately to the pineal gland (Altun and Ugur-Altun,

2007). In response to photic input, pinealocytes synthesize the amine melatonin (N- acetyl-5-methoxy-tryptamine) by way of a four-step process for which tryptophan is the precursor molecule. Melatonin is generated by the activity of AANAT

(arylalkrylamine-N-acetyltransferase), which converts serotonin (5- hydroxytryptamine) into melatonin and is the rate-limiting enzyme for melatonin synthesis (Altun and Ugur-Altun, 2007). This pathway produces serotonin during the day and melatonin in darkness (Murch et al., 2000)

In both nocturnal and diurnal species, circulating melatonin levels are much higher during the dark phase of the LD cycle (Altun and Ugur-Altun, 2007).

Variation in the timing and duration of melatonin excretion encodes information about day length and therefore time of year, and melatonin’s principle timekeeping role appears to be regulating seasonal changes in behavior, especially as those behaviors relate to reproduction (Arendt, 2005; Malpaux et al., 1999). However,

melatonin also plays a role in regulating circadian patterns of behavior such as core temperature rhythms and sleep-wake cycles (Arendt, 2005).

The suppression of nocturnal melatonin production due to light exposure is well documented in humans and many other species. Light at night suppresses pineal melatonin production in a variety of laboratory rodents (Brainard et al.,

1984; Depres-Brummer et al., 1995) as well as rhesus macaques (Reppert et al.,

1981) and the squirrel monkey (Hoban et al., 1990). The effect of light exposure is dose-responsive, meaning that higher light intensities lead to greater suppression of melatonin (Stevens and Rea, 2001). A greater intensity of light is needed to upset the circadian rhythm of melatonin production than to temporarily suppress or mask its expression (Hashimoto et al., 1996). Suppression of melatonin by light exposure in human subjects has been repeatedly demonstrated in controlled experiments as well as applied studies with shift workers (Khalsa et al., 2003; Laakso et al., 1993;

Lowden et al., 2004; Navara and Nelson, 2007; Reiter, 1991; Zeitzer et al., 2000).

Even exposure to ordinary room light before bed can delay the onset of nocturnal melatonin production and reduce its duration (Gooley et al., 2010; Zeitzer et al.,

2000).

As a result of their retinal morphology, nocturnal animals are much more sensitive to light-induced melatonin suppression than diurnal species (Reiter,

1991). Laboratory studies have focused on acute exposure to intense light at night, and the chronic effects of exposure to dimmer illumination are in need of further study (Stevens and Rea, 2001). Chronic dim lighting is probably the most comparable laboratory paradigm to current zoo conditions, in which animals

housed on reversed light cycles are exposed to light of some intensity all hours of the day.

The degree of melatonin suppression that occurs as a result of light exposure depends on light wavelength. Brainard et al. (1999) reviews mammalian variation in action spectra, defined as the degree of biological effect as a function of wavelength.

In humans, peak melatonin suppression occurs by exposure to light in the range of visible blue light (504-514 nm), but peak sensitivities in nocturnal rodents may occur in the ultraviolet range (as low as 330 nm) (Brainard et al., 1999). Even relatively dim blue light can suppress melatonin production; the threshold for intensity for blue light suppressing melatonin in horses has been measured at three to ten lux (Walsh et al., 2013). Selectively filtering short wavelengths successfully prevents light-induced melatonin suppression in rats (Rahman et al., 2008) and humans (Schobersberger et al., 2007). Falchi et al. (2011) compare action spectra for circadian regulation to the luminous output of common artificial lights. They recommend a total ban on outdoor lighting that emits wavelengths less than 540 nm in cities to prevent deleterious effects of light on circadian rhythmicity and melatonin production in humans and other animals.

Melatonin’s role as an endocrine timekeeper means that it has important effects on a wide array of physiological systems that undergo daily or seasonal change including the immune (Skwarlo-Sonta, 2002), metabolic, and reproductive systems (Pandi-Perumal et al., 2006; Reiter, 1991). Melatonin has general immunostimulatory and antioxidant properties, and relationships between illness

and melatonin suppression are likely mediated by the immune system (Navara and

Nelson, 2007).

A growing body of evidence implicates exposure to light at night and melatonin suppression in oncogenesis (Reiter et al., 2007). In laboratory studies, animals exposed to constant illumination develop mammary and other tumors at increased rates (Dauchy et al., 1997; Stevens and Rea, 2001). Epidemiological studies show increased rates of ovarian (Bhatti et al., 2013), breast, prostate, endometrial, and colorectal cancer in shift workers and other individuals who routinely experience circadian disruption (Stevens and Rea, 2001). Blask (2009) even suggests that uninterrupted darkness may be a natural form of cancer prevention.

Other major diseases associated with shift work that may be related to circadian disruption and the effects of light include cardiovascular disease, major depression, metabolic syndrome, and infertility (Arendt, 2005; Navara and Nelson,

2007). Melatonin regulates reproductive activity through multiple channels and can exert its effects through receptors on each level of the hypothalamic-pituitary- gonadal (HPG) axis (Luboshitzky and Lavie, 1999). Melatonin can alter the normally pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), leading to acyclicity (Abbott et al., 2004; Luboshitzky and Lavie, 1999). Melatonin ultimately downregulates gonadal estrogens, and suppression of melatonin is associated with elevated estrogen levels and infertility (Navara and Nelson, 2007). There are thus multiple endocrine-mediated routes through which chronically elevated light

exposure can have negative impacts on health and reproduction in captive mammals.

Melatonin is considered a primary biomarker for circadian dysregulation

(Mirick and Davis, 2008) and can be reliably measured in various matrices

(Middleton, 2006). Pineal melatonin is not stored but instead immediately enters circulation, so melatonin levels in plasma or serum are a reliable indicator of pineal activity (Altun and Ugur-Altun, 2007). Circulating melatonin passively diffuses into saliva through capillary beds in salivary glands, and salivary melatonin reliably reflects circulating melatonin concentrations in humans (Voultsios et al., 1997).

Finally, melatonin or its major metabolite, 6-sulphatoxymelatonin, can also be measured and assessed for rhythmicity in urine (Klante et al., 1997).

Because the expression of melatonin varies temporally, repeated sampling is necessary throughout the day (Middleton, 2006). Measurement of salivary melatonin, which is a reliable point measure yet also relatively non-invasive, has shown great utility in epidemiological studies. Nonhuman primates can be easily trained to provide saliva samples (Lutz et al., 2000; Tiefenbacher et al., 2003), and free-ranging primates will readily chew on saliva collection devices without training

(Higham et al., 2010). Salivary analysis therefore has great promise for assessment of circadian function in zoo-dwelling and free-ranging wildlife.

Cortisol

The steroid hormone cortisol exhibits a robust circadian rhythm and is therefore also a useful biomarker for circadian activity (Buckley and Schatzberg,

2005). Expression of glucocorticoids (GCs, including cortisol) is regulated by the hypothalamic-pituitary-adrenal (HPA) axis. Endocrine cells in the paraventricular nuclei (PVN) of the hypothalamus secrete corticotropin-releasing-hormone (CRH), stimulating release of pituitary adrenocorticotropin (ACTH) and GCs from the adrenal medulla (Van Reeth et al., 2000). The PVN receive input from the SCN and release CRH in a rhythmic manner (Blask, 2009). Like melatonin, cortisol can be easily measured in saliva (Lac, 2001), and this method has been successfully used to document the circadian rhythmicity of cortisol expression in several nonhuman primates (Cross and Rogers, 2004; Heintz et al., 2011; Quabbe et al., 1982).

Cortisol concentration, like melatonin, is strongly related to nocturnal light exposure and its health effects. In humans, rhythms of cortisol expression can be experimentally altered with light (Boivin and Czeisler, 1998). In rats, constant light exposure eliminates rhythms in the expression of corticosterone and 6- sulphatoxymelatonin (Claustrat et al., 2008). In humans, cortisol rhythms are altered in sleep disorders (Blask, 2009; Van Reeth et al., 2000) and in depression

(Germain and Kupfer, 2008). Rhythmic expression of melatonin and cortisol are also dampened in women with metabolic syndrome (Corbalan-Tutau et al., 2012). Causal relationships between circadian disruptions, altered rhythmicity of hormones, and illness are difficult to untangle and are made more complex by the role of cortisol in the stress response.

Cortisol is often described as a “stress hormone,” although it plays many roles in regulating the metabolic system during homesostasis (Sapolsky et al., 2000).

The relationship between cortisol excretion and stress has been widely employed—

with some controversy—to investigate the welfare of animals in captivity (Mormede et al., 2007; Rushen, 1991). ‘Stress’ is the biological response elicited by perceived threats to an individual’s homeostasis (Moberg, 2000). Even abiotic factors like lighting and sound can be a source of stress in the captive environment, leading to activation of the HPA axis (Morgan and Tromborg, 2007). The effects of GC release in response to stress are wide-ranging and include augmented cardiovascular activity, rapid activation of the immune system, increased circulating glucose, and inhibition of reproductive behavior (Sapolsky et al., 2000). Chronic stress can result in prolonged elevated GC levels and is associated with a variety of behavioral and health problems (Morgan and Tromborg, 2007). The chronic stress experienced by some animals as a result of captivity (Morgan and Tromborg, 2007) may therefore complicate attempts to utilize cortisol as a biomarker for circadian regulation.

Welfare Implications of Circadian Studies for Zoo Animals

Exploring captive behavior on a wider temporal scale may have additional benefits beyond understanding proximate factors that precipitate activity. Despite the clear relevance of nighttime behaviors, such as sleep disturbances, as indicators of stress (Abou-Ismail et al., 2007; Van Reeth et al., 2000) or sickness (Millman,

2007), few studies in zoos have incorporated the full daily pattern of activity in behavioral assessments of welfare. Richter (2006) examined activity patterns in zoo-housed koalas and found that evidence that the largely nocturnal animals were likely disturbed by early-morning husbandry routines. Laws et al. (2007) found that both fecal stress hormone metabolites and sleep disturbances increased in an

African elephant following relocation to a new herd. Yet, these approaches are rare and researchers have instead focused on daytime time budgets (the proportion of time observed devoted to different behaviors) as indicators of welfare in captive settings (McCann et al., 2007). As an alternative approach, studies that examine activity patterns in captivity offer the dual benefit of providing information both on behavioral rhythms as well as revealing specific after-hours behaviors, such as sleep disturbances, that are often neglected in zoo research but are likely indicative of animal welfare states (Anderson, 1998).

THESIS OBJECTIVES

The general hypothesis was that exhibit lighting design affects activity patterns and circadian physiology in nocturnal strepsirrhine primates. Specifically, the aims were threefold. The first aim was to evaluate the status of the current captive population of lorisid primates in North American zoos and related facilities, including common husbandry practices and major health concerns. The second aim was to develop methods for measuring circadian behavior and physiology in nocturnal strepsirrhines. The final aim was to document behavioral and endocrine patterns associated with lighting design in the captive environment.

Chapter Two asks how lorisid primates in North American zoos are actually housed in practice in comparison to current husbandry standards. To answer this question, 29 AZA accredited zoos and related facilities were surveyed about current husbandry practices, with a focus on lighting design.

Chapter Three examines health trends in captive lorisids. Death records for lorises and pottos housed in AZA zoos over the last thirty years were reviewed.

These data showed important trends in age-specific mortality and also identified the major causes of death for these species in captivity.

Chapter Four assesses the utility of an automatic behavioral tracking technique, actigraphy, for use in monitoring circadian activity rhythms in the pygmy loris and potto. Although this method was not pursued for the remaining studies here, the data indicate that actigraphy has great potential for future captive and field-based studies examining lorisid activity patterns despite their slow-moving locomotor style.

Chapter Five describes the development of techniques for collecting saliva from nocturnal strepsirrhines for hormone analysis. The results of chemical and biological validation experiments used to develop an assay for salivary melatonin in lorises and pottos are also reported. For these studies, 24-hour rhythms of salivary melatonin concentrations were examined as well as acute suppression of salivary melatonin concentrations by exposure to different wavelengths and intensities of light.

Chapters Six, Seven, and Eight report on the results of a multi-institutional experiment comparing the behavioral and hormonal effects of red and blue exhibit lighting in zoos. Chapter Six describes the dramatic effects of this manipulation on a single aye-aye subject at Cleveland Metroparks Zoo, while Chapter Seven delineates the behavioral effects of red and blue exhibit lighting in nocturnal strepsirrhines at

Cleveland Metroparks Zoo and Cincinnati Zoo and Botanical Gardens. Finally,

Chapter Eight reports on salivary melatonin and cortisol concentrations measured in pottos under blue and red light at three different zoos.

General results of these studies are discussed in Chapter Nine. The future and challenges of research using salivary biomarkers to understand circadian

(dys)regulation in applied settings are discussed. In closing, the implications of these studies for the welfare and population sustainability of nocturnal strepsirrhines in zoos are considered and recommendations are put forth for lighting design in nocturnal zoo exhibits.

Chapter Two

A Survey of Husbandry Practices for Lorisid Primates in North American Zoos and Related Facilities

INTRODUCTION

According to Groves (2001), the primate family Lorisidae (Loridae) consists of small, nocturnal prosimians in two groups: the Asiatic lorises and the African perodicticinae. Asian forms include the grey (Loris lydekkerianus) and red (Loris tardigradus) slender lorises, and the greater (Nycticebus coucang), Bengal (N. bengalensis), and pygmy (N. pygmaeus) slow lorises. A single species of potto

(Perodicticus potto) is recognized along with the golden (Arctocebus aureus) and

Calabar (A. calabarensis) angwantibos; however, it is considered likely that future investigations will describe several new species in the genus Perodicticus (Grubb et al., 2003).

North American zoos and related facilities currently house five lorisid species: the pygmy loris, red slender loris, greater and Bengal slow lorises, and potto. According to the current sustainability framework adopted by the Association of Zoos and Aquariums (AZA), none of these populations qualify as a green Species

Survival Plan® (SSP), the highest sustainability category defined by population size and genetic diversity. Only the pygmy loris received a yellow (intermediate) designation, while the remaining populations are all considered red programs because they contain fewer than fifty individuals (Kuhar et al., 2011).

The effects of premature death or reproductive failure can be profound for such small populations (Schulze, 1998), and indeed, several lorisid species generally

exhibit poor reproductive success in zoos (Debyser, 1995; Fitch-Snyder and Jurke,

2003). Debyser (1995) reviewed the sources of mortality for strepsirrhine juveniles across zoos and primate centers and found that of all the strepsirrhines studied, the lorisids (N. coucang, L. tardigradus, and P. potto) exhibited the highest frequency of juvenile deaths as measured by the percentage of total animals born that die prior to weaning (the cumulative mortality incidence). Information about basic husbandry is critical for mitigating problems with health and stress in captive animals that are related to inappropriate environments (Schulze, 1998), and population trends of captive lorisids suggest a need for investigation into their care.

Reproduction is perhaps the most studied aspect of lorisid husbandry to date.

Basic reproductive parameters have been described in captive colonies of pygmy and slow lorises (Izard et al., 1988; Weisenseel et al., 1998), slender lorises (Izard and Rasmussen, 1985), and pottos (Cowgill et al., 1989; Frederick and Campbell,

1995). Hormonal correlates of reproduction have been examined in the slow loris

(Perez et al., 1988) and pygmy loris (Jurke et al., 1997; Jurke et al., 1998), in which

Fitch-Snyder and Jurke (2003) also examined behavioral correlates of reproductive endocrinology. Some information is also known about reproductive behavior in the slender loris (Goonan, 1993) and mate preference in the pygmy loris (Fisher et al.,

2003a). In general there is more literature on captive reproduction in lorises than pottos.

Only a few aspects of enclosure design have been empirically evaluated for lorisid species. Daschbach et al. (1982/83) examined behavior in relation to cage size for N. coucang, while Frederick and Fernandes (1996) focused on exhibit

complexity rather than size to study the effects of naturalizing an exhibit occupied by a potto breeding pair. Trent et al. (1977) found an inverse relationship between the intensity of activity and lighting levels in three slow lorises at the Woodland

Park Zoo. Frederick and Fernandes (1994) also found greater activity and behavioral diversity in a potto pair after altering both light and dark phase illuminances and changing night lighting from blue to full-spectrum light. Together these studies suggest that a dark, naturalistic environment promotes active, natural behaviors in lorisid primates.

A comprehensive review of current knowledge of Asian loris captive care is provided in Fitch-Snyder and Schulze’s (2001) husbandry manual and is also available at www.loris-conservation.org. Guidelines for habitat design are briefly summarized from the manual as follows. The minimum cage size should be no less than 15.6 m3. Relative humidity should be maintained between 40-60% and the temperature between 65.5-85.5 °F. The animals require approximately 12 hours of daily light, with full-spectrum illuminance preferred during both light phases, and red light as a nighttime alternative. Frequent cage cleaning is not recommended as lorises may be stressed by the procedure, but accumulated urine marks should be regularly removed. Finally, an enriched environment should contain nest boxes, leafy branches that provide cover for the animals, other hiding places, and floor substrates which can be used for sleeping and olfactory stimulation.

Social and reproductive requirements are also outlined in detail in the husbandry manual (Fitch-Snyder and Schulze, 2001). Central to understanding lorisid social needs is that their largely solitary ranging system does not indicate

that animals are asocial; as for most nonhuman primates, opportunities for social interaction are an important aspect of the enriched environment. Lorises should be housed as breeding or mother-offspring pairs. Even more ideal would be separate cages that share a common area where lorises can interact; this design mimics the most common natural ranging system in which one male’s territory overlaps with several distinct female territories. Aggression among individuals should be monitored and is especially common surrounding breeding activity and parturition.

Mixed species housing may also provide a source of enrichment, and has the additional benefits of utilizing exhibit space more efficiently and creating public education opportunities (Ferrie et al., 2011; Fitch-Snyder and Schulze, 2001).

Although the current lorisid husbandry manual (Fitch-Snyder and Schulze,

2001) is thorough, the extent to which husbandry guidelines have been implemented across different institutions is largely unknown. Information about how animals are actually housed in practice is important for understanding the dynamics of health and reproduction in captive populations and for identifying risk factors for individual welfare (Barber, 2009). Thus, the goal of this study was to survey North American facilities housing lorisid taxa and to describe their current husbandry practices. We aimed to determine the extent to which current husbandry practices are consistent with established guidelines, as well as to identify issues of concern and gaps in knowledge for future investigation.

MATERIALS AND METHODS

Survey

We solicited all zoos and related facilities currently housing lorisid primates listed in the North American regional studbooks for participation in the survey. We first contacted each institution by sending a postcard to their institutional representative (IR) to the Prosimian Taxon Advisory Group. Some institutions did not have an IR listed; in these cases we contacted a primate curator or someone in an equivalent position listed in the AZA directory. We contacted non-AZA facilities using email addresses and phone numbers available on their websites. We then sent each individual a link to the online survey and a research proposal. We made multiple attempts via email to contact participants and followed up by phone when necessary as recommended by Plowman et al. (2006).

We collected data using an online survey created using SurveyMonkey®

(Portland, OR, USA). We collected responses between January and May of 2010. We asked respondents to complete a survey for each group of lorisid primates housed in their institutional collection, and we defined a ‘group’ as one or more animals that share the same exhibit or living space the majority of the time. The survey consisted of five sections: basic group information; primary and secondary enclosure design; lighting conditions in the primary enclosure; animal care practices; and a series of demographic questions about each group member. All multiple choice questions included an optional “other” category and a text field to explain responses. A list of survey questions is included in Appendix 1; a few questions were modified from

Tarou et al. (2005).

We instructed respondents to utilize the following definitions in completing the survey. The ‘primary enclosure’ was defined as the main space where the group is exhibited. In the case of groups that are not exhibited, this refers to the place where the animals are housed for the majority of the day. The ‘secondary enclosure’ was defined as an alternate exhibit or holding space which may be an enclosure occupied seasonally or an off-exhibit holding area. The ‘light phase’ was defined as daylight or part of the day during which bright lights are used to simulate daylight in the enclosure. The ‘dark phase’ was defined as nighttime or the part of the day during which the enclosure lights are off, dimmed, or shifted in wavelength to simulate nighttime conditions.

Data Analysis

We asked survey participants to submit a survey for each group as a matter of convenience, because many aspects of physical housing and animal care are the same for animals sharing an exhibit. However, our results revealed that a high number of ‘groups’ were actually solitary individuals. For this reason, we chose to analyze survey questions based on the percent of individuals for whom a particular response was chosen. All subjects were thus treated as independent data points for the purpose of data analysis. For some questions, multiple response options were permitted; for this reason, total values for categorical data do not total 100% where indicated.

RESULTS

Population Demography and Group Compositions

We received surveys from 29 institutions: 100% (n = 27) of those accredited by AZA; one certified related facility; and one of three other non-AZA facilities that currently house lorisid primates in North America. Our sample included 104 lorisid primates representing five species. The sample included eleven greater slow lorises

(n = 3.8.0, male.female.unknown) and four Bengal slow lorises (n = 3.1.0). Based on the uncertain hybrid status of most of these individuals (Kuhar et al., 2011), we combined them into a single category called ‘slow loris’ for data analysis. We received data for 90% of the North American population of pygmy lorises (n =

31.31.0) representing 42 groups at 23 facilities. We received surveys for 100% of slender lorises (n = 8.4.0 individuals, 8 groups at 4 facilities), slow lorises (n = 6.9.0,

12 groups at 10 facilities), and pottos (n= 8.6.1, 12 groups at 3 facilities).

Across sexes, the mean age in years (SD) for each species was: 8.3 (4.5) for pygmy lorises, 7.8 (5.3) for slender lorises, 14.7 (3.9) for slow lorises, and 10.9 (9.8) for pottos. The pygmy loris evinced the only potentially stable age pyramid (Figure

1), while the other three populations were much smaller and constrictive in nature.

For all species except the slow loris, the majority of individuals were living in breeding groups: 71% of pygmy lorises, 67% of slender lorises, and 53% of pottos resided in breeding groups compared to only 27% of slow lorises.

Table 1. Group compositions of lorisid primates in North American facilities.

Number of Pygmy loris Slender loris Slow Potto Groups (n = 42 groups) (n = 8 groups) loris (n = 12 (n = 12 groups) groups) 1.0 13 4 3 6 [one male] 0.1 [one 10 0 6 3 female] 1.1 16 2 3 2 [one pair] Other 3 2 1 [one group of 0.2.0*, one group [1.0.0 mixed with [one of 1.2.0, and one mixed species pygmy loris; and 0 group of group of 1.1.0 pygmy loris and one group of 0.1.1] 1.0.0 slender loris] 1.2.0] Mean 1.25 1.25 group size 1.50 (0.09) 1.57 (0.3) (0.13) (0.13) (SE) Group size 1-3 1-3 1-2 1-2 range * Notation indicates: (# males. # females. # unknown individuals)

Our sample included 73 lorisid groups with a mean of 2.5 + 0.4 (SE) groups per zoo, half of which (49%) were identified as breeding groups. Across species, group sizes ranged from one to three individuals (Table 1). The majority of ‘groups’ (61%) reported in the survey actually consisted of solitary individuals, including: 23 pygmy lorises (37% of individuals of this species), 4 slender lorises (33%), 9 slow lorises

(60%), and 9 pottos (60%). In most other cases, animals were housed in male- female pairs, which consisted of breeding groups, non/post-reproductive pairs, or parent-offspring pairs. Only one group included more than one lorisid species and consisted of 1.1.0 pygmy lorises and 1.0.0 slender loris.

Figure 1. Age pyramids for lorisid primates in North American institutions. Ages are calculated from the date of birth listed in the species studbook to the date the survey was distributed, January 19, 2010. At this time, the population consisted of a total of 104 lorisids: 62 (31 male: 31 female) pygmy lorises, 12 slender lorises (8.4), 15 slow lorises (6.9), and 15 (8.6.1 unknown) pottos. For the potto, the one unknown individual (a neonate) is not included in the age pyramid above but is included in the population total.

Twenty groups containing 28 individuals (27%) were identified as mixed- species groups, and 53 groups containing 76 individuals (73%) were described as non-mixed groups. Pygmy lorises were housed with northern tree shrews (Tupaia belangeri), aye-ayes (Daubentonia madagascariensis), Malagasy jumping rats

(Hypogeomys antimena), greater Malayan chevrotains (Tragulus napu), and mouse

lemurs (Mirza zaza). Pottos were housed with African hedgehogs (Atelerix spp.) and black and rufous elephant shrews (Rhynchocyon petersi). Slender lorises were housed with dwarf lemurs (Cheirogaleus medius) and greater Malayan chevrotains.

Slow lorises were found with the widest array of different species, including three- banded armadillos (Tolypeutes matacus), brush-tailed porcupines (Atherurus africanus), chevrotains, aye-ayes, Asian small-clawed otters (Aonyx cinerea),

Prevost’s squirrels (Callosciurus prevostii); and the only avian species currently housed with a lorisid primate, the blue-bellied roller (Coracias cyanogaster).

Enclosure Design

The majority of facilities maintained lorisid groups in a dedicated nocturnal building. The second most common housing arrangement consisted of indoor exhibits adjacent to diurnal exhibits rather than a specialized nocturnal area (Table

2). Only one facility housed any of these species (slender loris) outdoors. Overall, 26 lorisids (25%) had access to some secondary space; the remaining animals were all confined to a single primary exhibit which was usually indoors. Only pygmy lorises

(n = 9, 38%) had access to alternate enclosures on exhibit to the public. Off-exhibit holding spaces were slightly more common and were available to a single slender loris, a single slow loris, and 15 (63%) pygmy lorises. One slender loris and 13 pygmy lorises (54% of those with secondary enclosures) were allowed to move between the primary and secondary enclosure at will; otherwise secondary enclosures were utilized less frequently or only as needed.

Table 2. Enclosure design features of lorisid primates in North American facilities.

Question Response options Pygmy Slender Slow Potto All loris loris loris % (#) lorisids % (#) % (#) % of % of of (#) n = (n = n = 62 n = 12 of n = 15 104) 15 Where is the indoors, in a 45% 92% 47% 47% primary enclosure dedicated nocturnal 51% (28) (11) (7) (7) located? house indoors, in a 27% 8% 20% 0% nocturnal-only area 20% (17) (1) (3) (0) (a separate wing) indoors, in an area 27% 0% 27% 53% with both diurnal and 28% (17) (0) (4) (8) nocturnal exhibits 0% 0% 7% 0% outdoors 1% (0) (0) (1) (0) What best describes forced air circulation 76% 100% 87% 93% the ventilation running throughout 83% (47) (12) (13) (14) system for the the building enclosure? separate ventilation 24% 0% 7% 7% system from 16% (15) (0) (1) (1) public/keeper areas natural ventilation 0% 0% 7% 0% through windows or 1% (0) (0) (1) (0) outdoor access What best describes 39% 0% 40% 60% bare concrete 38% the substrate in the (24) (0) (6) (9) enclosure?^ 47% 33% 40% 40% mulch 43% (29) (4) (6) (6) 0% 0% 7% 0% dirt 1% (0) (0) (1) (0) variable, 15% 67% 13% 0% 18% combination, or other (9) (8) (2) (0) Does the enclosure 71% 100% 87% 80% nest boxes 78% contain any hiding (44) (12) (13) (12) or sleeping sites?* baskets, tubes, or 84% 100% 93% 80% other covered 87% (52) (12) (14) (12) structures 56% 8% 67% 13% areas of dense foliage 46% (35) (1) (10) (2) 21% 17% 13% 7% other hiding spots 17% (13) (2) (2) (1) ^ Response option for grass substrate was eliminated because it was never selected. * Multiple responses to this question were permitted.

Figure 2. Primary enclosure size for lorisid primates in North American facilities. Values are for n =104 animals: 62 pygmy lorises, 12 slender lorises, 15 slow lorises, and 15 pottos.

Physical conditions were fairly consistent across species. Enclosure sizes were comparable across species with a tendency toward larger enclosures for slow lorises, although exhibit sizes were highly variable within species (Figure 2). Only one facility, which housed a slow loris outdoors, did not control the exhibit temperature. Other lorisids were maintained at similar mean temperatures, given as

˚F (SE): 75.7 (0.3) for n = 60 pygmy lorises; 77.3 (0.3) for n = 12 slender lorises; 76.3

(0.9) for n = 13 slow lorises; and 75.3 (0.5) for n = 15 pottos. In most cases, exhibits shared a forced air ventilation system that ran throughout an entire building (Table

2). Relative humidity (RH) levels in exhibits were actively controlled only for nine

(15%) pygmy lorises and one slender loris. Mean (SE) reported values for exhibits were 49.7 (1.7) %RH for n = 56 pygmy lorises, 44.6 (2.2) % RH for n = 12 slender lorises, 46.9 (4.3) %RH for n = 11 slow lorises, and 46.8 (1.6) %RH for n = 14 pottos.

Exhibit furnishings are also outlined in Table 2. Most lorisids were housed in exhibits with either bare concrete or mulch on the ground. Other flooring was most often described as wire (presumably cage floors). Every individual received some type of hiding spot on exhibit; the most common refuges were nest boxes, followed by tubes or other covered structures, and areas of dense foliage.

Lighting Conditions

The most common lighting regimen was the same for all species and consisted of an artificially reversed light cycle (Table 3). The majority of animals in each species except the slender loris were kept on fixed lighting regimens, in which phase lengths remained constant throughout the year. The average length (SE) of the fixed dark phase was 11.6 (0.2) hrs across species. Other animals were exposed to seasonal changes in day length, either as a result of exposure to natural light (16%) or intentional manipulation of artificial lighting (23%). If these two cases are grouped together and natural day lengths are calculated based on zoo latitude, the upper and lower bounds of variable dark phase lengths are 9.7 (0.2) hrs and 12.8

(0.3) hrs. Artificial lighting was used to mimic twilight periods for only eight (13%) pygmy lorises and a single slow loris.

Table 3. Lighting design of the primary enclosure.

Question Response options Pygmy Slender Slow Potto All loris loris loris % (#) lorisids % (#) % (#) % of % of of (#) n = 15 (n = n = 62 n = 12 of 104) n =15 How are light and natural day/night 11% 67% 13% 0% dark phases created cycles with sunlight 16% (7) (8) (2) (0) in the exhibit? access artificially created 16% 0% 33% 33% day/night cycles 19% (10) (0) (5) (5) (un-reversed) artificially created 73% 33% 53% 67% 64% reversed light cycle (45) (4) (8) (10) Do the lengths of the no, phase lengths 63% 17% 60% 67% 58% light and dark are fixed (39) (2) (9) (10) phases change yes, phase lengths seasonally? vary naturally or 37% 83% 40% 33% 42% artificially mimic (23) (10) (6) (5) seasonal change What sources of 11% 67% 20% 0% sunlight 17% light are used to (7) (8) (3) (0) illuminate the 77% 100% 80% 100% fluorescent lights 84% enclosure during the (48) (12) (12) (15) light phase?* 26% 0% 27% 0% incandescent lights 19% (16) (0) (4) (0) 3% 0% 0% 0% LED lamps 2% (2) (0) (0) (0) high intensity 8% 0% 7% 13% 8% discharge lights (5) (0) (1) (2) What sources of 3% 0% 0% 0% no light at all 2% light are used to (2) (0) (0) (0) illuminate the 8% 67% 20% 0% moon or starlight 15% enclosure during the (5) (8) (3) (0) dark phase?* fluorescent lights 31% 17% 20% 60% 32% with filters (19) (2) (3) (9) incandescent lights 23% 25% 27% 7% 21% with filters (14) (3) (4) (1) 3% 0% 0% 0% LED lamps 2% (2) (0) (0) (0) high intensity 3% 8% 0% 0% discharge lights 3% (2) (1) (0) (0) with filters colored bulbs of any 37% 17% 33% 0% 29% type (23) (2) (5) (0) 6% 0% 13% 33% other light source 11% (4) (0) (2) (5)

Question Response options Pygmy Slender Slow Potto All loris loris loris % (#) lorisids % (#) % (#) % (#) of n = % (n = of n = of n = of n = 15 104) 62 12 15 What color(s) of 39% 40% 47% red 17% (2) 38% artificial light (24) (6) (7) illuminate the 35% 40% 33% blue 17% (2) 34% enclosure during the (22) (6) (5) dark phase?* 23% 20% 33% white 0% (0) 21% (14) (3) (5) other color/not 21% 47% 0% 67% (8) 26% applicable (13) (7) (0) * Multiple responses to this question were permitted.

Facilities reported a great deal of variation in exhibit lighting fixtures and design (Table 3). Many facilities used more than one light source during dark and light phases. Fluorescent lights were most commonly used during the light phase, and filtered fluorescent lights were often used to brighten the exhibit for visitor viewing during the dark phase. Many institutions reported using more than one light color to illuminate the exhibit during the dark phase. The majority of animals were exposed to some kind of artificial light for all 24 hours of the day.

Animal Care Practices

Facilities also reported on basic husbandry practices for their groups (Table

4). All lorisids were cared for by at least two animal keepers. In most cases exhibits were cleaned (or at least spot-cleaned) daily. For all species, the majority of individuals received their daily diet in a single meal. However, many institutions noted that diets were scatter-fed or otherwise presented to promote foraging, and daily diets were often supplemented with food used for enrichment. Although the

vast majority of animals were provided with daily enrichment, training was rarely or never conducted with most individuals.

Table 4. Animal care practices.

Question Response Pygmy Slender Slow Potto All options loris loris loris % (#) lorisids % (#) % (#) % (#) of n = % (n = of n = of n = of n = 15 104) 62 12 15 How many keepers work 53% 67% 53% 53% two 55% directly with this group (33) (8) (8) (8) during a given week?* 24% 17% 13% 47% three 25% (15) (2) (2) (7) more than 23% 17% 33% 0% 20% three (14) (2) (5) (0) How often is the primary daily (including 89% 92% 100% 87% 90% enclosure cleaned? spot cleans) (55) (11) (15) (13) 2-3 times per 3% 0% 0% 0% 2% week (2) (0) (0) (0) 8% 8% 0% 0% weekly 6% (5) (1) (0) (0) other 0% 0% 0% 13% 2% frequency (0) (0) (0) (2) In how many feedings 56% 92% 60% 100% one 67% per day is the main diet (35) (11) (9) (15) presented (not including 40% 8% 40% 0% two 31% food used for training)? (25) (1) (6) (0) 3% 0% 0% 0% three 2% (2) (0) (0) (0) How often is enrichment 79% 100% 67% 87% daily 81% presented for this group? (49) (12) (10) (13) 8% 0% 33% 0% weekly 10% (5) (0) (5) (0) 8% 0% 0% 13% monthly 7% (5) (0) (0) (2)

5% 0% 0% 0% rarely# 3% (3) (0) (0) (0)

Question Response Pygmy Slender Slow Potto All options loris loris loris % (#) lorisids % (#) % (#) % (#) of n = % (n = of n = of n = of n = 15 104) 62 12 15 How often are training 5% 0% 7% 0% daily 4% sessions conducted with (3) (0) (1) (0) this group? 18% 0% 20% 13% weekly 15% (11) (0) (3) (2) 10% 0% 0% 0% monthly 6% (6) (0) (0) (0) 55% 83% 60% 87% rarely 63% (34) (10) (9) (13) 13% 17% 13% 0% never# 11% (8) (2) (2) (0) Are specific members of pairs are 23% 17% 0% 53% the group generally usually 23% (14) (2) (0) (8) removed during separated# parturition and/or infant pairs are not 6% 0% 0% 0% rearing? usually 4% (4) (0) (0) (0) separated# the zoo has tried both of 15% 0% 0% 0% 9% the above (9) (0) (0) (0) strategies# 37% 42% 100% 47% not applicable# 48% (23) (5) (15) (7) 19% 42% 0% 0% no response# 16% (12) (5) (0) (0) How frequently do 37% 67% 60% 53% yearly 46% members of this group (23) (8) (9) (8) receive physical exams every other 31% 17% 20% 47% 30% by a veterinarian?^ year (19) (2) (3) (7) 31% 8% 13% 0% only as needed 21% (19) (1) (2) (0) other 2% 8% 7% 0% 3% frequency (1) (1) (1) (0) * Response option for “one keeper” was eliminated because it was never selected. # Response option category was added in data analysis based on write-in responses. ^ Response option for “twice a year” was eliminated because it was never selected on the survey.

For all species, most facilities reported giving individuals annual veterinary exams. Finally, breeding pairs were most commonly separated for parturition

and/or infant rearing; however, most institutions responded that this question was not applicable as their groups were non-reproductive.

Individual Characteristics

Finally, we asked respondents to estimate each individual’s reproductive success (Table 5). The only species reported to include a high percentage of reliable breeders was the potto, and only two slender lorises (a pair) were characterized as reliable breeders. A particularly large number of slow lorises were described as never having bred despite repeated attempts. This question was reportedly not applicable to most animals, although the reasons given for this response varied among species. Most slender lorises were not housed in breeding situations. One slow loris pair was used for education purposes, one female previously underwent a hysterectomy, and the others were housed alone or were not in breeding situations.

One aged female potto was presumed to be post-reproductive, and two males did not have available breeding partners other than direct relatives. Pygmy lorises appeared to show a different pattern. Most pygmy lorises were described as potential (but not proven) breeders that had recently been moved among institutions based on breeding recommendations, had only recently been paired with a new breeding partner, or were potentially pregnant at the time the survey was completed.

DISCUSSION

We surveyed all AZA zoos and related facilities (and one non-AZA facility) in

North America that currently house lorisid primates. Our data indicate that the typical lorisid is housed solitarily or in a pair; pair housing was more common for slender and pygmy lorises than other taxa. Most lorisids do not reside in mixed- species exhibits. Most animals are housed in buildings dedicated to exhibiting nocturnal animals and have access to only one living space. Animals are most commonly housed under a reversed light cycle, presumably so they are visible to the public during their active period. Most occupy complex environments and receive additional enrichment frequently. Animals are always cared for by at least two keepers, who feed animals and clean exhibits once a day, but rarely conduct training sessions. Reported practices were thus fairly consistent with existing husbandry recommendations for lorises (Fitch-Snyder and Schulze, 2001).

The Physical Environment

Institutional housing practices are consistent with most current standards for abiotic exhibit design (Fitch-Snyder and Schulze, 2001). Reported temperature and humidity ranges are consistent with those listed in the husbandry manual, and most animals receive at least 12 hours of light a day.

Pygmy and slow lorises commonly occupy spaces larger than the recommended minimum while pottos and slender lorises occupy slightly smaller spaces. Daschbach et al. (1982/83) reported that increased space stimulates activity

in the slow loris, although it should be noted that both cages tested in this study were smaller than current space recommendations.

It may be that the quality of space, rather than quantity, has more dramatic effects on lorisid behavior. Frederick and Fernandes (1996) found that naturalizing an exhibit by increasing the amount of vegetative covering and hiding spots produced an increase in activity and social behaviors in two pottos. Almost every institution in our survey provided nest boxes, other hiding places, mulch or other substrates, and daily enrichment to their lorisid groups. These results suggest that many facilities are taking advantage of the positive behavioral benefits of natural, enriched environments (Clark and Melfi, 2011; Lutz and Novak, 2005; Markowitz and Spinelli, 1986).

Table 5. Estimated reproductive success of lorisid primates.

Reproductive Success Sex Pygmy Slender Slow Potto All loris # (%) loris loris # (%) lorisids of n = 62 # (%) # (%) of n = % of n (31.31.0)* of n = of n = 15 = 96 12 15 (8.6.1) (8.4.0) (6.9.0) Reliable breeder Combined 16% 17% 0% 40% 17% (When given the sexes (10) (2) (0) (6) opportunity, an offspring 16% 12.5% 0% 37.5% Male - is almost always (5) (1) (0) (3) produced.) 16% 25% 0% 50% Female - (5) (1) (0) (3) 0% Unknown - - - - (0) Successful breeder Combined 6% 0% 0% 7% 5% (Does not necessarily sexes (4) (0) (0) (1) produce an offspring 6% 0% 0% 12.5% Male - after each opportunity.) (2) (0) (0) (1) 6% 0% 0% 0% Female - (2) (0) (0) (0) Unknown - - - 0% (0) -

Reproductive Success Sex Pygmy Slender Slow Potto All loris # (%) loris loris # (%) lorisids of n = 62 # (%) # (%) of n = % of n (31.31.0)* of n = of n = 15 = 96 12 15 (8.6.1) (8.4.0) (6.9.0) 50/50 breeder Combined 2% 0% 0% 0% 1% (Has bred successfully sexes (1) (0) (0) (0) but only produces an 0% 0% 0% 0% Male - offspring about half the (0) (0) (0) (0) time following an 3% 0% 0% 0% Female - opportunity.) (1) (0) (0) (0) 0% Unknown - - - - (0) Rare breeder Combined 11% 0% 7% 0% 8 % (Has only reproduced sexes (7) (0) (1) (0) one time or rarely done 13% 0% 0% 0% Male - so.) (4) (0) (0) (0) 10% 0% 11% 0% Female - (3) (0) (1) (0) 0% Unknown - - - - (0) Never bred Combined 11% 17% 40% 20% 17% (Despite repeated sexes (7) (2) (6) (3) attempts.) 10% 12.5% 50% 12.5% Male - (3) (1) (3) (1) 13% 25% 33% 33% Female - (4) (1) (3) (2) 0% Unknown - - - - (0) Not applicable or other Combined 53% 67% 53% 33% 44% sexes (33) (8) (8) (5) 55% 75% 50% 37.5% Male - (17) (6) (3) (3) 52% 50% 56% 17% Female - (16) (2) (5) (1) 100% Unknown - - - - (1) * Notation indicates: (# males. # females. # unknown individuals).

One aspect of the abiotic environment that appears to be less consistent among facilities is lighting design. Day lighting is fairly consistent among facilities, but a wide range of light sources and colors are employed to simulate night lighting.

Strepsirrhines may perceive blue light as brighter than red (Frederick and

Fernandes, 1994; Goffart et al., 1976), and therefore red or neutral-density filters are thought to be preferable for night lighting in exhibits (Fitch-Snyder and Schulze,

2001). Despite this, slightly more than half of captive lorisid groups are currently housed under blue light. Furthermore, most facilities we contacted were unable to provide light intensities for their exhibits, despite research suggesting that lorises are less active in brighter exhibits (Frederick and Fernandes, 1994; Trent et al.,

1977). Light color and intensity may also have implications for animal health and reproduction.

Exposure to light at night is known to suppress production of the timekeeping hormone melatonin in humans (Mirick and Davis, 2008) and other primates (Hoban et al., 1990; Reppert et al., 1981). The suppressive effects of nocturnal light exposure are more pronounced in nocturnal species (Reiter, 1991), as well as under greater light intensities and shorter (blue) wavelengths (Aral et al., 2006; Rahman et al., 2008). In humans, nocturnal melatonin suppression is associated with elevated cancer risk, cardiovascular disease, major depression, metabolic syndrome, and decreased fertility (Arendt, 2005; Navara and Nelson, 2007). In many mammals, melatonin also plays an important regulatory role in seasonal reproduction

(Malpaux et al., 1999). It is not clear if lorisids are seasonal breeders (cf. Fitch-

Snyder and Jurke, 2003; Izard et al., 1988; Radhakrishna and Singh, 2004) or if reproduction is under photoperiodic control, but captive lighting could conceivably impact reproductive success in these species through this hormonal pathway.

Clearly there is a need for further research on the behavioral and physiological

impacts of lighting on nocturnal primates, and evidence-based guidelines should be developed for nocturnal lighting intensity and color.

The Social Environment

Our survey results suggest that the social lives of lorisids present management challenges. Nearly half of the animals in our survey were housed solitarily. Although our survey did not directly address aggression, respondents often cited aggression during introductions as the reason why potential breeding pairs had not successfully reproduced. Anecdotal reports of groups being separated due to aggression are also common. Yet, wild pottos may be found in breeding pairs

(Pimley et al., 2005), and slender lorises at several field sites have shown high levels of gregariousness (Nekaris, 2001; Radhakrishna and Singh, 2002). It may be that many institutions lack the flexibility in design needed to replicate the dispersed, community style social structure of these species. Few institutions in our survey reported having secondary exhibits or holding areas for animals. Daschbach et al.

(1982/83) did not find a relationship between cage size and frequency of agonistic behavior in two slow loris pairs. However, perceived social density appears to moderate aggression in many primate species (Honess and Marin, 2006). It would be fascinating to see the results of research exploring how creative manipulation of exhibit structure and size affects the ability of zoos to manage lorisids in pairs or other groups, as well as if greater social familiarity promotes breeding success

(Fisher et al., 2003b).

Mixed-species exhibits may provide another source of social enrichment

(Leonardi et al., 2010). Only one group in our survey contained multiple lorisid species (pygmy and slender loris), although historically pygmy lorises have been housed with both slow and slender lorises (Ferrie et al., 2011). Overall, less than a third of the animals surveyed shared exhibits with non-lorisid taxa. Adequate off- exhibit holding space is considered important for creating successful mixed-species groups (Ferrie et al., 2011), so the lack of secondary spaces identified in our survey may explain why mixed groups are not more common. Our survey does confirm that lorisids tend to mix well with other strepsirrhines such as aye-ayes (Ferrie et al.,

2011); although, the success of any particular mixed group will no doubt depend on individual characteristics as well as environmental factors. Perhaps more facilities will take advantage of mixed species opportunities as other nocturnal primates become more common in zoos.

When animals cannot be housed socially, olfactory enrichment may provide social stimulation. Scents from other animals or even predator feces can stimulate activity in lorises (Fitch-Snyder and Schulze, 2001). Frequent cage cleaning may also interfere with olfactory communication, which may play an important role in the expression of reproductive behaviors. When experimentally given a choice between two males, female pygmy lorises were more likely to show preference toward a male whose scent was familiar (Fisher et al., 2003b), and males will compete with one another by countermarking over the scents of other males when given the opportunity (Fisher et al., 2003a). Our survey respondents indicated that cages were generally cleaned daily. However, we did not distinguish between intensive cleaning

measures, which would likely interfere with chemosensory signals, and spot cleaning. Nevertheless, it may be worthwhile for facilities to consider the adequacy of their habitat design for meeting olfactory needs, especially for lorisids in breeding situations.

Animal training and other types of keeper interaction may be another source of social stimulation for animals (Pizzutto et al., 2007), providing mental stimulation and acting as a means of environmental enrichment (Laule and Desmond, 1998).

The vast majority of institutions in our survey rarely or never conduct training with lorisids. Information on training is notably absent from the current husbandry manual (Fitch-Snyder and Schulze, 2001). Perhaps caretakers do not think that lorises respond well to training; however, our recent success with training pottos and pygmy lorises for saliva collection suggests this may not be the case. Training can have many positive benefits for animal care such as desensitizing animals to fear-invoking stimuli (which could be particularly helpful in shy loris species), improving social management, and promoting voluntary cooperation in husbandry and medical procedures (Laule and Whittaker, 2007; Young and Cipreste, 2004). It seems that institutions may be missing a valuable opportunity to use positive reinforcement training as a tool to promote the welfare of captive lorisids.

Population Status

Reproductive and demographic data collected in our survey indicate an uncertain future for most lorisid species in North American zoos. The age structures of these populations illustrate that slender lorises, slow lorises, and pottos are

essentially in demographic crisis. In response, the current nocturnal strepsirrhine breeding and transfer plan has recommended breeding for all available animals in these species (Kuhar et al., 2011). Our survey included questions intended to assess the reproductive potential of these populations. Few slender and slow lorises were described as reliable breeders. Many of these animals are not housed in situations to promote breeding, most likely due to their age or reproductive history. Exacerbating breeding problems is the naturally low reproductive rate of slender (Izard and

Rasmussen, 1985) and slow (Izard et al., 1988) lorises. More pottos were identified as reliable breeders in the survey, a designation which is somewhat at odds with the reproductive history of this species in captivity. Given how limited reproductive opportunities are for these species, it is important that institutions follow breeding recommendations to ensure that no prospects are missed. Research may also be beneficial to these populations, as improved knowledge of husbandry in these species may enable caretakers to maximize rare reproductive opportunities.

The demographic status of the captive pygmy loris situation is more stable, and pygmy lorises are currently the most common lorisid primate exhibited in

North American zoos. Pygmy lorises have qualities which promote population growth such as a higher twinning rates, faster maturation, and shorter lactation lengths than other Nycticebus species (Weisenseel et al., 1998). Although our survey indicated that many animals have not successfully bred at this time, it appears that management changes are in progress and that there is actually great reproductive potential in the current pygmy loris population.

We conducted a comprehensive survey of animal care and housing practices for slender, slow, and pygmy lorises, and pottos, in North American facilities.

Although our survey generally showed that institutional housing practices are consistent with current husbandry guidelines, there are aspects of the physical environment, such as lighting design, that show room for improvement or where additional knowledge is needed. Our survey also highlighted the need for further information on reproduction in captive lorisids, particularly on reproductive seasonality and its environmental triggers. The urgency of this need is underlined by the population declines revealed by our demographic and reproductive data.

Even if management of these species in AZA collections is eventually discontinued, lessons learned from their husbandry may inform future efforts to manage the pygmy loris population. As commercial demand for pet lorises grows (Nekaris and

Campbell, 2012), it is also likely that husbandry information will be of increasing importance for managing animals in situ in sanctuaries as they are recovered from the wildlife trade. In zoos, preservation of the captive pygmy loris population will ensure that lorisid primates remain as captive ambassadors for educating visitors about the dire threats faced by all wild lorisids, as well as the ecological niche occupied by lorises and their fellow creatures of the night.

Chapter Three

A Retrospective Review of Mortality in Lorises and Pottos in North American Zoos, 1980-2010

INTRODUCTION

Lorises (genus Loris and Nycticebus) are strepsirrhine primates that dwell in forested habitats across Southeast Asia. Slender lorises include at least two species in India and Sri Lanka (Brandon-Jones et al., 2004), and the slow lorises are a radiation of species extending through Indonesia into southern China. Ongoing investigations are revealing increased diversity within the genus Nycticebus (Munds et al., 2012), just as it is becoming clear that all loris species are declining in the wild

(Iseborn et al., 2011; Nekaris and Jayewardene, 2004; Nekaris and Nijman, 2007). In recent years the pet trade has had an increasingly devastating impact on wild slow loris populations (Nekaris and Campbell, 2012; Nekaris et al., 2010). Many animals rescued from this illegal trade have suffered physical injuries that make reintroduction to the wild impossible, and the need for sanctuary housing grows along with the popularity of lorises as pets (Nekaris and Jaffe, 2007). Zoos that have historically housed lorises thus may have the opportunity to contribute to in situ conservation efforts by providing sanctuaries with information on captive loris husbandry and veterinary care.

Zoo populations of slender and slow lorises are found in Europe, Asia, and

North America. North American zoos have historically housed the pygmy slow loris

N. pygmaeus, and a hybridized population of N. coucang and N. bengalensis managed under the coucang moniker. North American zoos have also housed two subspecies

of red slender loris: L. tardigradus tardigradus and L.t. nordicus, although the North

American population of the latter is now extinct. According to Fitch-Snyder and

Schulze (2001), the history of slow and slender lorises in North America dates back to late 19th/early 20th century exhibits at the Philadelphia and Bronx zoos, while a breeding population of pygmy slow lorises was established in North America in

1987. In addition to slender and slow lorises, North American zoos are home to a small population of an African lorisid: the potto, Perodicticus potto. Pottos are thought to be widespread throughout sub-Saharan Africa but their true taxonomic and conservation status is largely unknown (Grubb et al., 2003).

Pygmy loris numbers are increasing in zoos, and currently they are the only lorisid species nearing sustainability in North American collections (Kuhar et al.,

2011). Populations of other lorisid species are small (fewer than 50 individuals) and declining. Although L.t. nordicus is no longer found in captivity in North America, there are still some individuals managed in Europe. Research into reproduction, as well as veterinary and social needs, may play an important role in maintaining viable populations of these species in captivity (Schulze, 1998).

Stalis (2001) reviewed mortality in lorises from the San Diego Zoo (1982-1995) compared to Duke University Lemur Center (1980-1994). These data show that in

addition to trauma, significant contributors to captive loris morbidity and mortality include dental, renal, and respiratory disease. This study expands on this research

(with some sampling overlap) and seeks to describe more comprehensively the factors contributing to mortality in captive lorises and pottos.

We reviewed postmortem records for L.t. nordicus, L.t. tardigradus, N. coucang, N. pygmaeus, and P. potto born between 1980 and 2010 that lived in North

American zoos. Our primary aim was to describe major sources of mortality in these species. In a companion to this paper, we also surveyed facilities that are currently housing lorisid primates to determine how animals are actually cared for in practice

(Fuller et al., 2013). This study sets the stage for future hypothesis-driven research examining associations between environmental design, diet, health, and animal behavior. Ultimately, our goal is to identify husbandry concerns and areas of research most urgently needed to address the health, welfare, and population sustainability of lorises and pottos in the care of sanctuaries and zoos.

MATERIALS AND METHODS

Study Population

We requested full medical records from all North American zoos and related facilities for animals born between January 1, 1980 and December 31, 2010 representing five species: Loris tardigradus nordicus, L.t. tardigradus, Nycticebus coucang, N. pygmaeus, and Perodicticus potto. Animals and facilities included in these populations were identified by consulting North American Regional

Studbooks for each species as of February 2010. Species groups are based on

studbook data as well. L.t. nordicus and L.t. tardigradus have separate studbooks and were treated as separate populations here. In contrast, N. coucang and N. bengalensis have historically been grouped as one species in North American facilities and the population’s hybrid status is uncertain; for these reasons they are treated as a single population (referred to as N. coucang) here.

In total, one unaccredited zoo and 32 of 35 zoos (91%) accredited by the

Association of Zoos and Aquariums (AZA) where a lorisid primate died during this time period contributed medical records to this study, representing a total of 367 animals. The total number of records received for each species were: 20 (10.9.1)

(male.female.unknown) out of 25 L.t. nordicus (80%), 72 (31.29.12) of 80 L.t. tardigradus (90%), 109 (50.52.7) of 152 N. coucang (72%), 133 (59.57.17) out of

173 total N. pygmaeus (77%), and 33 (17.11.5) of 37 P. potto (79%). Across species, this sample included 167 males (45.5%), 158 females (43.0%), and 42 animals of unknown sex (11.4%).

The majority of records (241, 66%) were formatted using MedARKS or ARKS

(International Species Information Systems, Eagan, MN, USA) but the sample included other electronic formats and hand-written records. Additionally, one facility elected to provide summaries of necropsy and histopathology findings rather than submitting full records. Medical records included in this study varied greatly in detail and scope. Necropsy reports were available in 247 (67%) of the records received, and 214 (58%) included a histopathology report.

Organ System Classification and Cause of Death

Data for each animal were entered into a Microsoft Access® database. Age at death, sex, and death type (spontaneous, euthanasia, or unknown) were recorded for each animal. Based on their age at death, the animals within each species were categorized into one of four age groups: neonate, x ≤ 30 days old; juvenile, 30 days < x ≤ 1 year; and adult, 1 year < x ≤ geriatric; and geriatric. Age at which an animal was considered geriatric varied by species and was calculated as 75% of the mean lifespan of all animals in our study population that reached adulthood (i.e. excluding neonates and juveniles). L.t. nordicus were considered geriatric at 9 years old, L.t. tardigradus at 10y, N. coucang at 9y, N. pygmaeus at 8y, and P. potto at 12 years of age. One year was chosen as a common cutoff point between juveniles and adults based on published reports of age at first reproduction in these species (Charles-

Dominique, 1977; Fitch-Snyder and Schulze, 2001).

Diagnoses from necropsy and histopathology reports were used to determine which organ systems showed evidence of pathology at the time of the animal’s death. Organ system classifications were based on Hope and Deem (2006) and included the following categories: Cardiovascular and Hemolymphatic, Central and

Peripheral Nervous System (CNS), Dental, Endocrine & Metabolic, Ear, Nose &

Throat (ENT), Gastrointestinal, Hepatic & Biliary, Immunologic (including lymph nodes), Integumentary, Musculoskeletal, Ocular, Renal (including the urinary bladder), Reproductive, Respiratory, and Whole Body.

Records were also reviewed to determine the cause of each animal’s death or primary reason for being euthanized. If a cause of death was not clearly indicated in

the medical report, one was identified based on organ system pathology described in the record. In addition to the organ systems listed above, cause of death included the following categories specific to neonates: Maternal Neglect, Stillborn & Abortion, and Unexplained Neonate Death. Deaths for neonates were considered unexplained when no diagnosis was given, whether or not a necropsy and/or histopathology exam was performed (and available in the record). For all other age classes, the cause of death was only listed as Unknown if the record contained a necropsy and histopathology report but a primary cause of death was still unclear. When a histopathology report was not available and the cause of death was not clearly stated in the record, the cause of death was undeterminable and was listed as

Incomplete Record. For all age classes, in clear cases of environmental or social injury, the cause of death was listed as Trauma; even if death was more immediately attributable to septicemia or infection subsequent to the initial insult.

RESULTS

Age-Specific Patterns of Mortality

For all animals in this study, mean age of death, regardless of death type, was

7.7y (7.1 SD). The mean age at death for each species was: 8.9y (6.6 SD, range 0-

18.7) for L.t. nordicus, 8.0y (7.3, range 0-21.5) for L.t. tardigradus, 9.5y (6.8, range 0-

24.3) N. coucang, 6.4y (6.3, range 0-21.9) for N. pygmaeus, and 6.2y (9.0, range 0-

25.7) for P. potto. Taking into account only animals that reached adulthood, the mean age at death for each species was: 11.8y (4.7 SD, N=15) for L.t. nordicus, 12.7y

(5.1, N=45) for L.t. tardigradus, 11.8y (5.6, N=88) N. coucang, 10.1y (5.1, N=84) for

N. pygmaeus and 15.6y (7.3, N=13) for P. potto. Mean age at death for individuals that reached adulthood was nearly equal for males and females in every species tested, and there were no significant differences in lifespan based on sex.

For all the species in this study, death most commonly occurred spontaneously (Table 1) rather than as a result of euthanasia. Neonate mortality

(death within the first 30 days of life) was high for each species, most strikingly the potto, in which 60% of the sample population died by 30 days of age (Table 2). Of all the animals in this study, 30.2% (111/367) died as neonates. For all species, mortality during the juvenile period was relatively low. Generally, mortality was greatest in neonate and geriatric groups (Table 2).

Table 1. Recorded type of death for lorises and pottos in North American facilities 1980-2010.

Species Loris Loris Nycticebus Nycticebus All tardigradus tardigradus Perodicticus coucang pygmaeus Animals, nordicus tardigradus potto N=109 N=133, N=367, Death Type N= 20, % N= 72, % N=33, %(#) % (#) %(#) % (#) (#) (#) 30.0 Euthanasia 45.0 (9) 29.2 (21) 30.3 (33) 30.1 (40) 21.2 (7) (110) 65.4 Spontaneous 40.0 (8) 69.4 (50) 63.3 (69) 67.7 (90) 69.7 (23) (240) 4.6 Unknown 15.0 (3) 1.4 (1) 6.4 (7) 2.2 (3) 9.1 (3) (17)

Organ System Pathology and Cause of Death

For all the lorises and pottos in this study, multi-systemic disease was the most frequent cause of death or euthanasia, followed by renal disease and trauma

(Table 3). Many animals were stillborn or died as neonates due to maternal neglect or other unexplained reasons. Only 2-6% of deaths were attributed to diseases in each of the cardiovascular & hemolymphatic, gastrointestinal, hepatic & biliary, musculoskeletal, and respiratory systems. Less than 2% of deaths were attributed to each of the remaining organ systems.

Table 2. Distribution of loris and potto deaths by age class in North American facilities 1980-2010.

Species Loris Loris Nycticebus Nycticebus All tardigradus tardigradus Perodicticus coucang pygmaeus Animals, nordicus tardigradus potto N=109 N=133, N=367, Age Class N= 20, % N= 72, % N=33, %(#) % (#) %(#) % (#) (#) (#) Neonate 30.2 (x ≤ 30 25.0 (5) 31.9 (23) 15.6 (17) 34.6 (46) 60.6 (20) (111) days) Juvenile 5.2 (30 days < 0.0 5.6 (4) 3.7 (4) 2.3 (3) 0.0 (19) x ≤ 1) Adult (1 year 17.7 20.0 (4) 15.3 (11) 24.8 (27) 21.1 (28) 9.1 (3) < x ≤ (65) geriatric*) 46.9 55.0 (11) 47.2 (34) 56.0 (61) 42.1 (56) 30.3 (10) Geriatric (172) *Cutoff age for geriatric animals was based on 75% of the mean adult lifespan in this population: L.t. nordicus > 9y, L.t. tardigradus > 10y, N. coucang > 9y, N. pygmaeus > 8y, and P. potto > 12y.

For all animals (Table 4), the most common organ system showing lesions or abnormalities at death was the renal system, with 50% of all animals in the study affected. Over 40% of all lorisids sampled also showed pathologies in the respiratory and hepatic & biliary systems. Across species, organ systems in which more than 20% of animals showed signs of disease included the cardiovascular & hemolymphatic, endocrine & metabolic, gastrointestinal, and immunologic systems.

Specific diseases affecting more than 20% of animals in each species are detailed below.

Neoplasia was responsible for the deaths of 10.6% (39/367) of animals; all of these cases occurred in pygmy and slow lorises, with the exception of one potto

(Table 5). Medical records included 80 reports of distinct neoplastic growths in 66 animals; 39 of these were fatal. All neoplasms occurred in geriatric animals with the exception of two adult N. coucang and four adult N. pygmaeus. Both neoplasias reported for L.t. nordicus occurred in males as did five of six cases in L.t. tardigradus.

Neoplasia occurred equally between the sexes in N. pygmaeus (13 cases in males and 14 in females) and P. potto (1 male and 1 female), but was more common in female N. coucang (20 cases) than male (9 cases).

Most animals that experienced traumas were neonates that died as a result of bite wounds (Table 6). Only cases in which cannibalism clearly occurred prior to death were counted as traumas. Often cases were classified as unexplained neonate deaths because the carcass was scavenged but it was not possible to determine if this occurred pre- or postmortem. Several cases in which conspecific bite wounds led to necrosis, cellulitis, or other infection occurred, most commonly in Nycticebus

spp. All traumas thought to be related to mortality, even if cause of death was listed under another organ system, are detailed in Table 6.

Loris tardigradus nordicus

The primary cause of death was attributed to renal pathology in the majority of the L.t. nordicus in this study (Table 3). Records refer to renal failure (2/5) or glomerulonephritis, nephritis, and general glomerulopathy. There were no deaths attributed to neoplasia in this species, although some neoplastic growths were discovered in necropsy (Table 5).

The renal and cardiovascular systems were most commonly affected by disease in L.t. nordicus (Table 4). Animals suffering from renal disease had a group of diagnoses including nephritis, glomerulonephritis, fibrosis, renal infarcts, and distention. One case of cystitis was also included in the renal category.

Cardiovascular & hemolymphatic diagnoses included cardiomyopathy, leukocytosis, fibrosis, myocarditis, endocardiosis, and epicarditis. CNS diseases included two cases of hemorrhage and notes of age-related histological changes in the brain.

Ocular changes included synechiae, cataracts, blindness, and nerve fiber degeneration. Respiratory illnesses included pneumonia, serositis, mineralization, metaplasia, and one case of a benign bronchioalveolar adenocarcinoma. Whole Body changes of note included anorexia, serous atrophy of adipose tissue, and dehydration.

Table 3. Primary cause of death or reason for euthanasia in lorises and pottos housed in North American facilities 1980- 2010.

Loris Loris tardigradus Nycticebus Nycticebus Perodicticus Species tardigradus All Animals tardigradus coucang pygmaeus potto nordicus N=367 N=72 N=109 N=133 N=33 N=20 (167.158.42) (31.29.12) (50.52.7) (59.57.17) (17.11.5) (10.9.1) % (#) % (#) % (#) % (#) % (#) Cause of Death % (#) Cardiovascular & - 4.2 (3) 3.7 (4) 4.5 (6) 6.1 (2) 4.1 (15) Hemolymphatic Central Nervous System - 2.8 (2) - 2.3 (3) 3.0 (1) 1.6 (6) Endocrine & Metabolic - - 2.8 (3) 0.8 (1) - 1.1 (4) Ear, Nose, & Throat - - 2.8 (3) 0.8 (1) - 1.1 (4)

67 Gastrointestinal 5.0 (1) - 6.4 (7) 1.5 (2) - 2.7 (10)

Hepatic & Biliary 5.0 (1) 5.6 (4) 5.5 (6) 3.0 (4) - 4.1 (15) Immunologic - - - 0.8 (1) - 0.3 (1) Integumentary - 1.4 (1) - 1.5 (2) - 0.8 (3) Musculoskeletal - 2.8 (2) 2.8 (3) 5.3 (7) - 3.3 (12) Ocular 5.0 (1) - - 0.8 (1) - 0.5 (2) Renal 25.0 (5) 18.1 (13) 19.3 (21) 15.0 (20) 6.1 (2) 16.6 (61) Reproductive - - 3.7 (4) 0.8 (1) 3.0 (1) 1.6 (6) Respiratory 10.0 (2) 4.2 (3) 2.8 (3) 5.3 (7) 15.2 (5) 5.4 (20) Multi-systemic 5.0 (1) 19.4 (14) 22.9 (25) 15.8 (21) 15.2 (5) 18.0 (66) Trauma 5.0 (1) 13.9 (10) 10.1 (11) 10.5 (14) 9.1 (3) 10.6 (39)

Loris Loris tardigradus Nycticebus Nycticebus Perodicticus Species tardigradus All Animals tardigradus coucang pygmaeus potto nordicus N=367 N=72 N=109 N=133 N=33 N=20 (167.158.42) (31.29.12) (50.52.7) (59.57.17) (17.11.5) (10.9.1) % (#) % (#) % (#) % (#) % (#) Cause of Death % (#) Unknown - - 1.8 (2) 0.8 (1) - 0.8 (3) Incomplete Record 20.0 (4) 4.2 (3) 5.5 (6) 7.5 (10) 6.1 (2) 6.8 (25) Maternal Neglect 10.0 (2) 1.4 (1) 0.9 (1) 6.8 (9) 6.1 (2) 4.1 (15) (neonates) Stillborn/Abortion - 4.2 (3) 5.5 (6) 9.8 (13) 12.1 (4) 7.1 (26) (neonates) Unexplained Neonate 10.0 (2) 18.1 (13) 3.7 (4) 6.8 (9) 18.1 (6) 9.3 (34) Death (neonates)

Table 4. Percent of lorises and pottos with pathology diagnosed by organ system upon postmortem examination 1980- 2010.

Species Loris tardigradus Loris tardigradus Nycticebus Nycticebus Perodicticus All

nordicus tardigradus coucang pygmaeus potto Animals

N=20 N=72 N=109 N=133 N=33 N=367

% (#) % (#) % (#) % (#) % (#) % (#) Organ System Cardiovascular & 30.0 30.0 (6) 26.4 (19) 33.0 (36) 32.3 (43) 18.2 (6) Hemolymphatic (110) 16.6 Central Nervous System 20.0 (4) 16.7 (12) 15.6 (17) 16.5 (22) 18.2 (6) (61) 3.5 Dental 5.0 (1) 4.2 (3) 4.6 (5) 3.0 (4) 0.0 (13) 22.6 Endocrine & Metabolic 15.0 (3) 12.5 (9) 35.8 (39) 19.5 (26) 18.2 (6) (83) 9.3 69 Ear, Nose, & Throat 15.0 (3) 4.2 (3) 14.7 (16) 6.0 (8) 12.1 (4) (34)

23.4 Gastrointestinal 15.0 (3) 8.3 (6) 36.7 (40) 22.6 (30) 21.2 (7) (86) 43.3 Hepatic & Biliary 15.0 (3) 47.2 (34) 51.4 (56) 42.9 (57) 27.3 (9) (159) 22.1 Immunologic 5.0 (1) 9.7 (7) 35.8 (39) 23.3 (31) 9.1 (3) (81) 5.7 Integumentary 0.0 6.9 (5) 5.5 (6) 7.5 (10) 0.0 (21) 16.9 Multi-systemic 5.0 (1) 19.4 (14) 22.0 (24) 14.3 (19) 12.1 (4) (62) 13.1 Musculoskeletal 10.0 (2) 11.1 (8) 14.7 (16) 15.0 (20) 6.1 (2) (48) 9.3 Ocular 25.0 (5) 19.4 (14) 4.6 (5) 6.8 (9) 3.0 (1) (34) 49.9 Renal 50.0 (10) 41.7 (30) 57.8 (63) 51.9 (69) 33.3 (11) (183)

Species Loris tardigradus Loris tardigradus Nycticebus Nycticebus Perodicticus All

nordicus tardigradus coucang pygmaeus potto Animals

N=20 N=72 N=109 N=133 N=33 N=367

% (#) % (#) % (#) % (#) % (#) % (#) Organ System 15.0 Reproductive 15.0 (3) 9.7 (7) 22.9 (25) 12.8 (17) 9.1 (3) (55) 44.1 Respiratory 25.0 (5) 47.2 (34) 46.8 (51) 46.6 (62) 30.3 (10) (162) Whole Body 20.0 (4) 12.5 (9) 11.0 (12) 17.3 (23) 18.2 (6) 14.7 (54)

Loris tardigradus tardigradus

Cause of death in the red slender loris was most commonly attributed to multi-systemic disease (Table 3). Many of these deaths (5/14) were attributed to septicemia, one to hepatitis secondary to bacterial infection, and one to infection- associated granulomatous lesions attacking multiple organ systems. In the remaining cases, animals were geriatric or had problems affecting multiple organ systems with no obviously primary condition. Deaths involving the renal system were also common and were attributed generally to renal failure (6/13), fibrosis

(1/13), or glomerulonephritis or nephritis (6/13).

Trauma and neonatal mortality were significant in this group as well (Table

6). Of the ten deaths attributed to trauma, two were bite wounds (with subsequent infection in one case) that lead to juvenile deaths, and six others were neonate deaths. Traumatic deaths in neonates occurred due to traumatic bone injury (1/9), aggression from a neighboring animal (1/9), and within-group aggression leading to bite wounds and death (5/9). Additionally, one neonate death was attributed to maternal neglect and 14 were unexplained. Like L.t. nordicus, there were no deaths attributed primarily to neoplasia in L.t tardigradus.

Overall, the most common pathologies affecting L.t tardigradus occurred in the hepatic & biliary, and respiratory systems (Table 4). Liver disease was common and included cirrhosis, hepatitis, hyperplasia, congestion, fibrosis, lipidosis, vacuolar change and degeneration, and one presumptive case of hemosiderosis. Reports of liver neoplasias included one hepatoma and a hepatocellular adenoma. One animal reportedly suffered from an obstruction in the gall bladder and another from

cholangitis. In the respiratory system, pneumonia was commonly reported. Other respiratory changes included atelectasis, hemorrhage, congestion, hemosiderosis, edema, fibrosis, and bronchitis. Cardiovascular issues included congestive heart failure, myocardial atrophy, fibrosis, myocarditis, and congestion. Vascular problems were also common including vasculitis, histiocytosis, arteriopathy, atherosclerosis, and thrombosis. Renal disease was a common finding, and in addition to the common renal changes previously listed for L.t. nordicus, there was one report of a renal cyst and one of a renal cell carcinoma (Table 5).

Nycticebus coucang

Like the red slender loris, the most common causes of death for slow lorises were multi-systemic and renal diseases (Table 3). Neoplasia (Table 5) affecting multiple organ systems accounted for several (6/23) cases and included two adenocarcinomas and two lymphosarcomas. Septicemia was responsible for three deaths and infections for five. One animal reportedly died from toxicosis but a toxin was never identified, and the remaining cases were all attributed to non-infectious diseases processes (often age-related) affecting multiple organ systems. Renal diseases included two neoplasias, cases described generally as renal failure (7/21), and nephritis, including pyelonephritis and glomerulonephritis (12/21).

After renal and multi-systemic disease, trauma was the most frequent cause of death in slow lorises (Table 6). Most victims of trauma (6/11) were neonates. One animal was euthanized due to pathological maternal over-grooming, and the others died following bite wounds and neglect from parents. Two juveniles died of trauma

as a result of parental aggression, one from multiple skull fractures and the other from necrosis and cellulitis resulting from bite wounds. Three adults died as a result of traumatic events. One animal developed cellulitis secondary to wounds received from a conspecific, while another escaped an enclosure and was attacked by an ocelot. A four-year old male succumbed to acute heart failure following an immobilization made necessary because the loris had become entangled in a cargo net used as exhibit perching.

Table 5. Neoplasia reported for lorises and pottos in North American facilities 1980-2010. Cases marked * were cited as the primary cause of death or reason for euthanizing the animal.

Species Loris Loris tardigradus Nycticebus tardigradus Nycticebus coucang Perodicticus potto nordicus pygmaeus tardigradus 34 cases 2 cases 4 cases 34 cases Organ System 6 cases Cardiovascular & - - - - sarcoma (heart) - Hemolymphatic - astrocytoma (brain Central Nervous stem*) - - - ependymal (brain) - System - ependymoma (cerebrum*) - adenoma - 2 adenoma - adenoma (adrenal) - adenoma (thyroid) Endocrine and (parathyroid) (1 adrenal, 1 - carcinoma - sarcoma - Metabolic - myelolipoma thyroid) (pancreas*) (pancreas) (adrenal) 74 - squamous cell - squamous cell Ear, Nose & Throat - - carcinoma - carcinoma (tongue*) (larynx/pharynx*) - sarcoma - lymphosarcoma Gastrointestinal - - - (large intestine*) (small intestine*) - 2 adenoma (both - adenocarcinoma - adenoma liver) (liver) (liver) - 4 carcinoma Hepatic & Biliary - - adenoma (liver*) - - hepatoma (all liver***) - lymphosarcoma, (liver) - unspecified mass leukemic (liver*) (liver*) - lymphoma (spleen) Immunologic - - - - 2 lymphosarcoma - (both spleen*) Integumentary - spindle cell - - - - sarcoma (dorsum*)

Species Loris Loris tardigradus Nycticebus tardigradus Nycticebus coucang Perodicticus potto nordicus pygmaeus tardigradus 34 cases 2 cases 4 cases 34 cases Organ System 6 cases - fibroma (tarsal - adenocarcinoma region) Musculoskeletal - - (skeletal muscle*) - 3 fibrosarcoma (2 - - spinal mass lumbar**, 1 elbow*) osteosarcoma (rib*) - melanoma Ocular - - - - (iris) - leiomyosarcoma - adenocarcinoma - carcinoma (bladder*) - adenocarcinoma Renal - (kidney*) (kidney) - spindle cell (kidney) - carcinoma (bladder*) sarcoma (kidney*) - adenoma (seminal vesicle) - 2 adenocarcinoma (1 75 - 2 carcinoma (1

mammary, 1 uterus*) testis, 1 uterus) - carcinosarcoma -2 leiomyoma (both (uterus*) uterus*) - granulosa cell - 2 leiomyoma (both - carcinoma Reproductive - - granulosa cell tumor (ovary) uterus*) (endometrium*) tumor (ovary) - multiocular mass - Leydig cell tumor (mammary) (testis) - prostate mass - 2 unspecified - uterine mass* testicular mass

Respiratory

- 2 adenoma (both - carcinoma - lung) - carcinoma (lung*) - (bronchioalveolar) - carcinoma (lung)

Species Loris Loris tardigradus Nycticebus tardigradus Nycticebus coucang Perodicticus potto nordicus pygmaeus tardigradus 34 cases 2 cases 4 cases 34 cases Organ System 6 cases -unspecified mass - unspecified mass (abdominal*) (abdominal) - lymphoma* - 2 adenocarcinoma ** - lymphosarcoma - - - 2 lymphosarcoma** - Multi-systemic (leukemic) - sarcoma* - metastatic - transitional cell histiocytic round carcinoma cell tumor*

Neoplasia was common in slow lorises; including the cases above, 17.4% of animals (19/109) died or were euthanized due to neoplasia, meaning it was one of the leading causes of death in captive slow lorises (Tables 3,5). All three reproductive neoplasia affected females and were uterine in origin. Other notable causes of death were two probable cases of diabetes mellitus. Four animals died from focal infections: myocarditis, otitis, rhinitis, and a chronic abscess of the thigh muscle.

Overall, more than 45% of slow lorises showed some lesions or abnormality in their hepatic & biliary, respiratory, and renal systems. Common liver diseases reported were hemosiderosis, hepatic lipidosis, hepatitis, and vacuolar change. The most common renal lesions were due to nephritis, glomerular or pyelonephritis.

Diseases affecting the bladder occurred in eight animals, including cystitis (six cases), serositis, and two bladder cancers. Pneumonia was the most common respiratory finding, although several neoplasias were reported in this organ system as well (Table 5).

Several other organ systems showed more than a 20% prevalence of disease.

Cardiovascular disease noted at death included cardiomyopathy, fibrosis, endocardiosis, and myocarditis. Diseases of the endocrine & metabolic system targeted the adrenal glands (adrenalitis, amyloidosis, hemosiderosis), pancreas

(mainly hyperplasia), and thyroid (goiters, cysts). The most common gastrointestinal diseases were enteritis and gastritis, in addition to parasitism in the

GI tract. Immunological changes noted at death included histiocytosis, hyperplasia, and neoplasia of the lymph nodes, and splenitis and congestion affecting the spleen.

Nine animals showed evidence of hemosiderosis affecting the spleen and/or lymph nodes. Reproductive diseases affected females more than males and included cervicitis, pyometra, plancentitis, and ovarian cysts. Reproductive neoplasms occurred in several animals (Table 5), including cancers of the uterus, ovary, and prostate. Two geriatric animals- one male and one female- had mammary neoplasms. In general, cases of neoplasia were largely limited to geriatric animals.

Nycticebus pygmaeus

Neonatal mortality had a significant impact on the study group, accounting for 34.6% of deaths in our sample of pygmy slow lorises (Tables 2, 3). Trauma

(11/46) and maternal neglect (9/46) caused neonate deaths in 43.3% (20/46) of cases. When cases of unexplained neonate death that reference carcass scavenging are included, this total increases to 50%. Traumas experienced by neonates included head trauma (4/11), inter-group aggression (1/11) and intragroup aggression (6/11). In several cases neonates were cannibalized, and one infant was euthanized after losing several limbs. Four mature pygmy lorises died as a result of trauma. Two adults died from what was thought to be an acute toxicosis, but a toxin was never identified. One geriatric male died of septicemia following infection from an armadillo bite, and a 3y-old gravid female was killed by an ocelot after escaping her home enclosure (Table 6).

The most common causes of death in pygmy slow lorises were renal and multi-systemic diseases. Renal diseases cited as the cause of death were renal failure (6/20), nephritis (10/20), and one case in which a kidney abscess led to

peritonitis and eventual death. Two animals died from complications related to cystitis, which led to bladder necrosis in one case. Renal neoplasms were considered the primary cause of death for two animals (Table 5): one geriatric male (14y) developed a spindle cell sarcoma in the kidney, while an adult (7y) female was euthanized due to a leiomyosarcoma of the bladder. Three additional neoplasias were listed as multi-systemic deaths (3/19), all for geriatric animals. Other deaths coded as multi-systemic included cases of septicemia (7/19), other infectious processes (5/19), and non-infectious disease processes and aging (6/19).

Neoplasias reported as the primary cause of death were found in nearly every organ system and accounted for 14.3% (19/133) of pygmy loris deaths overall (Table 5).

The organ systems most frequently affected at death for the pygmy loris are described in Table 4. Organ systems which showed pathology for more than 20% of animals were the cardiovascular & hemolymphatic, gastrointestinal, hepatic & biliary, immunologic, renal, and respiratory systems. Neoplasia was common throughout all organ systems (Table 5).

Table 6. Circumstances surrounding traumas related to death in lorises and pottos in North American zoos 1980-2010.

Species Loris tardigradus Loris tardigradus Nycticebus Perodicticus Nycticebus coucang nordicus tardigradus pygmaeus potto Trauma type - 5 neonates (one - 1 neonate from conspecific - 5 neonates (1 killed by - 6 neonates (housed with dam neighbor, others dam, 2 suspect sire or (1 suspect sire; 1 and other (?) could have been dam Bite wounds and other male; 2 could have conspecific cagemates, bite or sire) - 1 neonate cannibalism been dam or sire) neighbor; housing wound and fall - 2 juveniles (one - 1 juvenile (suspect male situation unclear during intragroup from cagemate, cagemate) for others) aggression) other could have been dam or sire) Infection/disease secondary to bite - 1 juvenile (cellulitis and 80

wound necrosis on hands and - 1 neonate feet, bite from dam or (septicemia from male neighbor (?)) hand mutilated by - 1 adult - 3 adults (1 severe mother) (septicemia) necrotizing myositis of - 1 juvenile - 2 geriatric (1 bite wound from (cellulitis, history septicemia from cagemate; 1 cellulitis of chronic, poorly month-old bite - from cagemate bite; 1 healing bite - wound; 1 lung had chronic sinusitis wounds) edema possibly resulting from bite - 1 geriatric related to bite received as an infant (purulent abscess wound thought to from dam); from bite wound) have healed) - 1 geriatric (euthanized - 1 geriatric for neoplasia, also had (infected bite from other recent problems armadillo) including bite wounds)

Species Loris tardigradus Loris tardigradus Nycticebus Perodicticus Nycticebus coucang nordicus tardigradus pygmaeus potto Trauma type Cases of scavenging presumed to have - 1 neonate - 10 neonates - 1 neonate - 6 neonates - 2 neonates occurred postmortem - 4 neonates (3 incurred during intragroup aggression) - 1 neonate - 1 juvenile Head trauma/ - 1 adult (related to - - (possible fall - 2 neonates traumatic fall metabolic related to maternal condition?) neglect?) - 1 geriatric (spinal

81 fracture, age-

related?) - 1 adult with fetus (interspecific - 1 neonate (maternal predation) over-grooming) - 1 geriatric (bite - 1 adult (interspecific wounds Other - - predation) - contributed to - 1 adult (accident decision to involving cage euthanized, cause furnishings) of death listed as renal failure)

Perodicticus potto

Most of the pottos in this study died as neonates (Tables 2,3). All reported cases of trauma involved neonates, and two of seven unexplained neonate deaths involved scavenged bodies. Two additional neonate deaths were attributed to maternal neglect and one to a systemic infection. Additionally, three of five respiratory deaths occurred in neonates.

The majority of other deaths occurred in geriatric animals. Multi-systemic causes of geriatric death were all related to multiple organ failure with associated shock. Other geriatric deaths were attributed to cardiomyopathy, endocarditis, renal failure, and one endometrial carcinoma representing the only death attributed to neoplasia in all pottos (Table 5).

Lesions occurring in more than 20% of animals were found in the gastrointestinal, hepatic & biliary, renal, and respiratory systems (Table 4).

Gastrointestinal pathologies included esophagitis, enteritis, and colitis. Liver conditions reported were cirrhosis, hepatitis, congestion, and necrosis. Most renal pathologies were varieties of nephritis; however, a geriatric male and female were both diagnosed with polycystic renal disease. The majority of respiratory disease was pneumonia. Other interesting lesions discovered at death included four animals with rhinitis, and extensive arteriosclerosis in three animals.

DISCUSSION

We reviewed medical records for 367 lorises and pottos that died in North

American zoos over a 30-year period. Our results clearly show that poor neonate

survivorship is a major concern both for individual health and welfare, as well as population management. However, animals that survive the critical first month of life are likely to reach sexual maturity and live into the geriatric stage. Adult and geriatric animals are most likely to die of renal disease or multi-systemic issues such as systemic infections, neoplasia, or multiple organ failure. High neonate mortality was due to high stillbirth percentages as well as trauma, which was often inflicted by conspecifics. Adults also fall victim to trauma from cagemates, suggesting that greater efforts to address the social management of lorises in captivity would likely have positive impacts on population sustainability and animal health.

Other studies have also found exceptionally high infant mortality in lorisiform primates living in both zoo and sanctuary settings. Streicher (2004) summarized health problems experienced by pygmy lorises confiscated from the wildlife trade in Vietnam. Of the 15 deaths reported over an 8-year period, 11 were animals less than 1 year old; overall, 42% of the sanctuary animals died during their first year of life. Debyser (1995) found infant mortality to be higher among lorises and their close relatives compared to other strepsirrhines, and like others

(Streicher, 2004; Tartabini, 1991) speculated that captivity-induced stress was likely an underlying cause of infant deaths involving maternal neglect and trauma.

Information about social conditions was difficult to infer from our medical records, but it was clear that infants in this group were killed by sires, dams, and other conspecifics. Several different fitness benefits have been hypothesized to explain infanticide among nonhuman primates, including male-male competition and resource competition (Hrdy, 1979). Infanticide risk may be related to social

density, which is interesting considering that cage sizes for captive pottos and slender lorises in North America are currently smaller than the minimum size recommended by Fitch-Snyder and Schulze’s (2001) husbandry manual (Fuller et al.

2013). Deaths involving maternal neglect in this study hint at a pattern similar to that described for captive Galago crassicaudatus umbrosus by Tartabini (1991).

Failure to perform maternal behavior, for example due to poor socialization or stress related to the captive setting, leads to infant starvation and death, and the dead infant becomes a resource to consume (Tartabini, 1991). If this is the case, then careful attention to postpartum maternal behavior and if necessary, swift intervention may be important for saving infant lives. Maternal parity and litter sizes are also associated with infant survivorship (Pollock, 1986), and new mothers or multiple births will likely require special attention.

Management of group composition around the perinatal period is likely to have a large impact on infant survivorship. Nekaris (2003a) speculates that paternal care may be one factor promoting the relatively greater fecundity of slender than slow lorises, and paternal care has also been observed in captive pottos (Frederick,

1998). These kinds of socialization opportunities are important for the formation and maintenance of social groups, and the 2001 husbandry manual states that it is preferable to keep groups intact through births unless there is cause to suspect an individual needs to be removed (Fitch-Snyder & Schulze 2001). However, it is possible that social management practices surrounding parturition have changed in recent years, perhaps to address traumatic infant death. A 2010 survey of AZA facilities housing lorisids showed that very few males and females were housed

together during the period surrounding birth, although more facilities indicated that they had attempted this strategy in the past (Fuller et al. 2013). This survey also showed that a large number of animals in each species were solitarily housed on a perpetual basis (Fuller et al., 2013). It may be that efforts to increase the social wellbeing of captive lorisids by providing social partners may be in conflict with strategies needed to minimize infant mortality. Given the tenuous status of these captive populations, isolating gravid females is probably a sound strategy until the proximate causes underlying infanticide are better understood.

It was often not possible in our study to distinguish between cases of infanticide, neglect, or trauma, or other possible causes of death; because infant carcasses were cannibalized. Although recovery of nutritional resources is a possible explanation for cannibalism, this behavior has been rarely observed in wild primates; if cannibalism is truly more prevalent in captive and reintroduced animals, then it is likely pathological in nature (Dellatore et al., 2009; Tartabini,

1991). However, lack of tissues for necropsy renders it impossible to determine if infants actually died due to congenital or other disease states, or if infants were neglected by mothers because they were weak or ill. Greater efforts should be made to promptly remove deceased infants so that postmortem exams can clarify these issues.

Traumas were a significant contributor of mortality to adult as well as immature animals. In several cases, animals died following bite wounds that were chronically non-healing, leading to necrosis and cellulitis. In one case, a 3y-old adult slow loris died from chronic sinusitis from an infection that originated with a bite

wound sustained from his mother as a neonate. It has been speculated that a chemical produced by the brachial gland, mixed with slow loris saliva, may be the source of toxicity in bites (Krane et al., 2003). Necrotic bite wounds have also been reported in sanctuary-housed pygmy lorises (Streicher, 2004), and more information on the chemical structure and physiological role of loris venom may aid caretakers in the treatment and management of wounds.

Across the species in this study, renal disease was the second most common cause of death, and the renal system exhibited the greatest frequency of lesions at death compared to all other organ systems. Renal changes such as glomerulosclerosis and nephritis are known to be prevalent in captive strepsirrhines and are often age-related (Burkholder 1981, Fitch-Snyder & Schulze

2001, Junge 2003). Renal disease appears to occur more frequently in lorisoids

(lorises, pottos, and galagos) than lemur species (Boraski, 1981). Iron storage disease, or hemosiderosis, is a common pathology in lemurs (Benirschke et al.,

1985) but was not very prevalent in the lorises and pottos in this study. A review of necropsies of L. tardigradus housed at a German university revealed several cases of cholelithiosis or gallstones, all of which were composed of cholesterol and were speculated to be related to dietary factors like the presence of egg yolk and the reduced insect composition in the captive diet (Plesker and Schulze, 2006).

Of the two cases of gallstones in this study, one occurred in L.t. tardigradus and one in L.t. nordicus, but none were reported for slow lorises or pottos. We also recorded one case of diabetes mellitus in L.t. tardigradus, five in N. coucang, and two in N. pygmaeus. Captive lorises are prone to obesity (Ratajszczak 1998, Fitch-Snyder

& Schulze 2001), but body condition was not reported consistently at death so its role in the development of degenerative diseases is not evident here.

It seems likely that diet is a contributing factor to non-infectious and degenerative diseases in captive lorises. The low metabolic rate characteristic of lorises is associated with a natural diet high in toxic secondary compounds from insects and plants (Wiens et al., 2006). It is tempting to speculate that metabolic derangement may occur when a diet this specialized cannot be replicated in the captive setting. The Mysore slender loris (Loris lydekkerianus lydekkerianus) feeds almost exclusively on animal prey, many of which are toxic species (Nekaris and

Rasmussen, 2003). New data from the field has highlighted the importance of nectars and exudates in the slow loris diet (Nekaris, 2009; Tan and Drake, 2001); at one study site in India, N. bengalensis fed almost exclusively on exudates during the winter season (Swapna et al., 2010). Lorises often obtain gums by gouging at trees, and the lack of opportunities to perform this behavior in captivity may be associated with periodontal disease (Nekaris, 2009).

We were surprised by how rarely dental disease was indicated on histopathology and necropsy reports of the animals we studied. It seems likely that in this study, many necropsy reports did not include notes of missing teeth or gingival disease that may have occurred many years earlier and/or seemed unrelated to the immediate cause of death. Dental disease has previously been reported as a major source of morbidity for Asian lorisines in captivity (Sutherland

Smith and Stalis, 2001). In a sample of 25 L. tardigradus at a German university, 7 individuals had missing or loose teeth, or severe calculus at death, and another 4

animals showed evidence of inflammation and infection secondary to dental disease

(Plesker and Schulze, 2013). In this study, several cases were noted in which a retrobulbar or other facial abscess occurred secondary to dental infection. Dental disease may play an under-recognized role in loris health, and more efforts should be made to institute preventative care during routine exams (Plesker & Schulze

2013) and to identify dental abnormalities or infections at necropsy.

Around ten percent of the animals in our sample died as a result of neoplasia.

A malignant lymphoma that occurred in a slow loris in this sample has been previously described elsewhere and was thought to be caused by a herpes viral infection (Stetter et al., 1995). Our sample also overlaps with neoplasias reported for the Duke Lemur Center (DLC), which contributed records to this study, and at which cases of neoplasia have been thoroughly described (Remick et al., 2009;

Zadrozny et al., 2009). However, cases of hepatic neoplasia were reported from four additional facilities in this study, suggesting liver cancer may play an important role in mortality for AZA loris populations as a whole. Our results concur with those reviewed by Remick et al. (2009) that neoplasia in lorises and pottos, like other strepsirrhines, commonly occurs in the digestive (including liver), hematopoietic

(here these were generally classified as multi-systemic), and reproductive systems.

Little research has addressed the etiology of cancers affecting captive strepsirrhines, and further research into the role of diet (Bingham et al., 1976; Cowgill et al., 1989), exhibit lighting (Navara & Nelson 2007, Fuller et al. 2013), and other factors contributing to neoplasia is needed.

After reviewing death records spanning 30 years for all lorises and pottos in

North American zoos, it is clear that social management remains a challenge in these species. Deaths due to trauma are common and most likely to affect neonates and juveniles before they have the opportunity to reproduce. These trends suggest that targeting efforts to improve infant survival could have major benefits for the sustainability of captive populations. Animals that live to adulthood are likely to die as a result of infectious agents, neoplasia, or degenerative changes. Renal disease remains a major source of pathology for all lorises and pottos, and further investigation is needed to understand its etiology. A greater understanding of the role of diet in the development of non-infectious disease is also needed.

Chapter Four

Validating Actigraphy for Circadian Monitoring of Behavior in the Pygmy Loris (Nycticebus pygmaeus) and Potto (Perodicticus potto)

INTRODUCTION

Animals have evolved internal time keeping systems, or biological clocks, that allow them to predict and respond to regular patterns of environmental change that occur on a daily or seasonal basis (Rietveld et al., 1993). Biological clocks coordinate changes in physiology and behavior with environmental conditions to which they are synchronized by ambient light levels or other reliable ‘zeitgebers’

(German for ‘time keeper’) (Challet, 2007). Internal clocks also have the flexibility to adjust to acute, short-term environmental changes termed ‘masking’ agents (Erkert,

2008; Rietveld et al., 1993). The interactions between internal clocks, zeitgebers, and masking agents produce rhythmic outputs of behavior in the form of activity patterns (Fernandez-Duque, 2003). Investigating how biological clocks interact with ecological factors to shape animal activity patterns has been termed ‘chronoecology’

(Halle and Stenseth, 2000a). In primatology, this theoretical approach has illuminated how environmental conditions, including light and temperature, interface with predator behavior, morphology, metabolic needs, and other forces to structure primate activity patterns in a complex, species-specific manner (Bearder et al., 2002; Curtis and Rasmussen, 2006; Fernandez-Duque, 2003).

All species in the Family Lorisidae (Loridae) (Groves, 2001) are small-bodied, solitary, nocturnal foragers that consume high-energy diets comprised of fruits,

exudates, and small animal prey (Bearder, 1987). Lorisids are adapted to a slow way of life; they have a low basal metabolic rate, low activity rates, and low levels of energy expenditure (Wiens et al., 2006). Lorisids climb slowly along substrates in a quadrupedal stance, a locomotor pattern which may serve to aid in the detection of food or avoidance of predators, or promote olfactory marking behavior (Oates,

1984). Studies of captive slow lorises (Nycticebus coucang, Kavanau, 1976) and free-ranging pottos (Perodicticus potto, Charles-Dominique, 1977) indicate that patterns of activity in these nocturnal species are largely regulated by photic conditions. Additionally, their nighttime activity periods are often punctuated with periods of inactivity (Charles-Dominique, 1977); for example, Nekaris (2001) found that the Mysore slender loris (Loris tardigradus lydekkerianus), spends up to 46% of its nighttime active period inactive. In short, lorisids are slow, inactive animals that may be sensitive to light.

AZA-accredited zoos in North America currently house four lorisid species: the African potto and the Asiatic slender (L. tardigradus), slow, and pygmy

(Nycticebus pygmaeus) lorises. Several lines of evidence suggest that husbandry practices alter activity patterns in these species. Potto activity levels are affected by exhibit lighting design (Frederick and Fernandes, 1994) and complexity (Frederick and Fernandes, 1996), and slow lorises are also more active in larger, more enriched cages (Daschbach et al., 1982/83). Oswald and Kuyk (1978) found that activity in three slow lorises peaked during hours that visitors were present in their nocturnal house, and Kavanau (1976) found that the time of onset of nocturnal activity was affected by extreme temperatures in an outdoor colony of slow lorises. From a

chronoecological perspective, husbandry and housing practices represent both artificial zeitgebers and masking agents which interact to shape the activity patterns of captive animals (Richter, 2006).

Because a chronoecological approach requires behavioral monitoring across the 24-hour cycle, it is necessary from a practical standpoint to have an automated means of data collection. It is becoming more common in zoos to utilize video for data collection, and time-lapse video techniques can be used to examine behavior over long time spans, while infrared technology facilitates observations on nocturnal species (London et al., 1998). The utility of video may depend on the quality of the technology employed; Munoz-Delgado et al. (1995) were able to study behavioral sleep in stumptail macaques (Macaca arctoides) by using high sensitivity video equipment to record subtle behaviors such as eye movements and myoclonus.

High quality video equipment can be expensive, and in zoo exhibits, it may be difficult to visualize entire exhibits without installing multi-camera systems.

Another potential disadvantage of video observation is that a live observer is still needed to score behaviors from video recordings at a later time, so the procedure can be time intensive. In some cases, actigraphy may be a preferable option over video observation.

Actigraphs are small accelerometers which can be used to track animal activity patterns when worn by subjects (Mann et al., 2005). Actigraphs record gross movement patterns by means of an omni-directional sensor that records the degree and speed of motion over pre-determined sampling epochs. The average level of activity over a given epoch is calculated by integrating the intensity, number, and

duration of accelerations and is reported as an activity count (Muller and Schrader,

2003).

Actigraphs have been employed for activity monitoring in a variety of species, including African elephants (Loxodonta africana, Rothwell et al., 2012), elk

(Cervus elaphus nelsonii, Naylor and Kie, 2004), dairy cows (Muller and Schrader,

2003), and human children (Finn and Specker, 2000). They have been widely used with nonhuman primates such as rhesus monkeys (Macaca mulatta, Papailiou et al.,

2008), spider monkeys (Ateles geoffroyi, Munoz-Delgado et al., 2005), callitrichids

(Kantha and Suzuki, 2006b), and a single red-ruffed lemur at a zoo (Varecia variegata rubra, Sellers and Crompton, 2004). Fernandez-Duque and Erkert (2006) also measured lunar variation in patterns of activity in free-ranging owl monkeys

(Aotus azarai azarai) wearing accelerometers on collars. Actigraphs are also widely used for monitoring sleep patterns in a variety of species (Kantha and Suzuki,

2006b). However, the utility of actigraphy for behavioral monitoring in lorisids may be compromised by the exceptionally slow locomotor patterns of these species. The location where the device is placed on the animal can also affect activity recordings

(Muller and Schrader, 2003; Papailiou et al., 2008), so it is important to validate actigraph measures for each new species tested.

The goal of this study was to validate the use of actigraphy for monitoring behavior in the pygmy loris and potto. We first sought to assess if wearing the actigraph device compromised animal safety or produced changes in behavior. We then examined whether different intensities of activity recorded by the actigraph device were correlated with specific behaviors, in order to understand the

resolution with which this technique can be applied for behavioral monitoring.

Ultimately we aimed to validate methods that may be widely applicable to investigating topics such as captive animal welfare, mechanisms controlling behavioral rhythmicity, and environmental forces that shape animal behavioral patterns in captive and field settings.

MATERIALS AND METHODS

Subjects and Housing

We worked with several different individuals to conduct initial pilot tests of the actigraph harness. We first tested the harness alone using an 18-yr old female slow loris. We also attempted to use the actigraph harness with two additional male pygmy lorises, one of whom was three yrs old and the other eleven years. However, none of these animals wore the harness for an extended period of time, and these individuals are not included in any data analysis.

Our subjects were a single male pygmy loris (nine yrs old) and a male potto

(16 yrs), which both wore the actigraph harness for extended periods of time. Both individuals were born in captivity and parent reared. These animals were housed in separate exhibits in the Primate, Cat, and Aquatics building at Cleveland Metroparks

Zoo (CMZ) in Cleveland, Ohio, and tests were conducted during October and

November, 2009.

Figure 1. a) Attaching the actigraph harness to the pygmy loris subject; b) the potto wears the actigraph harness on exhibit.

The pygmy loris was housed alone in an exhibit measuring 2.6 x 3.0 x 2.9 m.

The exhibit contained a wooden nest box, gunnite rock fixtures, wooden perches covered with artificial vegetation, and mulch substrate. The loris was housed on a

12:12 light-dark (LD) cycle with the dark phase from 1000-2200 hrs. Light phase illumination was provided by fluorescent and high-intensity discharge lamps, and dark phase lighting with fluorescent fixtures covered by blue gel filters.

The potto was housed with a nulliparous female potto (a potential breeding partner) in a similarly furnished exhibit that measured 2.9 x 4.9 x 4.3 m. The exhibit also contained a breeding pair of black and rufous elephant shrews (Rhynchocyon petersi). Lighting conditions for the potto were the same as for the pygmy loris, except that dark phase onset occurred at 2200 hrs in this exhibit and fluorescent lights were covered with both red and blue gel filters. Both the potto and pygmy loris were fed a single meal each morning consisting of LabDiet New World Primate

Diet 5040 (PMI Nutrition International, St. Louis, MO), superworms (Zophobas

morio), and mixed produce including sweet potatoes, apples, carrots, bananas, and endive.

Activity Recording

We collected activity data using a tri-axial accelerometer, the ActiSleep

(ActiGraph, Pensacola, FL). The ActiSleep weighs 18 g and measures 43.2 x 38.1 x

14.73 mm. The ActiSleep records accelerations ranging from 0.05 to 2.5 G which are digitized and integrated into activity counts over a user-defined epoch. We used a

30-sec sampling epoch, which is slightly more frequent than prior research with small primates (Kantha and Suzuki, 2006b), to ensure adequate sampling of the infrequent, brief bouts of activity characteristic of our study subjects.

The study subjects were fitted with a small pet harness (Super Pet, Elk Grove

Village, IL) to which the ActiSleep monitor was attached dorsally using heavy-duty

Velcro and a fabric strap (Figure 1) (Kantha and Suzuki, 2006b; Sellers and

Crompton, 2004). Zookeepers fitted the actigraph harness onto the animals while they were awake using manual restraint. We attached the harness to the animals during the active (dark phase) period to avoid disrupting their sleep-wake cycles.

The total weight of the ActiSleep and harness was 31.4 g for the loris and

39.2 g for the potto. This weight represented 5.9% of total body weight for the pygmy loris (532 g) and 2.8% for the potto (1381 g). Although the general consensus among field researchers is that radio collars should represent no more than 5% of animal body weight, Gursky (1998) found that tarsiers (Tarsius spectrum), which are extremely small-bodied (~150 g) tolerated collars up to 7% of

their body weight without any detectable behavioral deficits. Thus, we considered the harness weights to be within an appropriate range for our animals, which were also continuously monitored for the first day that they wore the harness for signs of discomfort or compromised safety. The animals were also visually inspected to ensure that the harness did not cause abrasions or other irritation. This research was reviewed and approved by the Animal Care and Use Committee at CMZ.

Behavioral Data Collection

Behavioral data were collected by a live observer while the subjects wore the

ActiSleep harness. We compared baseline observations (no harness) to behavioral observations taken while each subject wore the harness. Continuous, focal-animal behavioral data were collected (Altmann, 1974) over 30-min sessions using a hand- held computer and Observer Software (Noldus Information Technology Inc.,

Leesburg VA). Observations were timed to coincide with the subject’s active period and occurred from 1000 to 1700 hrs for the pygmy loris and 2200 to 0800 hrs for the potto; observations were balanced across these time periods. Baseline observations were conducted on the potto over four days in October 2009 and observations with the harness occurred on two subsequent days. Baseline observations on the pygmy loris were conducted over six days in September 2009, and observations with the harness were collected over four days that November.

The ethogram (Table 1) was constructed so that behaviors could be grouped into inactive, low activity, and high intensity activities, with the aim of determining

whether it was possible to differentiate among these classes using the actigraph data (e.g. Muller and Schrader, 2003; Naylor and Kie, 2004).

Data Analysis

We analyzed data within subjects and compared each subject’s behavior while wearing the harness to baseline observations using t-tests for which equal variances were not assumed. For analysis of activity budgets, several behaviors were lumped into the following groups. Sleep and rest were combined into ‘rest’.

‘Other’ consisted of exploration, other (low), solitary play, and other (high) behaviors. ‘Move’ included move, climb up, climb down, and suspensory behavior.

Finally, the category ‘social’ included the behaviors of social touching, social play, reproductive behavior, and agonistic behavior.

Following Naylor and Kie (2004), discriminant function analysis was used to examine the predictive value of actigraph activity counts for identifying behaviors recorded during focal sampling. To do so, instances of behaviors that spanned the entire 30-sec recording epoch were first identified using behavioral data to ensure that only a single activity contributed to each data point. These analyses were conducted on individual behaviors as well as the consolidated behavior classes grouped by intensity in the ethogram (Table 1), and finally on gross differences between activity and inactivity. ActiSleep data were generated in ActiLife v. 4.2.0 and exported to Microsoft Excel®. Statistical analyses were conducted using SPSS

12.0 and p-values were set at 0.05.

Table 1. Ethogram for behavioral data collection for actigraph study

Behavior* Operational Definition# Level 0: Inactive Behavior Rest Animal is motionless and may be lying down, standing (upright with all four limbs), rearing (standing on two legs), sitting (body hunched but head erect), or hanging with one or two feet. Animal may be sleeping. Level 1: Low-Intensity Active Behaviors Exploration Sniffing, licking, or manually manipulating objects or stationary enclosure furnishings. Self-Directed Auto-grooming using tooth comb or tongue, scratching the self using grooming claw or nails, facial rubbing (rubbing snout, chin, cheeks, or neck on a substrate or object), or rubbing the head against the arms. Feed Ingesting food, normally by grabbing a food item with one hand and taking it to mouth. Includes drinking from surface or dipping hands in liquid and licking the hand. Social Allogrooming (licking or combing another animal’s face or fur) or social Touching exploration (sniffing the body of another animal). Other (Low) Any other behavior which does not result in movement of the animal by a distance of more than one body length. Level 2: High-Intensity Active Behaviors Move Quadrupedal motion in any direction, including climbing and backing up. Social Play Attempted mild bite or attack (partner does not attempt to flee), dangling by feet, wriggling body and wrestling between animals. Reproductive Female hangs while male mounts her while dorsally clasping her sides and Behavior making rapid thrusting movements, or attempted mount with no thrusting observed. Agonistic Attempted or successful bite or attack, lunging with open mouth, Behavior aggressive pursuit, or turning away or fleeing in response to received aggression. Other (High) Any other behavior which results in movement of the animal by a distance of more than one body length. Not Visible Behavior Not Visible The animal or its behavior is not visible.

*Behaviors are adapted from (Fitch-Snyder and Schulze, 2001).

#All behaviors are defined as states. Classifications of intensity level are based on motor patterns underlying each behavior (e.g Papailiou et al., 2008). Inactive behaviors involve little to no movement, low-intensity behaviors involve head/neck or arm movement only, while high-intensity active behaviors involve whole-body movement.

RESULTS

Animal Harnessing

The degree of difficulty in attaching the actigraph harness varied among the individual animals tested. Restraining the slow loris to apply the harness was difficult, and one keeper received a bite wound during the process. Once the harness was in place, the slow loris slipped out of the harness within 30 min, and no further attempts were made to work with this animal due to unrelated health issues. The eleven-yr old pygmy loris wore the actigraph and harness for only about an hour as well. The harness repeatedly slipped out of place on this animal, which also appeared to be agitated by the presence of the harness. While the harness was in place, the loris moved in rapid circles around his enclosure, a behavior that the keeper described as unusual for this individual. No further attempts were made to harness this animal, again, for unrelated health reasons. Finally, the three-yr old pygmy loris was successfully harnessed after two attempts. This animal was harnessed immediately prior to the onset of his light phase. He slept through the light phase with the harness in place, but when the dark activity period began, he immediately removed the harness by using his forelimbs to slide the apparatus over his head.

Ultimately, we were only successful in harnessing two of our five potential subjects for an appreciable amount of time, and only these individuals are included in further analyses. The nine-yr old pygmy loris repeatedly wore the harness for extended periods of time, although he only did so after multiple adjustments to the fit were made. In each trial, attaching the harness to this animal did not prove

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difficult (Figure 1a); however, he slipped out of the harness within 24 hours on the first two trials.

Figure 2. General activity budget for the potto subject with and without the actigraph harness in place. This individual did not have access to a nest box.

Two keepers successfully harnessed the potto using manual restraint immediately prior to the onset of the light phase in his exhibit (Figure 1b). The potto slept through the light phase while wearing the harness and observers did not report any unusual behavior during this time, suggesting that the presence of the harness did not disturb the potto’s sleep. Anecdotally, the potto did appear to have

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some difficulty moving around his exhibit when he first became active. However, he quickly adjusted his movements to utilize larger supports, avoiding smaller branches on which the harness more easily became entangled. The potto’s female cagemate persistently chewed and pulled on the male’s harness, and she ultimately assisted the male in removing the harness by pulling it over his head while they were hanging suspended from a perch.

All further data analysis is based on one extended time period wearing the harness for each of our two subjects. The data presented here represent 38 continuous hrs wearing the ActiSleep harness for the potto and 97 hrs for the pygmy loris. We did not notice any behavioral signs of discomfort in the animals during this time period, and physical inspections did not reveal any signs of irritation to the animals’ skin.

Animal Activity Budgets

A total of 30 hrs of behavioral data were collected on the potto: 20 hrs (10,

30-min samples) of baseline observations and 10 hrs of observation while the potto wore the ActiSleep. For the pygmy loris, 21 hrs of baseline (no harness) observations were conducted and 18 hrs with the harness, for a total of 39 observation hours.

The potto showed a few significant changes in behavior with the ActiSleep harness in place compared to baseline data (Figure 2). While wearing the harness, the potto spent significantly less time feeding (n= 60 observations, t (54) = 2.421, p

= 0.019) and not visible (n = 60, t (40) = 2.515, p = 0.016). The potto spent

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significantly more time engaged in ‘other’ (high and low combined) behaviors while wearing the harness compared to baseline (n = 60, t (53) = -2.164, p = .035). This difference was not due to solitary play (which was never observed in this subject) or exploration, which was not observed during the actigraph condition. Although the potto appeared to spend more time engaged in social behavior while wearing the harness compared to baseline, this difference was not statistically significant.

However, the apparent increase in social behavior was likely a result of the female’s attention to the male’s harness.

Figure 3. General activity budget for the pygmy loris subject with and without the actigraph harness in place. This individual had access to a nest box and was often not visible in this location.

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In all conditions, the only social behaviors observed in these subjects were social touching and play; reproductive and agonistic behaviors were never observed.

The pygmy loris subject’s behavior (Figure 3) did not differ as greatly as that of the potto between conditions. Like the potto, the pygmy loris spent significantly more time engaged in ‘other’ behavior while wearing the harness compared to baseline (n = 78 observations, t (35) = -3.353, p = 0.002). Time spent moving (n =

78, t (46) = -1.588, p = 0.119) and engaging in self-directed behavior (n = 78, t (45) =

-1.771, p = 0.083) both increased while the pygmy loris was wearing the ActiSleep harness; these differences approached but did not reach significance. The solitarily- housed loris was never observed to engage in social behavior or solitary play, and only a single bout of exploration occurred.

Circadian Actigraph Data

The potto’s actigraph data demonstrate a robust nocturnal activity pattern

(Figure 4). The onset of activity occurred almost immediately when the exhibit lights were extinguished each night at 2200 hrs. Bursts of activity during the light phase were brief and of low intensity. The average activity count reported during the dark phase was 116.2 (SD 233.2) compared to 10.2 (74.3) for the light phase.

Actigraph data for the pygmy loris (Figure 5) covers a longer time span than for the potto. The data are not directly comparable, but the pygmy loris’ activity does not seem to be as clearly confined to the dark phase as that of the potto. On several occasions, the loris exhibited prolonged bouts of activity during the light phase, although these were of lower intensity than activity bouts recorded during

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the dark phase. Activity counts averaged 3.6 (SD, 30.1) for the loris during the light phase and 21.6 (96.7) during the dark phase. The difference between activity count magnitudes recorded during the light versus the dark phase was therefore much smaller for the loris than the potto. Activity counts were generally higher for the potto than the loris, reflecting a greater intensity, frequency, and duration of movement by the potto.

Figure 4. Actigraph data for the potto subject. The graph shows activity counts reported at 30-second intervals for 36 of the 38 hours the potto wore the harness. The last two hours are omitted because the pottos were adjusting, and finally removing, the harness. The dark phase in the exhibit lasted from 2200-1000 hrs.

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Figure 5. Actigraph data for the pygmy loris subject. The graph shows activity counts reported at 30-second intervals for 96 of the 97 hours the loris wore the harness. The dark phase in the exhibit lasted from 1000-2200 hrs.

Correspondence between Actigraph and Behavioral Data

The magnitude of activity counts recorded during specific behaviors varied widely within behaviors for both the potto and pygmy loris (Figure 6). For all behaviors, activity counts were higher for the potto than the pygmy loris. We used discriminant function analysis to determine whether activity counts predicted concurrent behaviors. The discriminant function correctly identified rest 90% of the time for the potto and 99.1% for the loris, but action (i.e. anything but rest) was classified correctly only 44.9% and 39.1% of the time for the potto and loris, respectively. Significant results were not obtained using more fine-grained activity types such as feeding or self-directed behavior.

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Figure 6. Mean activity counts associated with behaviors observed in a potto and a pygmy loris. Only behaviors that spanned the entire 30-sec recording epoch of the ActiSleep were included in this analysis. The number of times each behavior was observed is indicated by the number above the bar on the histogram. No social behavior was observed in the pygmy loris, which was housed solitarily.

DISCUSSION

We attempted to validate actigraphy for automated activity monitoring in the pygmy loris and potto. We assessed potential impacts on the welfare of animals by comparing animal activity budgets with and without the harness in place, and monitoring other physical and behavioral signs of discomfort. We also explored whether the occurrence of specific behaviors could be reliably inferred from activity counts recorded by the actigraph. Our results demonstrate that actigraph data may

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provide useful information about the gross activity patterns of lorisid primates despite their slow-moving locomotor style; however, identifying individual behaviors on the basis of activity counts was not possible for our subjects. Although the harness did not cause the animals any obvious physical discomfort, behavioral changes associated with the presence of the harness warrant further investigation.

Our experiences attempting to harness lorises for this study also suggest that animal responses to the procedure are highly individual-specific.

By far the greatest challenge in using actigraphy for monitoring our study subjects was finding a way to safely and comfortably—but also firmly—attach the

ActiSleep to the animals. Successful techniques are likely to be species-specific. For ungulates, actigraphs are generally placed on anklets (dairy cows, Muller and

Schrader, 2003) or collars (Martiskainen et al., 2009; Naylor and Kie, 2004; Piccione et al., 2008; Van Oort et al., 2004). Collars have also been used to conduct actigraphy studies with several nonhuman primates, including common marmosets, (Callithrix jaccus, Mann et al., 2005), rhesus monkeys (Macaca mulatta, Papailiou et al., 2008), spider monkeys (Munoz-Delgado et al., 2004), and owl monkeys in both captive

(Kantha and Suzuki, 2006a) and field (Fernandez-Duque and Erkert, 2006) settings.

The harness style-design we employed was used previously for a zoo-housed red- ruffed lemur (Sellers and Crompton, 2004) and lab-housed callitrichids (Kantha and

Suzuki, 2006b). This approach is less common, but we were concerned about leaving animals unattended while wearing collars. Although we did not observe any safety issues with our harnesses, our subjects were able to remove them fairly easily using their forelimbs. Further testing may show that collars are more effective at

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keeping actigraphs in place. Other potential methods for future studies include the small “sweater” used with a zoo-housed koala (Phascolarctos cinereus, Takahashi et al., 2009) or even gluing actigraphs onto animals, as Byrnes et al. (2008) did to investigate gliding dynamics in the Malayan colugo (Galeopterus variegatus).

It was also difficult to fit our animals into their harnesses as they struggled under manual restraint, suggesting this procedure may be stressful in nature.

Studies of animal handling are few and have mixed implications. With repeated handling, wombats (Lasiorhinus latifrons, Hogan et al., 2011), sheep (Hargreaves and Hutson, 1990), and rabbits (Podberscek et al., 1991) all exhibited reductions in flight distance or other behavioral signs of stress associated with handling. Although animals appeared to habituate behaviorally, adrenocortical responses remained at levels indicative of stress despite repeated handling trials in sheep and wombats

(Hargreaves and Hutson, 1990; Hogan et al., 2011). An alternative approach may be to apply the harness while animals are under anesthesia, as did Kantha and Suzuki

(2006b) with marmosets; although, this approach presents its own challenges and concerns. However the actigraph is worn, its acceptance will likely vary based on individual animal temperament (Juri Suzuki, personal communication). In each case, the means by which the actigraph is attached to the animal presents welfare trade- offs that vary for individuals and species under investigation.

Further research is also needed to examine the duration and extent of behavioral impacts associated with actigraph harnesses. In our study, the potto spent less time feeding with the harness in place compared to baseline, which could be of concern over longer time periods. However, Gursky found that free-ranging

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tarsiers did not show deficits in body weight or prey capture rates after six months of wearing radio collars. Thus, it seems unlikely that provisioned, captive animals would experience nutritional deficits associated with harnesses. In our study, the increased time spent performing self-directed behavior by the pygmy loris may also indicate some discomfort caused by the harness. The animal may habituate or the harness could become increasingly uncomfortable over time. It is common in actigraph research to discard the first 24 hours of data collected with the animal wearing the device to allow for habituation time (Juri Suzuki, personal communication), but whether or not behavioral adjustment occurs after 24 hours— or at all—should be empirically evaluated before utilizing actigraphy for long-term animal monitoring.

The circadian data collected during our actigraph trials illustrates both the promise and difficulty of this method for animal monitoring. Rarely is behavioral data collected on such extended time scales in zoos. It was fascinating to get a glimpse into long-term patterns of our lorisids’ behavior, as well as activity occurring outside normal staff hours. The potential applications for animal monitoring are numerous. However, for research purposes, it is currently recommended that at least 72 hours of continuous data be collected for analysis of circadian rhythms in human actigraphy (Littner et al., 2003). After multiple trials, we were only able to collect data meeting these criteria on a single pygmy loris. We were also unable to link specific behaviors to reported activity counts (cf.

Martiskainen et al., 2009; Naylor and Kie, 2004), a result consistent with the finding that accelerometers are useful mainly as a measure of whole body movement in

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rhesus monkeys (Papailiou et al., 2008). For these reasons, and due to concerns about animal handling, we ultimately decided not to pursue this method in future studies examining behavioral impacts of lighting on lorisid primates. Yet, there are many reasons to consider further this approach in future research.

Animal welfare is notoriously difficult to define and measure (Mason and

Mendl, 1993; Rushen and Depassille, 1992). A common approach to behaviorally evaluating captive welfare is to calculate the percent of time animals spend performing particular behaviors (the activity budget), including stereotypic or abnormal behaviors indicative of negative welfare, and compare the time budget to that of free-ranging conspecifics (McCann et al., 2007; Novak and Suomi, 1988).

Perhaps for highly inactive species, such as lorisids, behavioral rhythmicity and its response to environmental change would be a more informative measure.

Information about quotidian patterns of behavior can also inform captive managers of the best time of day to observe their animals, which can be important for monitoring relatively inactive strepsirrhine species (Wright et al., 1989).

Actigraphy also enables investigators to examine patterns of animal sleep, an often overlooked behavior that may be extremely informative about animal welfare

(Abou-Ismail et al., 2007; Anderson, 1998). Circadian patterns of behavior provide insight into the functioning of the hypothalamic-pituitary-adrenal (HPA) axis

(Buckley and Schatzberg, 2005; Kant et al., 1995; Van Reeth et al., 2000), a system which is often the target of welfare investigations in which glucocorticoid secretion is measured as a proxy for stress (Rushen, 1991; Shepherdson et al., 2004). Indeed, changes in sleep architecture may be directly indicative of chronic stress (Van Reeth

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et al., 2000). In humans, sleep patterns measured via actigraphy are a reliable measure of circadian disruption (Ancoli-Israel et al., 2003). In turn, poor sleep quality and circadian disruption are associated with a plethora of maladies that may negatively impact welfare, including depression (Germain and Kupfer, 2008), cardiovascular disease (Malhotra and Loscalzo, 2009), and inflammation (Patel et al., 2009).

In conclusion, although the method has its limitations, actigraphy presents many opportunities for better understanding the behavior and welfare of lorisid primates and other captive taxa. Future investigators will need to weigh these potential benefits against the welfare concerns presented by fitting animals with monitoring devices. It may well be worth the effort; actigraphy offers the potential to reveal new connections between behavioral outputs and the physiological systems underlying them in an innovative, holistic manner.

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Chapter Five

Methods for Measuring Salivary Melatonin in the Potto, Perodicticus potto, and Pygmy Loris, Nycticebus pygmaeus

INTRODUCTION

Non-invasive methods for monitoring animal hormones have created a myriad of opportunities for understanding animal health, physiology, and hormone- behavior interactions (Whitten et al., 1998). Non-invasive measures are of particular importance for understanding the physiology of animal stress, which can be complicated by standard blood collection procedures that are inherently stressful

(Mormede et al., 2007). Non-invasive measures of fecal or urinary glucocorticoid metabolites (i.e., “stress” hormones) have been widely applied in zoo and other settings for the assessment of animal welfare (Shepherdson et al., 2004). Many of the same steroid hormones that are measured in feces and urine also can be measured in saliva, along with a variety of amines and peptides (Groschl, 2008).

Saliva has become an important matrix from which to extract information about endocrine activity in both research (Kirschbaum and Hellhammer, 1994) and clinical settings (Lac, 2001; Papacosta and Nassis, 2011).

Saliva has multiple functions that include the facilitation of swallowing and digestion of food (Groschl, 2008). Cytokines in saliva contribute to oral immune defense and regulate oral tumorogenesis, while steroid hormones in saliva may act as pheromones, playing a role in animal social behavior (Groschl, 2009). According to Groschl (2008), human saliva is comprised of secretions from three paired salivary glands in the oral cavity. Steroid and amine hormones circulating in blood

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are introduced into saliva via passive diffusion through capillary beds, whereas peptide hormones like insulin are usually actively transported into saliva.

Consequently, salivary levels of steroid and amine hormones more accurately reflect circulating hormone levels in plasma than peptide hormones. For steroids and amines, salivary measures have the additional advantage of providing a relatively instantaneous measure of circulating hormone levels, in contrast to fecal metabolite measurements that provide data on aggregate hormone levels over a period roughly equal to the animal’s gut transit time (Whitten et al., 1998). Thus, saliva can be used to quickly and accurately assess hormonal responses to acute stimuli.

Methods for collecting saliva have been investigated in several primate species. Lutz (2000) compared two apparatuses for collecting saliva samples for cortisol analysis from laboratory-housed rhesus macaques (Macaca mulatta). The

“lick” technique consisted of monkeys licking a sheet of gauze covered with wire, while for the “pole” method, the monkeys chewed on a rope attached to a PVC pole.

There were no significant differences found in cortisol values between these two methods, and flavoring the ropes with juice to entice chewing also did not affect cortisol values. Cross et al. (2004) examined salivary cortisol levels in marmosets

(Callithrix jacchus), which were trained to lick a cotton bud coated with a film of banana. Although measured cortisol concentrations were depressed by the banana flavoring, the effects were linear and sample concentrations could be easily corrected for the flavor’s effects. Using these or similar measures, salivary cortisol has been measured in a variety of nonhuman primate species, including squirrel monkeys (Saimiri sciureus) (Fuchs et al., 1997; Tiefenbacher et al., 2003), rhesus

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macaques (Boyce et al., 1995), hamadryas baboons (Papio hamadryas) (Pearson et al., 2008), western lowland gorillas (Gorilla gorilla gorilla) (Kuhar et al., 2005), and chimpanzees (Pan troglodytes) (Heintz et al., 2011).

The most commonly measured amine in saliva is melatonin. Melatonin is the primary hormone that conveys information about time of day to bodily systems

(Arendt, 2005) and is therefore frequently utilized in circadian rhythm research

(Groschl, 2008; Laakso et al., 1993; Voultsios et al., 1997) and as a biomarker of circadian disruption (Mirick and Davis, 2008). Humans (Kennaway and Voultsios,

1998) and other animals exhibit true circadian rhythms in melatonin production, and circulating levels of melatonin are much higher in the dark than the light phase for all species that have been studied, regardless of activity pattern (Arendt, 2005).

Human subjects can be trained easily to collect their own saliva samples for melatonin analysis, facilitating large-scale epidemiological studies on the effects of shift work, travel, sleep disorders, and other conditions associated with altered circadian rhythms (Arendt, 2005; Blask, 2009; Grundy et al., 2009)

Exposure to light at night has been widely demonstrated to suppress pineal melatonin production in humans (Hashimoto et al., 1996; Reiter et al., 2007;

Shanahan et al., 1997; Stevens and Rea, 2001; Zeitzer et al., 2000) and other animals

(Dauchy et al., 1997; Depres-Brummer et al., 1995; Hoban et al., 1990; Reppert et al.,

1981). Light toward the blue end of the spectrum also has a greater effect on melatonin suppression than light of longer wavelengths (red light) (Boulos, 1995;

Rahman et al., 2008; Schobersberger et al., 2007). In turn, melatonin suppression is associated with a wide variety of maladies, including breast and other endocrine-

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related cancers, cardiovascular disease, major depression, metabolic syndrome, and decreased fertility (Arendt, 2005; Navara and Nelson, 2007; Stevens and Rea, 2001).

Melatonin or its metabolites can be measured in most bodily fluids including blood, urine, and saliva (de Almeida et al., 2011). Salivary measures rarely have been utilized with nonhuman animals, although Stark et al. (1997) measured salivary melatonin responses to radio signals in dairy cattle. Surprisingly little research has been conducted on melatonin expression in nonhuman primates.

Aujuard et al. (2001) examined the effects of photoperiod on cellular aging in gray mouse lemurs (Microcebus murinus) by measuring urinary sulfatoxymelatonin.

Nocturnal light exposure has been documented to suppress melatonin concentrations in squirrel monkey plasma (Hoban et al., 1990) and in the cerebrospinal fluid of rhesus macaques (Reppert et al., 1981). However, to our knowledge salivary melatonin has not been investigated in any nonhuman primate species.

Our ultimate goal was to develop a biomarker that would be useful to assess the physiological effects of lighting design for zoo-housed pygmy slow lorises (PSL;

Nycticebus pygmaeus) and pottos (Perodicticus potto). Saliva sampling also enabled us to take melatonin measurements at several time points throughout the day, which would be impossible in such small animals using blood sampling. Thus, our specific aims were to create a procedure for saliva collection from these species and condition animals to provide samples, and to develop laboratory assays for quantifying melatonin concentrations in loris and potto saliva. To biologically validate the melatonin assay, we also examined acute suppression of melatonin due

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to nocturnal light exposure as well as 24-hour rhythms of melatonin expression in lorises and pottos.

MATERIALS AND METHODS

Saliva Collection

Saliva samples were collected using a pole apparatus similar to that described by Lutz et al. (2000), which consisted of a 1.5 m (1.27 cm diameter) fixed length of PVC (Figure 1). The lorises and pottos were trained for saliva collection using positive reinforcement techniques. The subjects were habituated to the presence of the pole and the researcher using super worms (Zophobas morio) presented on a hook attached to the pole. Although the potto subjects habituated to the pole in only one or two training sessions, it took many weeks of training to get the pygmy lorises to interact with the pole. Once the animals would regularly take worms off the pole, they were presented with the collection swabs. During sample collection, the animals were given 10 min to chew on the swab. Afterwards, the chewed portion of the swab was immediately cut off the pole using scissors and placed in a saliva collection vial (Salimetrics LLC, State College, PA). Saliva was recovered from the swabs by centrifuging the vials for 15 min at 2500 rpm.

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Figure 1. Collection of saliva from the female potto.

Various collection media were tested for this study, including cotton rope and salivettes (SARSTEDT AG & Co, Nümbrecht, Germany). The most successful collection swabs were children’s swabs (Salimetrics LLC, State College, PA). The children’s swabs are composed of an inert polymer that is durable but also soft. The length of the children’s swab also allowed the animals to hold it while they chewed, seemingly a preferred behavior (Figure 1). Although we tried many different flavoring agents—including various juices, mashed banana, and mashed super worms—the animals responded most favorably to honey and sucrose solutions. The final collection media developed for these experiments consisted of children’s swabs quickly dipped in a 1:3.5 dilution of honey in water. The swabs were then dried under forced air for 48 hrs to remove excess liquid. The decision to use honey rather

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than sucrose was based on the results of standard laboratory validations such as parallelism of pooled samples to the standard curve and accuracy/recovery values.

Laboratory Analysis of Melatonin Concentrations

We tested two different laboratory methods for quantifying melatonin in saliva: an enzyme-immunoassay (EIA) for direct measurement of melatonin in human saliva (RE54041, IBL International, Hamburg, Germany) and a radio- immunoassay (RIA) for direct determination of melatonin in human serum, plasma, or saliva (BA R-3300, Labor Diagnostika Nord GmbH & Co. KG, Nordhorn, Germany).

Initial results using both the EIA and RIA indicated that a component of the saliva sample was interfering with melatonin measurement. Possible sources of interference included the saliva matrix, flavoring agent, or collection medium. To identify possible sources of matrix interference, we compared serially collected saliva samples using cotton rope soaked in diluted honey or sucrose in pygmy lorises, pottos, and human volunteers by RIA. An organic solvent extraction was also tested to eliminate matrix interference in the RIA. Samples were mixed with diethyl ether in a 1:11 dilution, vortexed, centrifuged for 10 minutes at 2500 x g, and immediately frozen at -80 °C. After one hour, the ether was decanted and the supernatant was dried under air overnight. The samples were then reconstituted in distilled water equal to the original sample volume.

We also tested several different saliva extraction techniques in an attempt to eliminate matrix interference in EIA measurements (Harumi and Matsushima,

2000). Using saliva from the same loris and potto pools, we compared a liquid-liquid

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extraction technique (methanol-chloroform) (de la Puerta et al., 2007) to a methanol-solid phase extraction (SPE) protocol (Iriti et al., 2006; Kolar and

Machackova, 2005; Romsing et al., 2006). For the methanol-chloroform extraction, samples were first mixed with methanol in a 1:11 dilution, shaken on a multi-tube vortexer for 30 minutes, centrifuged at 3000 x g, frozen at -80 °C, decanted, and the supernatant was dried under air. Samples were then reconstituted in phosphate buffered saline (PBS) and mixed with chloroform in a 1:3 dilution. These samples were then frozen and decanted, and the supernatant was dried under air. For the methanol-SPE procedure, samples were extracted using methanol following the methods described above and then re-suspended in a 1:10 dilution of methanol to

PBS. Samples were processed through C-18 Sep-Pak cartridges, alternately eluted and washed using methanol and water, and reconstituted in water prior to analysis.

To assess the effectiveness of the extraction technique, parallelism to the standard curve was measured and compared to values obtained using un-extracted, dilute saliva.

BIOLOGICAL VALIDATION EXPERIMENTS

Experiment One: Acute Suppression of Melatonin by Light Exposure

The purpose of this experiment was to demonstrate suppression of melatonin production as a result of acute light exposure during the dark phase. This effect was expected to be dose-responsive, meaning that the degree of suppression would increase with light intensity. We also expected short wavelength (blue) light to have a greater suppressive effect than longer (red) wavelengths.

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Subjects and Housing

The subjects for this experiment were one female potto (Jahzira, 4 yrs) and

1.1 pygmy lorises. The adult female (PSL1, Hermione, 5 yrs) was housed in an exhibit measuring 3 x 2.7 x 2.9 m. Her male offspring (PSL2, Harry, 2 yrs) was housed alone in a nearby exhibit measuring 3 x 2.6 x 2.9 m. The potto was housed with a male (18 yrs) who did not regularly provide saliva samples and was therefore excluded from the experiment. The potto exhibit measured 2.9 x 4.9 x 4.3 m. The three exhibits were similarly designed, including gunnite rockwork, natural wood perching, and leafy cover. None of the exhibits contained a nest box during this experiment.

All the exhibits were located in a nocturnal wing of the Primate, Cat, and

Aquatics house at CMZ. The public floor space in this area was illuminated with dim red light, in addition to some diffuse white light from nearby diurnal exhibits.

During the experiment, the animals were housed on a 12:12 Light-Dark (LD) cycle with dark phase onset at 1800 hrs. The exhibits were illuminated by white halogen bulbs during the L phase. No direct artificial light was provided during the D phase, except for whatever small amount bled into exhibits from nearby hallways. To minimize uncontrolled light exposure, the public viewing glass at the front of each exhibit was covered with a wooden board and a canvass tarp from 1700 to 0700 hrs every evening during the study.

Light intensities were measured using a SPER Scientific light meter

(#840020, Scottsdale AZ, USA). L phase values (lux) for each animal were: 505.0 for

PSL1, 384.2 for PSL2, and 122.7 for the potto. D phase intensities in the exhibits

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were less than one lux. During the D phase, animals were acutely exposed to red, blue, and full-spectrum (FS) light from fluorescent bulbs. The fluorescent lights were covered with gel filters (Rosco Laboratories Inc., Stamford CT, USA) to create the red (filters #2001, 4690, 4660, and 4630), blue (#4290, 4260, and 4230), and FS

(#3404, 3403, 3402) conditions.

Data Collection

Data were collected December 2011 through January 2012. For this experiment, the subjects were experimentally exposed to 105 min of FS, red, or blue light during the dark phase; and saliva samples were collected for analysis of salivary melatonin. Saliva samples were collected using the pole technique and honey-flavored children’s swabs previously described. Sample collection was staggered among the three subjects. First, a baseline saliva sample was collected at two hrs after D phase onset for the PSL subjects (2000 hrs) and three hrs for the potto (2100 hrs). Next, the fluorescent lights were switched on in the exhibit, and saliva samples were then collected after 60 and 90 min of light exposure.

The animals were exposed twice to each of eight conditions. Animals were tested under two control conditions: darkness and high intensity FS light (no filter;

400-550 lux), in addition to dim (0-1 lux) or bright (70-100 lux) FS, red, or blue light. Only a single test was conducted each night, and the two tests for each condition were conducted serially. An interval of 48 hrs was observed between each test to allow animals to re-entrain to their baseline lighting regimen. This interval was chosen based on research showing that in humans experiencing jetlag or other

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circadian disruptions, a 24-hr interval is needed to adjust sleep patterns by 1 to 2 hrs (Kolla and Auger, 2011). The order in which the lighting conditions were tested was randomized among the exhibits.

Samples were immediately refrigerated following collection. The swabs were centrifuged the same day for 15 min at 2500 rpm to collect saliva, and the samples were frozen at -18 °C. Melatonin concentrations were measured in un-extracted saliva in a 10/90 dilution using the EIA methods previously described.

Experiment Two: 24-Hour Melatonin Rhythms

For this experiment, saliva samples were collected regularly throughout the day to look for evidence of rhythmicity in melatonin expression. Melatonin levels were expected to be significantly higher during the D phase compared to the L phase.

This study was conducted over a two-week period in April 2012. Saliva samples were collected from subjects using the pole method described for experiment one. Samples were collected every three hours, at: 1000, 1300, 1600,

1900, 2200, 0100, 0400, and 0700 hrs. Samples were stored and analyzed using the same methods described for experiment one, except that sample swabs were immediately frozen after collection at -18 °C. The swabs were thawed and centrifuged to collect saliva immediately prior to EIA analysis.

The subjects for this experiment included the same animals from experiment one with the addition of an unrelated adult male pygmy loris (PSL3, Tai, 6 yrs). PSL3 was housed alone in an exhibit next to PSL1 measuring 2.9 x 2.6 x 2.9 m. Exhibit

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conditions for PSL3 were comparable to those described for the other exhibits in experiment one, and this animal did not have access to a nest box. PSL1 and 2 both had access to a nest box in their exhibits during the experiment.

During the experiment, the animals were housed on a 12:12 reversed LD cycle with dark phase onset at 1000 hrs. The exhibits were illuminated by white halogen bulbs during the L phase and red-filtered fluorescent lights (#2001, Rosco

Laboratories Inc.) during the D phase. Intensities (lux) in the exhibits were as follows: 23.5 L: 0.7 D for the potto; 129.2 L: 1.1 D for PSL1; 129.2 L: 1.9 D for PSL2; and 163.4 L: 0.9 D for PSL3.

Data Analysis

For both experiments, data were analyzed using Microsoft Excel® and SPSS

12.0. Circadian variables were calculated using Cosinor software available at www.circadian.org (©Refinetti 2013; Refinetti et al., 2007). Period length was defined as length of time to complete one oscillation of a rhythmic variable (Halle and Weinert, 2000).

RESULTS

Comparison of Melatonin EIA and RIA

Serial dilutions indicated greater levels of matrix interference using sucrose than honey in potto and pygmy loris samples. Human melatonin values also more closely approached unflavored values using honey-flavored ropes than those soaked in sucrose. Although it was not possible to determine from these experiments

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whether the source of interference was the cotton in the rope or the sugar flavoring, we ultimately decided to use synthetic collection swabs (Salimetrics children’s swabs) to eliminate cotton as a potential inhibiting factor.

Pooled samples extracted with ether demonstrated the most consistent control values using the RIA. Serially diluted extracts displayed parallelism with the standard curve (t=2.103, p=0.054 for pygmy loris; t=2.046, p=0.063 for potto).

Pooled samples were used to measure recovery of high (20 pg/ml) and low (6 pg/ml) melatonin concentrations. Recoveries for high concentrations were 109.2% for PSL and 95.56% for potto, and recoveries for low concentrations were 100.8% for PSL and 104.92% for potto. Although the RIA met parallelism and recovery validations, we recorded unexpectedly high melatonin levels on a control (water- soaked) swab. As we had generally been measuring values toward the low end of the standard curve using this assay, we ultimately determined that the assay lacked the sensitivity to accurately measure melatonin concentrations in our subjects.

To assess the effectiveness of the different extraction techniques using the

EIA, recoveries of hormone from pooled samples spiked with 2.5 pg/ml or 27.5 pg/ml of standards were compared to values generated from un-extracted pools analyzed at a 1:10 dilution. For pottos, recoveries for pools extracted using methanol-chloroform were 130.0% (low) and 66.9% (high). SPE recoveries for pottos were 160.3% (low) and 60.71% (high), and recoveries from un-extracted pools were 68.0% (low) and 100.1% (high). For pygmy loris, methanol-chloroform recoveries were 179.1% (low) and 72.8% (high), SPE recoveries were 141.5% (low) and 60.9% (high), and recoveries from un-extracted saliva were 109.0% (low) and

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103.5% (high). Parallelism to the standard curve was only achieved using un- extracted samples, not with either extraction technique.

Ultimately, we determined that analysis of melatonin concentrations in these species was most successful using the EIA with samples analyzed at a 1:10 dilution to eliminate matrix interference. Serially diluted samples displayed parallelism to the standard curve (t=-0.839, p = 0.434 for pygmy loris; t=-1.111, p=0.303 for potto). The EIA detected no measureable melatonin in a control sample prepared by soaking a honey-flavored swab in a volume of distilled water equal to that of a typical saliva sample. We also directly assayed the diluted honey solution used to flavor swabs and found no detectable melatonin content using the EIA. Given that the EIA also demonstrated greater cross-reactivity with both species than the RIA, we determined EIA to be the preferred approach to melatonin determination in the potto and pygmy loris.

Experiment One: Acute Suppression of Melatonin by Light Exposure

Melatonin measurements taken on the potto were comparable under darkness and red light, and higher melatonin values were measured in these conditions compared to the blue and bright control conditions (Figure 2a). Although both intensities of blue light appeared to affect the potto in the same way, higher levels of melatonin were measured under dim red light than following exposure to bright red light. Surprisingly, the highest mean melatonin levels were measured following dim red light exposure, not in the dark (0 lux) control condition.

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Figures 2 a-c. Melatonin concentrations measured following nocturnal exposure to test lights in a potto (a), and pygmy lorises PSL1 (b) and PSL2 (c). The subjects were exposed to 105 minutes of full-spectrum, blue, or red light of varying intensities. Light wavelength is indicated by bar color and intensity is given on the y–axis. Light exposure began three hours after dark phase onset for the potto and two hours for each PSL. Samples collected after 60 and 90 minutes of light exposure on two test presentations (three for the 0.0 lux measurements) are combined for each light condition. Values above standard error bars indicate sample number.

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Melatonin levels in the potto were also higher in darkness and in dim FS light compared to both medium and high intensity FS light.

Of all three subjects, only the potto demonstrated an overall significant effect of lighting condition in a repeated measures ANOVA (F7,27 = 3.184, P=0.031), although the female loris approached significance (F2,27=2.218, P=.097). Paired t- tests approached significance for the potto with higher melatonin under dim red light compared both to dim blue (t2=-2.364, P=0.142) and bright blue (t2=-3.415,

P=0.076) light.

Visual inspection of results for the pygmy lorises suggests opposing trends.

The male pygmy loris (Figure 2c) had higher melatonin levels under dim red light than all other conditions, although overall intensity differences were negligible. The female pygmy loris (Figure 2b) consistently demonstrated patterns antithetical to our predictions: her melatonin levels were highest in the high intensity control condition, followed by the bright light conditions, and lower in the dim and dark conditions.

Experiment Two: 24-Hour Melatonin Rhythms

For this study, between one and four useable saliva samples were collected per subject at each time point. Mean melatonin concentrations varied significantly with lighting phase for the potto (41.44 + 4.02 pg/ml + SE for the D phase (N=16) vs.

20.28 + 1.80 for the L phase (n=13), t29 = -5.010, p< 0.001). However, for all three pygmy lorises, L and D phase melatonin values were not significantly different:

88.76 + 10.92 D (N=17) vs. 85.23 + 16.26 L phase (N=6) for PSL1; 88.43 + 9.49 D

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(N=16) vs. 83.92 + 12.18 L (N=11) for PSL2; and 46.42 + 4.72 D phase (N= 16) vs.

43.77 + 3.97 L (N=16) for PSL3. For PSL1 and 2, melatonin measurements did not appear to vary depending on whether the subject was in the nest box at the time of sample collection.

Melatonin values varied among subjects and were often quite variable at a given time point within a single subject (Figure 2). Using a repeated measures

ANOVA, only the potto demonstrated a significant effect of time of day on melatonin

(F2,27= 7.875, p =0.001). The cosinor model identified a period of 22.8 hrs for melatonin expression the potto (F2,27 = 9.169, p= 0.001), with an amplitude of 15.8 pg/ml. A significant period for melatonin expression was not identified in any of the

PSL subjects using the cosinor model.

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Figure 3. 24-hour patterns of salivary melatonin expression in a potto and three pygmy lorises (PSL). Between one and four samples were collected per subject at each time point, for a total of N= 29 for the potto, N = 23 for PSL1, N = 27 for PSL2, and N = 32 for PSL3. Animals were housed on a 12:12 LD cycle with dark phase onset at 1000 hrs.

DISCUSSION

We aimed to develop methods for using the hormone melatonin as a biomarker for the health effects of lighting design on nocturnal strepsirrhines exhibited in zoos. We conditioned several pygmy lorises and a potto for voluntary saliva collection and tested the subjects’ receptiveness to various flavoring and collection media. We compared laboratory results between a commercial EIA and

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RIA and ultimately determined the best results were obtained analyzing honey- flavored collection swabs by EIA. Chemical validations of the EIA were successful for both the potto and pygmy loris, but biological validation experiments examining acute suppression of melatonin by nocturnal light exposure and 24-hour melatonin rhythms produced unexpected results in the pygmy loris. Ultimately, we concluded that the assay is effective for salivary melatonin analysis in the potto but further testing is required for the pygmy loris.

The nature of the saliva matrix can create challenges for interpreting salivary hormone levels, even in the absence of added flavoring. The composition of saliva and the concentration of hormones within it are dependent upon a variety of factors. Saliva composition can be affected by disease states such as diabetes mellitus, kidney dysfunction, and epilepsy, among others (Aps and Martens, 2005).

Saliva is made up of secretions from several glands, which may vary in their contributions to mixed saliva at a given time, and gland-specific saliva cannot be easily harvested outside of medical settings (Groschl, 2008). Saliva secretion is largely moderated by the autonomic nervous system, and saliva composition can depend on the specific autonomic receptors that are activated (Papacosta and

Nassis, 2011).

Furthermore, saliva flow rate exhibits regular circadian variation and is also affected by the type of stimulation used to collect samples (Aps and Martens, 2005).

Steroid hormone concentrations in saliva are thought to be largely unaffected by saliva flow rate because they are passively diffused into saliva (Papacosta and

Nassis, 2011); however, whether this same argument applies to amines is unclear.

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In humans, melatonin concentrations measured in samples collected by gentle chewing on parafilm were higher compared to samples collected after high saliva flow rate was induced using citric acid (Voultsios et al., 1997). Although we did not directly control for salivary flow rate, during the course of our experiments we limited sample collection time to ten minutes per sample. Future investigators may want to account for saliva dilution by incorporating sample volume into statistical comparisons. Another possible control would be to use a measure of the total protein concentration of saliva as an indicator of saliva dilution, much as creatinine concentrations are used to assess urine dilution in endocrine analyses (Lac, 2001).

Non-specific interference may have also varied between the two assays we tested. Andersson et al. (2000) compared two commercial RIAs for measurement of melatonin in porcine plasma, both of which passed standard laboratory validations for parallelism and accuracy/recovery. However, they found only one of the RIAs detected expected low/absent levels of melatonin during the light phase, while the other assay showed comparable melatonin values at midday and midnight. These investigators speculate that non-specific binding of plasma proteins may have influenced results; however, they also found that extraction of samples prior to analysis did not eliminate this effect (Andersson et al., 2000). It may be possible that other substances in the saliva samples (in addition to the flavoring) interfered with melatonin measurement to different extents in the two assays we tested. We also found better accuracy/recovery values with potto rather than loris samples, and it is tempting to speculate that lorises, as the only known venomous primate (Kalimullah

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et al., 2008; Krane et al., 2003), may have substances in their saliva that cause greater assay interference than pottos.

The results of our lighting experiments demonstrate the importance of properly validating hormone measures both analytically and physiologically

(Touma and Palme, 2005). Results with the potto largely met our expectations, as this subject demonstrated an identifiable rhythm in melatonin concentrations, a clear difference between mean melatonin concentrations between the dark and light phase, and differential suppression of melatonin by acute exposure to lights at different intensities and wavelengths. In contrast, the pygmy loris subjects did not exhibit a melatonin rhythm or suppression resulting from light exposure.

There are several explanations for why the biological validation experiments were ultimately unsuccessful in the pygmy loris. In mammals, a relatively narrow band of wavelengths cause melatonin suppression, and the specific wavelengths responsible vary among species (Brainard and Hanifin, 2002). It may be that case that the experimental lighting we designed did not target the action spectra responsible for melatonin suppression in pygmy lorises. However, if these action spectra were known, it might be possible to design custom lighting that filters out only the wavelengths that suppress melatonin, without shifting the overall appearance of the light color (Rahman et al., 2008; Schobersberger et al., 2007).

Another possible explanation is that the lorises in our study were experiencing chronic melatonin suppression. When we tested 24-hour melatonin rhythms, the animals were housed on a reversed light cycle, meaning they were exposed to some intensity of light all 24 hours of the day. Housing rats under

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constant dim red light causes circadian rhythms of melatonin, activity, and body temperature to become desynchronized (Aguzzi et al., 2006). However, the potto, whose exhibit had the smallest difference in intensity between the light and dark phase of any exhibit, showed clear differences in melatonin concentrations based on time of day. There are some species that do not show the expected nocturnal rise in melatonin production or for whom this may vary on a seasonal basis (Reiter et al.,

1987). Whether these trends represent true species differences will remain unclear until additional pottos can be tested, as well as lorises and pottos under different lighting regimens.

The results of our efforts to validate methods for measuring salivary melatonin in the pygmy loris and potto show both the potential and challenges of applying salivary hormone analysis to nonhuman animal experiments. An alternative validation to perform in future experiments may be to orally administer melatonin and then test its subsequent concentrations in saliva (Kovacs et al., 2000).

In spite of such challenges, salivary hormone analysis shows great promise for understanding a variety of health and physiological issues in captive animals.

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Chapter Six

A Case Study Comparing Hormonal and Behavioral Responses to Red and Blue Exhibit Lighting in the Aye-Aye, Daubentonia madagascariensis

INTRODUCTION

Nocturnal mammals are often housed on reversed light cycles in zoos so that visitors can observe active behavior. Zoo exhibits historically utilized red light based on the reasoning that this would appear darker to nocturnal species with rod- dominated retinas (Conway, 1969; Davis, 1961). However, a recent survey of facilities housing lorisid primates in North America revealed that institutions exhibit animals under red and blue dark phase lighting equally (Fuller et al., 2013).

Currently, little empirical evidence informs the debate over light color for nocturnal exhibits.

Light signals play an important role in the mammalian circadian system, and artificial light cues may have dramatic effects on behavior and physiology (Erkert,

1989). Several studies have demonstrated that captive nocturnal strepsirrhines are more active at lower light intensities during the dark phase (Frederick and

Fernandes, 1994; Randolph, 1971; Trent et al., 1977). However, there is currently no research examining the effects of wavelength on nocturnal primate behavior.

Exposure to light at night disrupts the body’s timekeeping system by suppressing production of the hormone melatonin (Hoban et al., 1990; Zeitzer et al.,

2000), altering daily rhythms of cortisol and other hormones as well as activity patterns (Depres-Brummer et al., 1995; Mirick and Davis, 2008; Reiter, 1991). In human studies, chronic melatonin suppression is linked to maladies including

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cancer, cardiovascular disease, and immunosuppression (Navara and Nelson, 2007;

Reiter et al., 2007). Shorter wavelength (blue) light has a greater suppressive effect on melatonin than longer wavelengths (red) in humans (Brainard and Hanifin,

2002; Schobersberger et al., 2007) and other species (Rahman et al., 2008; Walsh et al., 2013). Color-specific melatonin suppression thus has important husbandry implications for zoos.

The aye-aye (Daubentonia madagascariensis) is a nocturnal strepsirrhine endemic to Madagascar (Groves, 2001). The goal of this study was to systematically investigate the effects of nocturnal light color in a single aye-aye subject. We hypothesized that the aye-aye would demonstrate signs of circadian disruption under blue but not red light.

MATERIALS AND METHODS

The subject was an 18-yr old, captive-born, female aye-aye housed at

Cleveland Metroparks Zoo (CMZ) in Cleveland, OH. The enclosure measured 3.6 x

4.0 x 4.4 m with gunnite rockwork and natural log perching. The exhibit contained a cardboard box or bag for nesting, nesting materials, puzzle feeders, and other enrichment items. A smaller off-exhibit room was accessible most days and every evening after 1700 hrs.

Data were collected October 2012—February 2013, during three study conditions: (1) baseline red light (5 weeks); (2) experimental blue light (5 weeks); and (3) a second red light baseline (3 weeks). In humans experiencing jetlag or other circadian disruptions, a 24-hr interval is needed to adjust sleep patterns by 1

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to 2 hrs (Kolla and Auger, 2011). To allow for entrainment, there was a two-week break in data collection between the first two conditions. This interval was extended to four weeks between the last two conditions to accommodate another experiment.

All aspects of lighting were constant across conditions except for dark phase light color. The aye-aye was housed on a light-dark cycle matched to Madagascar photoperiod, with dark phase onset at 1000 hrs and light phase onset between

2100-2200 hrs. The exhibit was illuminated with full-spectrum compact fluorescent lights, half of which were covered with red (#2001) or blue (#4290) gel filters

(Rosco Laboratories Inc., Stamford CT, USA) to simulate the darkness. Light intensities were measured using a SPER Scientific light meter 840020 (Scottsdale

AZ, USA) and were held constant at 4.5 lux during the dark phase (2.7 lux off- exhibit) and 234 lux during the light phase (200 lux off-exhibit).

For conditions one and two, behavior was observed all 24 hours of the day, while only dark phase observations were made during condition three. A continuous behavior sampling protocol was employed in ten-minute sessions using a simple ethogram (Table 1) and The Observer 5.0 (Noldus Information Technology,

Wageningen, The Netherlands). HOBO® data loggers (Onset Computer Corp., Cape

Cod MA, USA) were placed in exhibits to monitor light intensity, temperature, and relative humidity. Supplemental information on behavior and health was obtained from daily keeper reports and medical records.

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Table 1. Ethogram for behavioral data collection on the aye-aye.

Behavior Operational Definition Social Allogrooming, social exploration, social play, reproductive behaviors, or agonistic behavior. Move Motion in any direction, including climbing and backing up. Feed Ingesting food, normally by grabbing a food item with one hand and taking it to mouth. Includes drinking from surface or dipping hands in liquid and licking the hand. Self-Directed Auto-grooming using tooth comb or tongue, scratching the self using grooming claw or nails, facial rubbing (rubbing snout, chin, cheeks, or neck on a substrate or object), or rubbing the head against the arms. Object Animal is physically manipulating an object using hands or teeth or is Examination actively sniffing an object. Rest Animal is motionless and is lying down, sitting, or in another sleeping posture. The animal’s eyes may be open or closed. The animal may be actively scanning the environment. Other The animal is exhibiting any other behavior than those defined above. Not Visible The animal is not visible for an extended period of time (more than one (Nest box) minute) and inspection of the exhibit suggests it is resting in the nest box or another hiding location. Not Visible The animal or its behavior is not visible because it has briefly gone out of (Out) sight but has NOT retreated to the nest box.

Saliva samples were collected for hormone analysis at 1000, 1300, 1600,

1900, and 0100 hrs using voluntarily chewed swabs (Salimetrics, State College PA,

USA) flavored with diluted honey and dried. Swabs were centrifuged at 2500 rpm for 15-min. Saliva was aliquoted and frozen at -18 °C until analysis.

Salivary melatonin concentrations were measured with a commercial enzyme immunoassay (EIA) (IBL International Corp., Toronto ON, Canada). Salivary cortisol was quantified by EIA using methods by Munro and Lasley (1988), and an anti-cortisol antiserum (R4866) and cortisol-horseradish peroxidase (HRP) ligand obtained from Coralie Munro (University of California, Davis, CA). The polyclonal antiserum cross-reacts with cortisol (100%), prednisolone (9.9%), prednisone

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(6.3%), cortisone (5%) and < 1% with androstenedione, androsterone, corticosterone, desoxycorticosterone, 11-desoxycortisol, 21-desoxycortisone, and testosterone (Munro and Lasley, 1988). For both assays, serially diluted extracts were parallel to the standard curve (tmelatonin = -0.358, p = 0.727; tcortisol = -1.383, p =

0.190). Recoveries of hormone from saliva spiked with 2.4 pg/ml or 15.0 pg/ml of melatonin standard were 79% and 83%, respectively. Recoveries from saliva spiked with 0.4 ng/ml or 8.0 ng/ml of cortisol standard were 98.0% and 109.0%. Inter- and intra- assay coefficients of variation were less than 17% for melatonin and 15% for cortisol, and all samples were analyzed in duplicate.

Data were analyzed using Microsoft Excel. Circadian variables were calculated using Cosinor software available at www.circadian.org (©Refinetti 2013;

Refinetti et al., 2007). Period length was defined as length of time to complete one oscillation of a rhythmic variable (Halle and Weinert, 2000).

RESULTS

Condition two ended prematurely due to delayed waking time (Figure 1) and other unusual behaviors (squatting, lethargy, lack of interaction with enrichment) beginning 19 days after entering the blue light condition. A veterinary exam was conducted at day 23 of blue light. Blood, fecal, and urine analyses were in normal ranges, but ultrasound revealed a fluid-filled cyst adjacent to the bladder. Two cracked premolars were also extracted, resulting in a ten-day course of Clamavox.

The aye-aye returned to the exhibit but continued to demonstrate abnormal behavior, prompting the premature change of the lights back to red. The aye-aye’s

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waking time grew gradually earlier, returning to 1000 hrs after ten days under red light. By this point caretakers described her behavior and appetite as back to normal.

Figure 1. Daily time of emergence from the nest box by the aye-aye subject based on keeper reports. Dark phase lights in the exhibit were changed from blue to red at 0 days after 31 days of blue light. Dark phase onset occurred at 1000 hrs, which was considered the “normal” onset of activity for this animal. Open circles represent days that keepers intentionally awoke the aye-aye; on other days she emerged spontaneously from the nest. The triangle indicates the date the aye-aye was anesthetized for a veterinary exam.

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Ultimately, 179 observations (29.8 hrs) were conducted during condition one, in which 10 behavior samples/hr were taken in the dark phase and 5/hr in the light phase. During condition two, 91 observations were conducted (15.2 hrs): 7-9 observations/hr from 1000-1800 hrs, and 2-4 observations/hr from 0800-0900 and

1900-2200 hrs. During condition three 121 observations (20.2 hrs) were conducted,

10/hr of the light phase. We suspended observations for five days following the veterinary exam.

Figure 2. Time spent performing active behaviors (move, feed, self-directed, or object examination) by the aye-aye subject during the baseline red and experimental blue lighting conditions. Data for all 24 hours was only available during the first baseline. Dark phase in the exhibit occurred from 1000-2200 hrs. Sample size refers to the number of ten-minute behavior observations.

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The aye-aye was much less active during the blue condition than either red condition (Figure 2). Inactivity consisted of time in the nest box or resting, and all other behaviors were considered active. On multiple days during condition two, the aye-aye moved around the exhibit during the light phase, which was never observed during red conditions. The subject spent less time moving and examining objects, and slightly less time feeding, under blue light compared to red (Figure 3). There were no fluctuations in temperature or relative humidity corresponding to behavioral changes under blue light.

Figure 3. Dark phase activity budget (1000-2200 hrs) for the aye-aye subject during the baseline red and experimental blue lighting conditions. Sample size refers to the number of ten-minute behavior observations.

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Hormone concentrations were similar under red conditions but differed appreciably under blue light. Dark phase salivary melatonin concentrations were lower under blue light than red (Figure 4). The mean dark phase melatonin concentration under blue light (N=12, 17.96 + 1.65 (SE) pg/mL) was more comparable to light phase melatonin values (N=5, 19.17 + 3.23) than to dark phase values measured under red light (N= 75, 31.25 + 1.40). Period length for melatonin expression was 24.0 hr and 24.8 hr under red conditions one and three, respectively; compared to 25.1 hr for blue condition two. Salivary cortisol concentrations varied more with time of day under red than blue light and were higher overall under red (Figure 5). Period lengths for cortisol expression were 21.8 hr and 24.1 hr under red light and 22.6 hr under blue light. The amplitude of cortisol rhythms was lower under blue light (13.00 ng/ml) compared to both red conditions

(27.50 ng/ml and 36.00 ng/ml).

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Figure 4. Dark phase salivary melatonin concentrations in the aye-aye during the baseline red and experimental blue lighting conditions. Sample sizes are indicated above bars. Samples were collected at T0 (1000 hrs), T3 (1300 hrs), T6 (1600 hrs), and T9 (1900 hrs). For condition one, total samples = 10 each at T0, T3, T6, and T9. For condition two, total samples = 2 at T0, 5 at T3, 3 at T6, and 2 at T9. For condition three, total samples = 10 at T0, T3, and T6 and 5 at T9.

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DISCUSSION

Under blue light, the aye-aye was lethargic and spent less time moving and investigating enrichment than under red light. The delayed onset of activity and presence of irregular light phase activity observed during the blue condition are reminiscent of symptoms travelers experience as jet lag that occur while the circadian system adjusts to abruptly altered light cues (Kolla and Auger, 2011). The relatively rapid re-entrainment to red light that occurred after the blue condition, compared to the delayed response to the initial change to blue, suggests that it was not the change of color but properties of blue light itself that accounted for the erratic behavior observed during the blue condition. An alternative explanation for these findings is that the aye-aye experienced illness; however, she required no further treatment and there were no signs of infection associated with her extracted teeth.

Salivary hormone measurements supported the interpretation that the aye- aye’s endocrine timekeeping system was compromised under blue light (Mirick and

Davis, 2008). Lower dark phase melatonin under blue light suggests these wavelengths induced melatonin suppression. However, melatonin expression is influenced by both light intensity and wavelength (Rea et al., 2001), and the differential suppression we observed may be more or less pronounced at other light intensities. Cortisol regularly demonstrates a strong diurnal rhythm, and the changes to this rhythm during the blue condition also point to possible circadian dysregulation (Boivin and Czeisler, 1998; Corbalan-Tutau et al., 2012).

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Figure 5. Salivary cortisol rhythms in the aye-aye during the baseline red and experimental blue conditions. Samples were collected at T0 (1000 hrs), T3 (1300 hrs), T6 (1600 hrs), and T9 (1900 hrs). For condition one, total samples = 10 each at T0, T3, T6, and T9 and 5 at T15. For condition two, total samples = 2 at T0, 5 at T3, 3 at T6, and 2 at T9. For condition three, total samples = 10 at T0, T3, and T6 and 5 at T9.

Although the results of this experiment suggest that blue exhibit lighting may have deleterious effects, the behavioral effects of blue light may depend on the specific visual capabilities of the species under investigation. As Melin et al. (2012) explain, many nocturnal primates possess only a single cone opsin and therefore experience monochromatic vision. However, the aye-aye retina contains an additional photosensitive opsin with maximal sensitivity to blue wavelengths. The aye-aye’s ability to see blue is thought to be an adaptation to activity during twilight,

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which is rich in blue wavelengths. Thus, it is possible that blue exhibit lighting may be especially aversive to aye-ayes compared to other nocturnal strepsirrhines.

However, the suppressive effects of blue light on melatonin are not thought to be mediated by cone photopigments and are still evident in species that do not perceive the color blue (Rahman et al., 2008).

Although this study included only one subject, our results suggest that exhibit lighting design can have important implications for behavior and health in nocturnal primates. Even in humans, scientists are just beginning to unravel mechanisms responsible for the ill health effects of exposure to light at night

(Navara and Nelson, 2007; Rea et al., 2008). These effects are dramatic enough that the World Health Organization categorizes shift work as a possible carcinogen

(Straif et al., 2007). Nocturnal animals are the shift workers of zoos, and further researcher is greatly needed to design nocturnal lighting for successfully exhibiting nocturnal species without compromising their health or welfare.

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Chapter Seven

A Comparison of Nocturnal Strepsirrhine Behavior in Exhibits Illuminated with Red and Blue Light

INTRODUCTION

There is little consensus among zoos today regarding best lighting practices for nocturnal primates in captivity. Housing animals on reversed light cycles is common and ideally allows visitors to observe nocturnal species in an active state.

However, illuminating the exhibit during the dark phase means that its nocturnal occupants are exposed to artificial light all 24 hours of the day. Furthermore, institutional practices regarding the type and quality of nocturnal lighting vary considerably (Fuller et al., 2013). Information on the consequences of lighting design for animal health and behavior is needed to formulate evidence-based guidelines for the exhibition of nocturnal animals in zoos.

The first major nocturnal zoo exhibits utilized red light based on the belief that the rod-dominated retinas of nocturnal species would make exhibits appear darker to them (Conway, 1969; Davis, 1961). Many nocturnal primates possess morphological features that confer improved vision at night. These features include large eyes (Ross and Kirk, 2007) and a greater ratio of rods to cones in the retina relative to diurnal species (Silveira, 2004). For example, humans have an estimated

5 or 6 cones for every 100 rods in the retina, while in the potto this ratio is 1 to 300

(Goffart et al., 1976). Rod cells contain a single (RH1) opsin, meaning that vision under low-light conditions is essentially monochromatic. Because the spectral sensitivity of rods is shifted toward the blue end of the spectrum, electrical activity

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in the retina appears similar in response to blue and white light but is attenuated in response to red light. This effect has been demonstrated via electroretinogram in the potto, Perodicticus potto (Goffart et al., 1976).

The structure of the mammalian retina also indicates that most species do not perceive the color red. Most mammals are functionally dichromatic, possessing the short wavelength sensitive type one (SWS1) opsin and the middle to long wavelength (M/LWS) opsin; although, many haplorhine primates have evolved trichromatic vision by diversification of the M/LWS gene (Kawamura and Kubotera,

2004). Color vision abilities among strepsirrhine primates varies from full trichromatic vision in some diurnal lemurs (Tan and Li, 1999), to dichromacy in mouse lemurs (Microcebus murinus) (Dkhissi-Benyahya et al., 2001) and aye-ayes

(Daubentonia madagascariensis) (Melin et al., 2012). Lorisiform primates express only a single M/LWS opsin and are therefore monochromatic (Deegan and Jacobs,

1996; Kawamura and Kubotera, 2004). Thus, the perceptual evidence suggests that for both rod-mediated (scotopic) and cone-mediated (photopic) vision, nocturnal strepsirrhines are relatively insensitive to red light.

Current guidelines for illumination of nocturnal primate exhibits are limited.

The husbandry manual for Asian lorisines (Fitch-Snyder and Schulze, 2001) recommends full-spectrum light during the light phase and full-spectrum or red light during the dark phase. However, a recent survey of 29 North American zoos and related facilities housing lorisid primates showed that animals were exhibited under red and blue dark phase lighting at nearly equal rates, and full-spectrum lighting was rarely used during the dark phase (Fuller et al., 2013). Given the

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differential effects of blue light on vision under low-light conditions, in addition to the role of blue wavelengths in light pollution (Falchi et al., 2011), the practice of housing nocturnal primates under blue light needs assessment.

Although studies have repeatedly shown an inverse relationship between light intensity and dark phase activity in captive nocturnal strepsirrhines

(Randolph, 1971; Trent et al., 1977), there is little empirical evidence examining wavelength-dependent behavioral effects. Frederick and Fernandes (1994) simultaneously increased the intensity of simulated day lighting, decreased the intensity of night lighting, and changed night lighting from blue to full-spectrum lighting for two pottos at Franklin Park Zoo. They found increased activity and behavioral diversity following these changes, but the number of simultaneous lighting changes makes it difficult to attribute any causal effects to wavelength

(Frederick and Fernandes, 1994).

In addition to directly affecting behavior via visual perception, light signals can impact behavior through alternate neural routes (Bedrosian et al., 2013). The mammalian retina contains intrinsically photosensitive retinal ganglion cells

(IpRGCs) that project to neural targets involved in regulation of circadian rhythms, as well as areas involved in mood and cognition (Bailes and Lucas, 2010).

Information about time of day is transmitted to the neurological seat of the biological clock, the suprachiasmatic nuclei of the hypothalamus (SCN), by these ganglion cells (Reiter, 1991).

Exposure to light at night disrupts the body’s internal timekeeping system, altering daily rhythms of melatonin and other hormones, in addition to disrupting

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sleep and activity patterns (Depres-Brummer et al., 1995; Mirick and Davis, 2008;

Reiter, 1991). Nocturnal light exposure is consequently associated with a multitude of health problems including cancer, cardiovascular disease, and infertility (Navara and Nelson, 2007). The association between circadian disruption and behavioral dysfunction is evident in human sleep disorders and jetlag (Reid and Zee, 2009), as well as seasonal affective and other depressive disorders (Germain and Kupfer,

2008).

Behavioral responses to light exposure mediated by IpRGCs are also wavelength dependent. IpRGCs contain a photosensitive opsin called melanopsin that responds strongly to short wavelengths but little to red light (Bailes and Lucas,

2010). Consequently, blue light has a greater effect on circadian entrainment than other wavelengths, an effect that has been demonstrated in hamsters (Boulos,

1995), nocturnal mouse lemurs (Perret et al., 2010), and humans (Brainard et al.,

2008). Hamsters (Phodopus sungorus) chronically exposed to dim blue—but not red—light exhibited behavioral signs of depression as well as structural changes to hippocampal neurons involved in cognition and learning (Bedrosian et al., 2013).

We aimed to systematically explore the behavior of nocturnal strepsirrhine primates exhibited under blue and red dark phase illumination. This study was conducted with nocturnal strepsirrhines in the genera Potto, Nycticebus, and Galago and the cathemeral Hapalemur griseus at two facilities, one at which the standard practice was to house nocturnal primates under blue light, and the other red. We hypothesized that our subjects would show overall lower levels of activity under blue light compared to red, and we also expected activity rhythms to appear more

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irregular under blue light. Wavelength-dependent behavioral suppression may have important implications for animal health and welfare, as well as animal care guidelines in zoos and related facilities.

MATERIALS AND METHODS

Subjects and Exhibit Design

The subjects for this experiment consisted of nine nocturnal strepsirrhines representing four species housed at Cleveland Metroparks Zoo (CMZ) in Cleveland,

OH and Cincinnati Zoo and Botanical Garden (CZBG) in Cincinnati, OH (Table 1).

Subjects at CMZ consisted of 1.1 (male.female) Perodicticus potto housed together;

0.1 pygmy slow loris (PSL, Nycticebus pygmaeus) housed with an aye-aye

(Daubentonia madagascariensis) during condition one only; a breeding pair of moholi bushbabies (Galago moholi), which gave birth to an infant midway through the second condition; and 1.0 PSL housed with the bushbabies. The bushbaby exhibit additionally contained a black and rufous elephant shrew (Rhynchocyon petersi). Data collected on the aye-aye for this experiment has been reported elsewhere (Fuller et al., in review). Subjects at CZBG consisted of 1.0 potto housed with a female gray bamboo lemur (Hapalemur griseus) and 0.1 potto that was housed with a breeding male during the third study condition only.

Exhibit designs were similar for all subjects. All six exhibits featured gunnite rockwork, natural wood perching, and leafy cover. Every exhibit contained at least one wooden nest box or sleeping tube. All the exhibits contained bare concrete floors except for the bushbaby exhibit at CMZ, which had wood shavings as

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substrate. Exhibit temperatures were maintained at 24 °C at CMZ and 22-23 °C at

CZBG. Neither facility artificially controlled relatively humidity, which ranged from

44-50% at CMZ and 35-70% at CZBG. Additionally, HOBO® data loggers (Onset

Computer Corp., Cape Cod MA, USA) were placed in exhibits to monitor light intensity, temperature, and relative humidity at CMZ only.

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Table 1. Nocturnal strepsirrhine subjects and housing conditions for the multi-zoo study. CMZ= Cleveland Metroparks Zoo, CZBG= Cincinnati Zoo and Botanical Garden.

ID House Species Sex Age Zoo Group Changes Exhibit Lighting Light Red Dark Blue Name (y) Enclosure during Dimensions Regimen Phase Phase Dark (Regional Experiment (lxwxh m) (L:D hrs) Intensity Intensity Phase Studbook) (lux) (lux) Intensity (lux) female potto Jahzira Perodicticus PP1 F 6 absent with (1311) potto medical problem part CMZ Potto of condition 2.9 x 4.9 x 12:12* 9.25 0.34 0.65 Exhibit one; male 4.3 Ringo Perodicticus PP2 M 19 Otolemur (1236) potto garnettii present condition three 154 approx. 14:10 CZBG Potto new male potto Tiombe Perodicticus 0.91 x 0.91 (D phase PP3 F 11 Exhibit present - 38.7 37.6 (1266) potto x 1.22 varies condition three from 9 to 14 hrs) Amare Perodicticus PP4 M 5 (1312) potto

CZBG approx. Potto/ 14:10 no changes in HG Bamboo 0.91 x 0.91 (D phase group - 16.12 15.3 Lil Bit Hapalemur Lemur x 1.83 varies F 13 composition (T100068) griseus Exhibit from 9 to 14 hrs)

ID House Species Sex Age Zoo Group Changes Exhibit Lighting Light Red Dark Blue Name (y) Enclosure during Dimensions Regimen Phase Phase Dark (Regional Experiment (lxwxh m) (L:D hrs) Intensity Intensity Phase Studbook) (lux) (lux) Intensity (lux) CMZ Aye- Aye Exhibit 3.6 x 4 x 4.4 234.0 4.5 4.51 (condition PSL1 moved same for one) Sing Nycticebus from the aye- both NP1 F 16 CMZ PSL (1115) pygmaeus aye and housed exhibits, Exhibit alone 12:12* (conditions 3 x 2.7 x 2.9 199.5 1.43 1.57 two and three) Nox Nycticebus GM1 and GM2 NP2 M 6 (2496) pygmaeus added to Chachi Galago exhibit midway GM1 F 2 (1140) moholi CMZ PSL/ through

155 Bushbaby condition one; Mojo 3 x 2.6 x 2.9 12:12* 256.15 2.26 2.32 (1139) Exhibit GM1 gave birth Galago to a male infant GM2 M 2 moholi midway through condition two * The length of the dark phase was gradually shortened over the course of the study by ~ 7 min every two weeks.

With the exception of the previously mentioned social changes (Table 1), exhibit conditions and husbandry practices were largely constant throughout the course of the experiment. However, at CMZ, live crickets were regularly placed in exhibits during condition three only, and the animals at CMZ were also transitioned to a low-starch primate biscuit during this time period.

The CMZ exhibits were all located in the nocturnal wing of a building mainly featuring diurnal primates, while the CZBG exhibits were located in a nocturnal area adjacent to exhibits with artificial day lighting. At both zoos, animals were housed on a reversed light cycle in which conditions were designed to simulate nighttime during the day when visitors were at the zoo. Animals at CMZ were on a 12:12 LD

(light:dark) light cycle with dark phase onset at 1000 hrs. At CMZ the length of the dark phase was gradually shortened to simulate seasonal change, although this difference amounted to a change of less than 30 min over the course of the experiment. Exhibits at CZBG were not on a fixed light cycle, and daily lighting regimens could range from 15:9 to 10:14 LD. Keepers serviced exhibits in the morning and then initiated the dark phase between 0700-0900 hrs.

At both facilities, exhibits were illuminated with bright, full-spectrum light during the light phase, while baseline dark phase lighting was red at CMZ and blue at CZBG. Additional uncontrolled sources of illumination in exhibits came from white lights in keeper areas, red (CMZ) or blue (CZBG) lights illuminating public spaces outside the exhibits, illuminated monitors displaying graphics at CMZ, and occasionally, visitors with cameras or flashlights. Light intensities in the exhibits were measured using a SPER Scientific light meter (#840020, Scottsdale AZ, USA).

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At CMZ, the fluorescent lights were covered with gel filters (Rosco Laboratories Inc.,

Stamford CT, USA) to create the red (#2001) and blue conditions (#4290, 4260, and

4230). Dark phase lights were filtered using red or blue plastic tubes at CZBG.

Data Collection and Analysis

This study was conducted to compare red and blue light using an ABA experimental design at two institutions. Data were collected at CMZ October 2012—

February 2013 and at CZBG from February—May 2013. All aspects of lighting were constant across conditions except for dark phase light color (Table 1). At both zoos, the study consisted of three conditions: (1) baseline light (red at CMZ and blue at

CZBG); (2) experimental color change; and (3) a second baseline. In humans experiencing jetlag or other circadian disruptions, a 24-hr interval is needed to adjust sleep patterns by 1 to 2 hrs (Kolla and Auger, 2011). To allow for entrainment, there was a two-week break in data collection following a lighting change before data collection began for the next condition. Data collection for conditions one and two lasted five weeks at CMZ and three weeks for condition three; all conditions were two weeks in duration at CZBG. Therefore, total time spent under the experimental lighting regimen was seven weeks for subjects at CMZ and four weeks for subjects at CZBG.

Behavioral sampling schemes varied slightly by condition and facility. At CMZ behavior was observed all 24 hours of the day for conditions one and two, while only dark phase observations were made during condition three. Observations at

CZBG were only conducted during a portion of the dark phase, from 0900 to 1800

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hrs. The number of observations was balanced by time of day for each interval (24 or 12 hours for CMZ and 8 hours for CZBG). At both zoos, 20 hrs of observations were conducted in each condition for every subject during the dark phase; additionally, 10 hrs/condition of data were collected for each animal during light phase observations at CMZ.

Table 2. Ethogram for behavioral data collection.

Behavior Operational Definition Allogrooming, social exploration, social play, reproductive Social behaviors, or agonistic behavior. Move Motion in any direction, including climbing and backing up. Ingesting food, normally by grabbing a food item with one hand Feed and taking it to mouth. Includes drinking from surface or dipping hands in liquid and licking the hand. Auto-grooming using tooth comb or tongue, scratching the self using grooming claw or nails, facial rubbing (rubbing snout, chin, Self-Directed cheeks, or neck on a substrate or object), or rubbing the head against the arms. Object Animal is physically manipulating an object using hands or teeth or Examination is actively sniffing an object. Animal is motionless and is lying down, sitting, or assuming the Rest sleeping ball posture. The animal’s eyes may be open or closed. The animal may be actively scanning the environment. The animal is exhibiting any other behavior than those defined Other above. The animal is not visible for an extended period of time (more than Not Visible one minute) and inspection of the exhibit suggests it is resting in (Nest box) the nest box or another hiding location. Not Visible The animal or its behavior is not visible because it has briefly gone (Out) out of sight but has NOT retreated to the nest box.

A continuous behavior sampling protocol was employed in ten-min sessions using a broad ethogram (Table 2) and The Observer 5.0 (Noldus Information

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Technology, Wageningen, The Netherlands). For data analysis, the ethogram was divided into inactive (rest and not visible in nest box) and active behaviors (all other visible behaviors). This simplified ethogram (active vs. inactive) was used for data collection instead of the full ethogram on the bushbaby subjects.

For analysis of data, descriptive statistics were calculated using Microsoft

Excel®. The percent of time performing each behavior was calculated after correcting observation durations for time spent not visible in the exhibit for all subjects except the pottos at CMZ, which spent a large proportion of time in an area of their exhibit difficult to observe.

Subjects’ behaviors were using a mixed-model repeated measures ANOVA in

IBM® SPSS® v. 21, with condition as the within-subjects variable and individual and species as the between-subjects factors. Because the study design was inverted between the two facilities, data from each zoo were analyzed separately to compare the three study conditions. Facility data were combined to compare behavior under red vs. blue light. Because of the modified ethogram used to observe the bushbabies, data for these two subjects was analyzed separately as well. Data were tested for homoscedasticity using Mauchly’s Test of Sphericity, and degrees of freedom were corrected using Roy’s Largest Root for multivariate tests and Greenhouse-Geisser estimates of sphericity for univariate tests for behaviors that violated the assumption of sphericity. Paired t-tests were used for post-hoc analyses, and all significance values were set at p < 0.05.

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RESULTS

In general, the nocturnal strepsirrhines in this study spent less time moving around their exhibits under blue light compared to red; although, this change was more readily apparent in some subjects compared to others. The male potto (PP4) housed with the bamboo lemur at CZBG exhibited the most obvious changes. After the exhibit lights were changed to red, this animal went from spending most of his time huddled toward the back of a nest hollow in a tree to being noticeably more active moving around and exploring different areas of the exhibit.

Although this was not captured in the ethogram, the pygmy loris subjects exhibited a change in the quality of their movements around the exhibit. Each loris had a preferred resting spot in its exhibit, and much of the movement recorded during the study consisted of travel between that spot and a source of food or water.

Under blue light, the lorises would move either extremely rapidly or slowly toward the food, and they would quickly retreat to their rest spot after eating. In contrast, the lorises demonstrated a more relaxed locomotor style under red light and tended to move around the exhibit at a more moderate pace.

All subjects, regardless of facility or species, spent less time performing active behaviors under blue light compared to red. The mixed-model ANOVA revealed a main effect of light color (F9, 858 = 29.125, p < 0.001) as well as a significant interaction between subject and light color (F9, 863 = 52.431, p < 0.001).

Univariate analyses revealed significant changes in social behavior (F1, 866 = 7.535, p

= 0.006), moving (F1, 866 = 124.138, p < 0.001), self-directed behavior (F1, 866 =

19.624, p < 0.001), examining objects (F1, 866 = 39.908, p < 0.001), time spent in the

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nest box (F1, 866 = 122.630, p < 0.001), and the summary measure of time spent active (F1, 866 = 126.086, p < 0.001). Differences in time spent feeding, visibly resting in the exhibit, and engaged in other behaviors were not significantly different under red and blue light. The interaction between light color and subject was significant for all behaviors except other. A paired samples t-test showed a significant difference in overall activity under blue compared to red light for the bushbabies as well (t227= 11.228, p < 0.001).

Comparing all three conditions separately for each facility, the mixed-model

ANOVA showed a significant main effect of condition at CMZ (F9, 947 = 20.346, p <

0.001) and CZBG (F8, 628 = 22.453, p < 0.001), as well as a significant interaction between subject and condition at each facility (CMZ: F9, 951 = 16.465, p < 0.001;

CZBG: F8, 630 = 37.615, p < 0.001). There was a main effect of condition on activity level in the bushbabies at CMZ (F1.68, 47.004 = 7.450, p = 0.003), but the interaction between subject and condition was not significant in this case. Mean activity levels for the female bushbaby were similar before (38.47 + 5.31 (SE) % of time, n = 72 observations) and after (40.92 + 6.35%, n = 42) she gave birth during condition two.

Both the pottos and lorises at CMZ spent less time moving around their exhibits in the blue condition compared to red (Figure 2 a-b). Both species also spent more time investigating objects in their exhibits under red light, particularly during condition three. The pottos at CMZ also spent more time not visible in their exhibit in the blue light condition (92.07 + 4.81% of time not visible) compared to the red conditions (condition one, 76.62 + 9.71%; condition three, 80.67 + 8.06%).

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Figure 1 a-b. Percent of time spent performing active behaviors during the dark phase by animals at (a) Cleveland Metroparks Zoo and (b) Cincinnati Zoo and Botanical Garden. Study condition and light color are indicated on the x-axis. Note that the study design was inverted between the two facilities, one of which used red light as a baseline and the other blue. Legend indicates subject ID (see Table 1). Active behaviors included social, move, feed, self-directed, object examination, and other. *Mean percentages are corrected for time spent not visible for all subjects except PP1 and PP2. Standard error indicates number of observations, which varies among subjects but is approximately 120 observations per animal per condition.

Univariate tests revealed a significant effect of condition on moving (F1.946,

928.197 = 55.405, p < 0.001), self-directed behavior (F1.954, 932.212 = 5.734, p = 0.004), object examination (F1.255, 598.862 = 52.961, p < 0.001), resting (F1.985, 946.798 = 22.766, p

< 0.001), and overall activity level (F1.997, 952.636 = 30.943, p < 0.001) in subjects at

CMZ, and a significant interaction between subject and condition for self-directed

(F5.863, 932.212 = 11.137, p < 0.001) and resting (F5.955, 946.798 = 10.587, p < 0.001) behavior. Effects on social behavior were still not significant after removing the solitarily housed PSL from the analysis at CMZ. Tests of between-subjects effects

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showed significant differences among subjects in all behaviors except object examination.

The pottos and bamboo lemur at CZBG spent a greater proportion of time performing all active behaviors under red light compared to the blue conditions

(Figure 2 c-d). At CZBG, univariate tests revealed a significant effect of condition for moving (F1.963, 622.236 = 37.138, p < 0.001), feeding (F1.946, 616.750 = 7.828, p = 0.001), self-directed (F1.985, 629.176 = 11.149, p < 0.001), object examination (F1.864, 591.005 =

14.014, p < 0.001), and resting (F1.959, 621.120 = 15.602, p < 0.001) behaviors, as well as overall activity level (F1.959, 610.084 = 55.203, p < 0.001). However, interactions between subject and condition were significant for moving (F3.926, 622.236 = 19.503, p

< 0.001), object exam (F3.729, 591.005 = 6.910, p < 0.001), and resting (F3.919, 621.120 =

12.387, p < 0.001) behavior, as well as overall activity (F3.849, 610.084, = 11.889, p <

0.001). There was also a significant effect of condition on social behavior for the potto and bamboo lemur housed together at CZBG (F1.780, 373.778 = 4.224, p = 0.019).

Tests of between-subjects effects were significant for all behaviors except other.

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Figure 2 a-d. Changes in behavior across the three study conditions (C1-3) comparing red and blue light for pottos (a) and pygmy slow lorises (b) at Cleveland Metroparks Zoo (CMZ); and pottos (c) and a bamboo lemur (d) at Cincinnati Zoo and Botanical Garden (CZBG). Conditions are compared using dark phase observations only. Standard errors are based on n = 2 pottos at CMZ and CZBG and n = 2 lorises at CMZ. Standard errors for the bamboo lemur are based on observation number (n = 106 for C1, n = 118 for C2, n = 119 for C3). Values for social behavior are based on a single loris at CMZ and a single potto at CZBG, because only these animals had available social partners. Here again standard errors are calculated by observation number.

Using species as the between-subjects factors, we compared the pottos to the

PSL subjects at CMZ, and the pottos to the bamboo lemur at CZBG. The mixed-model

ANOVA revealed a significant main effect of condition (CMZ: F9, 951= 22.413, p <

0.001; CZBG: F8, 630 = 11.199, p < 0.001) and a significant interaction between species and condition at both zoos (CMZ: F9, 951 = 13.097, p < 0.001; CZBG: F8, 630 =

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8.473, p < 0.001). At CMZ, the interaction between species and condition was only significant between the pottos and PSLs for self-directed behavior (F1.956, 936.832 =

4.669, p = 0.01), resting (F1.982, 949.371 = 14.936, p < 0.001), and time spent in the nest box (F1.786, 855.430 = 8.120, p = 0.001). At CZBG, the interaction between species and condition was significant between the pottos and bamboo lemur for all behaviors except other.

Because of the species interaction, post-hoc comparisons of behavior by condition were conducted within species using paired t-tests. Time spent moving by the pottos at CMZ varied significantly between condition one and two (p < 0.001), and two and three (p < 0.001), but not between the two baseline conditions. Time spent examining objects varied significantly between condition three compared to both condition one (p < 0.001) and two (p < 0.001). There were no significant differences in time spent socializing between this pair among conditions. Overall activity level varied significantly between all three conditions for the pottos at CMZ

(p < 0.001 for C1 vs. C2 and C2 vs. C3; p = 0.046 for C1 vs. C3).

Paired t-tests also revealed significant differences in time spent moving (p <

0.001) and overall activity level (p = 0.001 for C1 vs. C2; p < 0.001 for C2 vs. C3; and p = 0.021 for C1 vs. C3) between all three conditions for the loris subjects at CMZ.

Time spent examining objects also varied between all three conditions (p < 0.001).

Paired t-tests for the pottos at CZBG showed a significant difference in time spent moving among all three conditions (p < 0.001 for C1 vs. C2 and C2 vs. C3; and p = 0.031 for C1 vs. C3). Time spent feeding varied significantly between conditions one and two (p = 0.016) and two and three (p = 0.018), but not between the two

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baselines. Self-directed behavior also varied significantly between all three conditions (p < 0.001 for C1 vs. C2; p = 0.024 for C2 vs. C3; and p = 0.029 for C1 vs.

C3). Object examination varied significantly between condition one and conditions two (p = < 0.001) and three (p = 0.001) but not between conditions two and three.

Overall time spent active varied significantly in each condition (p = < 0.001 for C1 vs. C2 and C2 vs. C3; p = 0.005 for C1 vs. C3).

Social behaviors between the potto and bamboo lemur varied significantly between conditions one and two (p = 0.027) but not between conditions one and three, or two and three. The bamboo lemur showed a significant difference in time spent moving between conditions two and three only (p = 0.041). No significant differences in the amount of time spent examining objects were found for this subject. Overall activity level for the bamboo lemur varied significantly between conditions one and two (p = 0.001) and two and three (p = 0.014) but not between conditions one and three.

Twenty-four hour behavioral data were available for the male potto and both pygmy loris subjects at CMZ from conditions one and two. All three subjects rarely exhibited active behaviors during the light phase, except for the period from 0800-

1000 when keeper staff were often present in exhibits cleaning and providing food.

The male potto (PP2; Figure 3a) and female loris (NP1; Figure 3b) showed consistently higher activity levels over time in the red condition compared to the blue condition. The male loris was somewhat more active during the light phase than the other subjects and also showed the smallest difference in behavior in response to red and blue light (NP2; Figure 3c).

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Figure 3 a-c. 24-hour activity level data for potto PP2 (a) and pygmy slow lorises NP1 (b) and NP2 (c) at Cleveland Metroparks Zoo. See Table 1 for subject IDs. Dark phase onset occurred at 1000 hrs in each exhibit. The y- axis shows the mean percent of time spent performing active behaviors in condition one (red light; red lines) and condition two (blue light; blue lines). Active behaviors included social, move, feed, self-directed, object examination, and other. Standard errors indicate variation between observations. 180 ten-min observations were conducted for each condition for each animal.

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DISCUSSION

The results of these experiments suggest that nocturnal strepsirrhines perform more active behaviors when they are housed in exhibits illuminated by red light than blue. At least some suppression of activity under blue light was documented in each subject of this study, regardless of species, facility, or other individual characteristics. The magnitude of these effects varied among our subjects, and the small-scale nature of these studies should caution against overgeneralizing the results. However, the consistent change of activity in the expected direction between the facilities using different baseline light colors is at the very least a compelling argument for further investigation of the behavioral and physical effects of exhibiting nocturnal primates under blue light.

Behavioral Differences Observed under Red and Blue Light

Taking all factors into account, the most compelling change that occurred in the strepsirrhine subjects was the increased time spent moving around the exhibit under red light compared to blue. This change in locomotor behavior largely accounts for the differences in overall activity level observed among the study conditions. While many behaviors occur rhythmically, locomotor behavior has long been the standard measure of activity in laboratory studies of circadian rhythms, and its neurological substrates are better understood than that of other behaviors

(Bartness and Albers, 2000). Given the role that blue wavelengths play in entraining the circadian system, it seems likely that the change in movement observed across study conditions is a true experimental effect.

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Somewhat complicating the interpretation of the effects of study condition on locomotor behavior are differences observed between baseline conditions at each zoo. As expected, time spent moving differed significantly between each baseline period compared to blue light, but not between the baseline conditions, for the pottos at CMZ. However, time spent moving varied between all three conditions for the lorises at CMZ and the pottos at CZBG.

Several husbandry changes likely account for the variability we observed among conditions. First, the animals at CMZ received live crickets as part of their diet during condition three only. In a prior experiment, providing a cricket dispenser greatly increased activity levels in two zoo-housed pottos by encouraging hunting and stalking behaviors (Frederick and Fernandes, 1996). The crickets clearly stimulated more foraging in the animals at CMZ, particularly the female loris.

The behavioral effects of the crickets are also reflected in the increased object examination observed during condition three for both the pottos and lorises at CMZ, and this husbandry change likely accounts for some of the apparent experimental effects of light color on investigative behavior.

The pottos at CZBG also displayed different amounts of locomotor behavior in each of the three study conditions. The female potto in particular spent much more time moving in condition three compared to the first baseline under blue light.

This change is likely due to the introduction in condition three of the male mate, which the female spent a large amount of time following around the exhibit. Free- ranging pottos often forage alone at night, and spatial proximity is the most commonly observed affiliative behavior between male and females (Pimley et al.,

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2005). This pair was observed breeding during condition three, so it is likely the female was in estrus and this also increased locomotor behavior (Fitch-Snyder and

Jurke, 2003).

The male potto at CZBG spent more time moving and feeding under red light compared to both blue conditions, but elevations in these behaviors continued into condition three. The male potto and bamboo lemur had been moved from a larger, darker exhibit containing a breeding pair of aye-ayes to a new exhibit only about four weeks before the study began. Keepers at CZBG reported a drastic reduction in locomotor behavior in this individual after switching exhibits. In the new, brighter exhibit, this potto spent a large percentage of his time resting in a hollow tree.

Captive slow lorises are known to be more active in larger cages, so baseline levels of locomotor behavior may have been depressed during condition one as a result of this pair being moved into a smaller exhibit (Daschbach et al., 1982/83). The increase in his activity after the lights were changed to red was very noticeable and seems to be at least partly an effect of light color. However, perhaps these active behaviors persisted at a higher baseline level after the return to blue light as this pair became more adjusted to their new exhibit space over time.

The male potto also spent more time engaged in social behavior with the bamboo lemur in red light compared to blue. Most social interactions between this pair took the form of allogrooming. After grooming one another, these animals would often groom themselves, which may account for their elevated levels of self- directed behavior. Although it would be interesting to document an effect of red light in the only cathemeral lemurid in this study, the increase in overall activity

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level in this animal seems to have resulted largely from increased social behavior. It appears that cathemerality may be relatively common in lemurs (Curtis and

Rasmussen, 2006), and it would be fascinating to examine how they adapt their behavior to diurnal compared to nocturnal lighting conditions in a zoo setting in future research.

The amount of time spent visibly resting, occupying the nest box, and not visible in the exhibit all differed significantly between study conditions, but in a species-dependent manner that was likely related to exhibit design. For example, the potto exhibit at CMZ was constructed in such way that the potto subjects in this study were able to spend an enormous amount of time out of sight during observations. This complicates interpreting the data for these animals, but it is also interesting in terms of our hypothesis that the pottos were visible more to the observer when the exhibits lights were red. On the other hand, the male pygmy loris was generally found resting in sight, because of the perching in his exhibit and the bushbaby group monopolizing the nest box. Differences in time spent resting or in the nest box are thus difficult to interpret between subjects and may or may not represent true experimental effects. Differences in self-directed behavior observed between study conditions are likely to be a consequence of altered visibility and species differences in grooming behavior rather than an experimental effect as well.

The effect of the bushbaby birth during the experimental condition two at

CMZ also complicates interpretation of the results for the individuals sharing that exhibit. The bushbabies showed the most dramatic reduction in activity level under the experimental blue light, but this effect is likely complicated by the birth that

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occurred during this time period. In captive Galago garnetti, activity levels are low for both members of mother-infant dyads immediately following birth and steadily increase over the next 14 weeks (Ehrlich and MacBride, 1990). The relative inactivity of the bushbabies during this time period in turn might have provided more opportunities for the male loris to use the exhibit, possibly explaining why this individual showed the least dramatic change in activity level under blue light.

Effects of Light Color on Circadian Function and Recommendations

Investigations into primate circadian rhythms suggest that there are two means by which light may affect locomotor behavior. A true circadian rhythm is the result of the interaction between endogenous rhythms and factors in the environment that entrain the rhythm to local conditions. For mammals, light is the primary agent entraining activity rhythms (Rietveld et al., 1993). Exposure to light during the dark phase has been documented to have a masking or suppressive effect on locomotor behavior in a variety of species (Redlin, 2001). Therefore, light plays a dual role in both entraining the locomotor rhythm and acutely altering it through masking effects.

The 24-hour data collected on the animals at CMZ seems to indicate that blue light may have had a greater masking effect on locomotor behavior during the dark phase than red. However, the animals largely limited their behavior to the dark phase under both lighting regimens, so they seemed to be equally entrained to both light cycles. Therefore, it seems likely that blue light did not lead to circadian disruption in our subjects. It is unfortunate that we were unable to collect 24-hour

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behavioral data on subjects at CZBG as well. The behavioral effects of nocturnal light exposure are dependent on light intensity as well as wavelength (Tokura and Hirai,

1980); in fact, dark phase intensity level is thought to be the primary factor regulating activity levels in nocturnal primates (Erkert and Cramer, 2006).

Therefore, we may have seen more evidence of circadian disruption in the animals housed in the brighter exhibits at CZBG.

There are several implications of these results for husbandry practices for nocturnal primates in zoos. Erkert (1989) proposes that artificial lighting regimens for nocturnal primates are effective when animals show evidence of stable circadian rhythms without any apparent masking of activity during the dark phase. Because blue light appeared to mask activity in our subjects, it follows that red would be a better choice. However, there may be other options.

Moonlight is reflected sunlight, and nighttime light is similar in color composition to sunlight with some added longer wavelengths from stellar sources

(Melin et al., 2012). Partly for this reason, dim white light is sometimes recommended for exhibiting nocturnal primates (Fitch-Snyder and Schulze, 2001;

Walker, 1968). Alternatively, lighting could be designed which essentially appears full-spectrum but selectively filters the narrow bands of wavelengths that activate

IpRGCs that communicate with the circadian system (Bailes and Lucas, 2010). This approach has been tried in shift workers; however, the subjects in this study did not report changes in their activity levels, ability to concentrate, or feelings of fatigue while working in an office at night with the experimental lighting (Schobersberger et al., 2007).

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In terms of environmental health risks, nocturnal animals are much like the shift workers of the modern zoo. The World Health Organization considers shift work a probable carcinogen (Straif et al., 2007), and in 2012 the American Medical

Association (AMA) released a statement calling for further investigation of the health effects of nocturnal light exposure, including its role in sleep disorders and cancer (AMA, 2012). These effects of nocturnal light exposure are commonly attributed to the blue light emitted from phones and other electronic devices, and the AMA recommends using dim red lights in the bedroom at night to ameliorate these effects (AMA, 2012). After examining the spectral output of common lighting sources, Falchi et al. (2011) recommend avoiding white LEDs and metal halide lamps for nighttime use. As our understanding of the effects of light on human and animal health grows, so too should the sophistication of lighting designs in controlled zoo exhibits.

This study demonstrated a small but consistent suppression of locomotor activity in nocturnal strepsirrhines exhibited under blue light compared to red. The extent to which this effect is biologically meaningful, or has implications for animal health or welfare, has yet to be determined. Also complicating the issue is the great variability and inherent flexibility of primate visual (Ankel-Simons and Rasmussen,

2008) and circadian (Erkert, 2008) systems. Future studies examining the effects of lighting on endocrine functioning and health should provide further insight for designing the most health-promoting light conditions for captive primates.

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Chapter Eight

Endocrine Responses to Exhibit Lighting in the Potto, Perodicticus potto

INTRODUCTION

The previous chapter compared behavioral responses to red and blue exhibit lighting in nocturnal strepsirrhines. For this study, saliva samples were also collected to measure concentrations of melatonin and cortisol in pottos, Perodicticus potto, living in the two lighting conditions. The hormones melatonin and cortisol are both biomarkers for circadian disruption (Corbalan-Tutau et al., 2012; Mirick and

Davis, 2008). Additionally, melatonin plays a critical role in transmitting information about time of day to the circadian system (Arendt, 2005). Exposure to light at night suppresses nocturnal melatonin production in a manner dependent on both light intensity (Zeitzer et al., 2000) and wavelength (Brainard et al., 2008). The damaging effects of light-induced melatonin suppression include infertility, metabolic syndrome, and cancer (Navara and Nelson, 2007).

Our hypothesis was that the type of lighting used to illuminate exhibits during the dark phase would affect the rhythmicity and amplitude of melatonin and cortisol expression in nocturnal strepsirrhines. We expected that melatonin suppression would occur in animals living in blue but not red light, and the degree of suppression would be greater at brighter luminosities. We also expected that the subjects would show a strong diurnal rhythm in cortisol in the red lighting condition but not under blue lights.

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MATERIALS AND METHODS

Subject and Housing

The subjects for this experiment consisted of 5.3 (#male:#female) pottos: 1.1 pair at Cleveland Metroparks Zoo (CMZ) in Cleveland, OH; 3.2 pottos at Cincinnati

Zoo and Botanical Garden (CZBG) in Cincinnati, OH; and 1.0 potto at Franklin Park

Zoo (FPZ) in Boston, MA (Table 1).

Housing conditions for the subjects at CMZ and 1.1 of the CZBG pottos were described in the previous chapter. The remaining pottos at CZBG were housed in a nocturnal building. One pair (PP6 and PP7) occupied a triangular-shaped exhibit with large glass walls on two sides. One window faced the public viewing area while the other looked into a mixed-species exhibit occupied by aardvarks, galagos, and bats. The other male potto (PP5) was housed solitarily in a wire cage in an off- exhibit area containing a variety of nocturnal animals. The male potto at FPZ (PP8) was housed alone in an exhibit located in an alcove of a greenhouse structure predominantly housing diurnal species.

Lighting conditions for the additional subjects in this study varied. The pair

PP6 and PP7 was on a fixed 12:12 LD (light:dark) reversed light cycle with dark phase onset at 0900 hrs. During the dark phase, blue fluorescent fixtures illuminated both their exhibit and the one adjacent to it, while the public area of the building was lit by blue LEDs. Light phase illumination was provided by halogen fixtures. The CZBG male in holding (PP7) was on a fixed 12:12 LD reversed light cycle with dark phase onset at 1200 hrs. The holding room was illuminated by red-

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filtered fluorescent fixtures during the dark phase and full-spectrum fluorescents during the light phase.

The potto at FPZ (PP8) was housed on a 12:12 reversed, fixed LD cycle with dark phase onset at 0800 hrs. During the dark phase, the exhibit was illuminated by

25W halogen sealed glass beam lamps covered with a mixture of red and blue filters.

In addition to these lights, six cool white fluorescent bulbs were on during the light phase. The red and blue conditions for this subject were created using the same filters previously described (Chapter 6,7).

Light intensities in the exhibits were measured using a SPER Scientific light meter (#840020, Scottsdale AZ, USA).

Study Design

This study was conducted to compare red and blue light using an AB(A) experimental design at three institutions. Data were collected at CMZ October

2012—February 2013, at CZBG from February—May 2013, and May—July 2013 at

FPZ. All aspects of lighting were constant across conditions except for dark phase light color (Table 1). At both zoos, the study consisted of three conditions: (1) baseline light (red at CMZ and FPZ; blue or red at CZBG); (2) experimental color change; and (3) a second baseline or a higher intensity blue light condition.

The general within-subjects design of the study comparing red and blue light was described in the previous chapter. There were some variations to this design necessitated by the baseline housing conditions of the additional subjects added here. The solitary male in holding at CZBG was housed under red light at baseline,

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while the light in the exhibit was blue. The blue light on exhibit was also a lower intensity than the exhibits housing the other pottos. Instead of altering the exhibit lights in the nocturnal building, the male from the holding area was moved into the exhibit for the blue condition two and the pair on exhibit was moved into his holding space for their red condition two.

An ultrasound during condition two revealed that the female PP6 was pregnant, and in preparation for the birth, her mate PP7 was moved into the exhibit with PP3. As a result, PP7 was moved from a dim red enclosure in the holding area to a blue exhibit with greater light intensity than he experienced on exhibit in condition one. In attempt to replicate the effects of this change in intensity level, the male at FPZ was moved from dim blue light in condition two to brighter blue light in condition three rather than returning to the baseline lighting condition.

Saliva samples were collected for hormone analysis using methods described in Chapters 5 and 6. Samples were collected at: 0 (T0), 3 (T3), 6 (T6), 9 (T9), and 15

(T15) hours after the onset of the dark phase at CMZ; T0, T3, and T6 at CZBG; and T6 only at FPZ. During each condition, we attempted to collect 45 saliva samples from each potto at CMZ and 20 for each potto at CZBG. Keepers at FPZ collected 15 samples per condition from their potto. Because only a single measurement was taken per day at FPZ, these samples were analyzed for melatonin concentrations only.

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Data Analysis

Salivary melatonin and cortisol were analyzed by enzyme-immunoassay using the methods described in Chapter 6. For pottos, serially diluted extracts were parallel to the standard curve for both assays (tmelatonin = -1.111, p = 0.303; tcortisol = -

1.338, p = 0.204). Recoveries of hormone from saliva spiked with 2.4 pg/ml or 15.0 pg/ml of melatonin standard were 68.0% and 100.1%, respectively. Recoveries from saliva spiked with 0.4 ng/ml or 8.0 ng/ml of cortisol standard were 98.0% and

84.0%. Melatonin samples for pottos were analyzed at a 1:10 dilution while cortisol samples were analyzed at a 1:2 dilution. Melatonin samples from the subject at FPZ were consistently low on the standard curve. For this reason, extrapolated values were adjusted to the lowest detectable limit for the assay prior to analysis.

We also created potto-specific controls for both assays using pooled samples.

Inter-assay coefficients of variation were less than 16% for the potto control in the melatonin EIA and less than 14% for controls included in the EIA kit. Inter-assay coefficients of variation were under 12% for in-house cortisol controls and were

14% for the potto control. All samples were analyzed in duplicate and only values with a coefficient of variation less than 12% were used for analysis.

For analysis of data, descriptive statistics were calculated using Microsoft

Excel®. Melatonin concentrations were compared under red and blue light for all individuals (regardless of study design) using a mixed-model repeated measures

ANOVA in IBM® SPSS® v. 21, with light color as the within-subjects variable and individual as the between-subjects factor. To standardize measures, only samples collected at T6 were used for this analysis. The values recorded for each individual

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under higher intensity blue light (condition three for PP7 and PP8) were not included in this comparison.

Differences in melatonin concentrations among the three conditions were analyzed using a mixed-model ANOVA with condition as the within-subjects variable and individual as the between-subjects factor. Because the study design varied among subjects, data from FPZ were analyzed separately and animals were grouped by study design (RBR = red-blue-red or BRB = blue-red-blue) for other analyses to compare conditions.

For analysis of rhythmicity, melatonin and cortisol concentrations were compared within each condition using a mixed-model repeated measures ANOVA with sample collection time (T0, T3, T6) as the within-subjects variable and individual as the between subjects factor. This analysis included the additional time points at T9 and T15 for the male potto only.

Data were tested for homoscedasticity using Mauchly’s Test of Sphericity, and degrees of freedom were corrected using Roy’s Largest Root for multivariate tests and Greenhouse-Geisser estimates of sphericity for univariate tests when necessary. Paired t-tests were used for post-hoc analyses, and all significance values were set at p < 0.05.

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Table 1. Nocturnal strepsirrhine subjects and housing conditions for the multi-zoo study. CMZ = Cleveland Metroparks Zoo, CZBG = Cincinnati Zoo and Botanical Garden, FPZ = Franklin Park Zoo.

Subject House Sex Age General Zoo Group Changes Exhibit Lighting Light Red Dark Blue ID Name (y) Study Enclosure during Dimensions Regimen Phase Phase Dark (Regional Design Experiment (lxwxh m) (L:D hrs) Intensity Intensity Phase Studbook) C1:C2:C3 (lux) (lux) Intensity (R= red, (lux) B = blue) female potto Jahzira PP1 F 6 R:B:R absent with (1311) medical CMZ Potto problem part of 2.9 x 4.9 x 12:12* 9.25 0.34 0.65 Exhibit condition one; 4.3 Ringo PP2 M 19 R:B:R male Otolemur (1236) garnettii present condition three approx. 181 CZBG 14:10 Tiombe Potto PP7 present 0.91 x 0.91 (D phase PP3 F 11 B:R:B - 38.7 37.6 (1266) Exhibit condition three x 1.22 varies from 9 to 14 hrs) CZBG approx. Potto/ 14:10 no changes in Amare Bamboo 0.91 x 0.91 (D phase PP4 M 5 B:R:B group - 16.12 15.3 (1312) Lemur x 1.83 varies composition Exhibit from 9 to 14 hrs)

swapped CZBG enclosure with PP5 Gabriel Nocturnal PP6/PP7 for 0.91 x 0.91 M 16 R:B:R 12:12 - 1.32 1.03 (1248) Building condition two x 1.83 Holding

Subject House Sex Age General Zoo Group Changes Exhibit Lighting Light Red Dark Blue ID Name (y) Study Enclosure during Dimensions Regimen Phase Phase Dark (Regional Design Experiment (lxwxh m) (L:D hrs) Intensity Intensity Phase Studbook) C1:C2:C3 (lux) (lux) Intensity (R= red, (lux) B = blue) Lucy swapped PP6 F 12 B:R:B (0085) enclosures with PP5 for CZBG condition two; Nocturnal PP6 was gravid; 2.9 x 1.8 x B:R: 12:12 - 1.32 1.03 Jabari Building PP7 moved in 2.3 PP7 M 11 brighter (1273) Exhibit with PP3 to B prepare for PP6’s upcoming parturition 5.4 for R:B: Rendille FPZ 1.83 x 1.22 C2; PP8 M 24 brighter no changes 12:12 249.7 5.2

182 (1215) Exhibit x 1.52 36.9 for B C3 * The length of the dark phase was gradually shortened over the course of the study by ~ 7 min every two weeks.

RESULTS

Some pottos more readily provided saliva samples than others, but individual tractability did not vary by study condition. Inadequately chewed samples were generally too thick with honey to assay and were discarded. In total, melatonin concentrations were measured in 447 saliva samples and cortisol in 384 samples. Total sample numbers for each individual are written as # melatonin, # cortisol: 93,93 for PP1; 89,73 for PP2; 61,60 for PP3; 10,0 for PP4; 54,56 for PP5;

35,41 for PP6; 61,61 for PP7; and 44, 0 for PP8.

Melatonin Suppression during the Dark Phase

Comparing melatonin concentrations measured at T6 under red and blue light for all animals, the mixed-model ANOVA did not show a main effect of light color (p = 0.663), and there was no significant interaction between light color and subject (p = 0.567).

Melatonin concentrations at T0, T3, and T6 were compared across conditions using a mixed-model ANOVA. For animals with the RBR study design (Figure 1a), the ANOVA demonstrated a significant effect of condition on melatonin concentration (F3, 17 = 4.476, p = 0.017), as well as a significant interaction between condition and subject (F4, 18 = 24.502, p < 0.001). However, univariate tests were not significant for melatonin measured at T0 (p = 0.403), T3 (p = 0.231), or T6 (p =

0.068). Interactions between subject and condition were significant at all three time points (F(T0)2.595, 11.678 = 3.715, p = 0.048; F(T3)4,18 = 8.982 , p < 0.001; F(T6)4,18 =

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3.314, p = 0.034). However, paired t-tests at T0, T3, and T6 were not statistically significant for this group overall for melatonin differences between any condition.

For animals in the BRB study design, there were only enough samples available at T0 and T3 to compare melatonin concentrations for two individuals

(PP3 and PP7). The mixed-model ANOVA showed a significant main effect of condition (F2, 14 = 38.628, p < 0.001) as well as a significant interaction between subject and condition (F2, 14 = 15.807, p < 0.001). Melatonin concentrations differed significantly by condition at T3 (F1.168, 8.176 = 6.019, p = 0.036) but not T0 (p = 0.450); however, univariate tests for the interaction between subject and condition were not significant at either time (T0, p = 0.446; T3, p = 0.236).

For all four subjects in the BRB study design (Figure 1b), the mixed-model

ANOVA showed a significant main effect of condition on T6 melatonin concentrations (F2, 40 = 5.958, p = 0.005) as well as a significant interaction between condition and subject (F6, 40 = 2.540, p = 0.035). The animals housed in the brightest exhibits (PP3 and PP4) showed no change in T6 melatonin concentrations, while the other two pottos in this group trended toward higher melatonin under red than blue light (Figure 1b).

For all four animals taken together in the BRB study design, post-hoc tests revealed a significant difference in melatonin concentrations between the two blue conditions (C1 vs. C3, t9 = -4.042, p = 0.003) at T3. T6 melatonin values differed significantly between conditions one and three (t23 = -2.398, p = 0.025), as well as two and three (t24 = 4.452, p< 0.001), but not between the first two conditions (p =

0.125).

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Figure 1a-c. Melatonin concentrations measured in potto saliva six hours after dark phase onset in red and blue light. See Table 1 for individual IDs. Each experiment consisted of exposure to three lighting conditions, in three different orders: (a) red-blue-red, (b) blue-red-blue, and (c) red-dim blue- bright blue. Sample number are as follows for C1, C2, C3: n = 4,10,10 for PP1; n = 5,8,9 for PP2; n = 8,7,9 for PP5; n = 10,10,10 for PP3; n = 1,1,2 for PP4; n = 9,8,6 for PP6; n = 10,10,10 for PP7; and n = 15,15,14 for PP8.

For all four animals taken together in the BRB study design, post-hoc tests revealed a significant difference in melatonin concentrations between the two blue conditions (C1 and C3, t9 = -4.042, p = 0.003) at T3. T6 melatonin values differed significantly between conditions one and three (t23 = -2.398, p = 0.025), as well as two and three (t24 = 4.452, p < 0.001), but not between the first two conditions (p =

0.125).

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One of the subjects in the RBR group (PP7) moved from an exhibit with dim blue light in condition one to a brighter exhibit for blue condition three. When this individual is removed from the previous analysis, only the difference in T6 melatonin concentration between the experimental red (C2) and second blue baseline (C3) conditions remains significant (t14 = 2.286, p = 0.038). Even without this individual included, mean melatonin levels were higher under the experimental red light compared to blue light in condition three, during which the gravid female showed a large decrease in melatonin concentrations (Figure 1b).

Figure 2. Scatterplot of light intensity compared to salivary melatonin concentrations measured in pottos at six hours after dark phase onset. Melatonin concentrations were measured in eight pottos in red (red diamonds) and blue (blue squares) lighting. The open blue squares represent two measures for the same individual (PP7) at different intensities, as do the open blue triangles (for PP8).

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For the male PP7 that experienced different intensity conditions (Figure 1b), post-hoc tests showed that he exhibited significantly higher melatonin values at T3 under dim blue compared to brighter blue light (t4 = 6.991, p = 0.002). At T6, his melatonin levels were higher in dim red light compared to both dim blue (t9 = -

2.446, p = 0.037) and bright blue (t9 = 4.870, p = 0.001) light. However, the difference between dim and bright blue light was not significant (p = 0.068).

There was also a significant main effect of condition on melatonin concentrations measured at T6 in the potto at FPZ (Figure 1c; Figure 2; F2, 26 = 8.858, p = 0.001). Post-hoc tests showed significant differences in melatonin concentrations comparing dim red to both dim (t14 = 2.446, p = 0.028) and bright

(t13 = -4.064, p = 0.001) blue light; as well as near significance comparing dim blue to bright blue (p = 0.05).

Biomarkers of Circadian Regulation: Melatonin and Cortisol

Melatonin concentrations were compared at different time points within each condition to assess rhythmicity. All five times were only available for the male potto at CMZ (PP2) for the first two conditions only. There was no significant main effect of time on melatonin concentration for this potto under red (p = 0.350 or) or blue (p = 0.392) light. Comparing all animals in the RBR condition using samples from TO, T3, and T6, the mixed-model ANOVA showed no main effect of time on melatonin levels under red (p = 0.403 or p = 0.880) or blue (p = 0.733) light. There were also no significant interactions between subject and time.

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Figure 3. Salivary cortisol concentrations measured in pottos living in blue and red dark phase lighting conditions. See Table 1 for individual IDs. Samples were collected at T0 (dark phase onset), T3, T6, T9, and T15. Each subject was exposed to three lighting conditions in one of two orders. Condition order was red-blue-red for subjects PP1, PP2, and PP5; and order was blue-red- blue for PP3, PP6, and PP7. Sample numbers are as follows for C1, C2, C3: n = 18,45,30 for PP1; n = 20,29,24 for PP2; n = 19,18,19 for PP5; n = 20,20,20 for PP3; n = 16,11,14 for PP6; and n = 20,21,20 for PP7.

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For animals in the BRB condition, there was a main effect of time on melatonin concentration in the first blue condition (F2, 16 = 1.845, p = 0.190) but the interaction between time and subject was not significant (p = 0.874). There was no main effect of time on melatonin concentration under red light (p = 0.059) or in the second blue light condition (p = 0.054).

Cortisol data were analyzed for rhythmicity using the same approach as for melatonin with the time points T0, T3, and T6 only (Figure 3). For animals in the

RBR condition, the mixed-model ANOVA did not show a significant main effect of time on cortisol concentrations in either red condition (C1, p = 0.198; C3, p = 0.339) or the blue condition (C2, p = 0.140). For animals in the BRB condition, the ANOVA did not show a significant main effect of time on cortisol concentration in either blue condition (C1, p = 0.818; C3, p = 0.096) or the red condition (C2, p = 0.141).

Two pairs of animals exhibited simultaneous elevations in salivary cortisol concentrations specific to a single condition. Both PP1 and PP2 showed markedly elevated cortisol concentrations during the red condition (C3) at CMZ. The CZBG pair of PP6 and PP7 both showed dramatically increased cortisol levels in the experimental red condition (C2) compared to either blue condition.

DISCUSSION

The results of these experiments do not support the hypothesis that blue dark phase exhibit lighting is associated with melatonin suppression and circadian dysregulation in pottos. Given the now overwhelming evidence for the selective role of blue light in circadian regulation in humans and other animals (AMA, 2012;

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Brainard et al., 2008; Falchi et al., 2011), these results are puzzling. However, we did document suppression of melatonin in animals exposed to higher luminosities of blue light. It seems likely that the negative results of these experiments were due to methodological issues and husbandry variations in our study population, rather than a legitimate non-effect of blue light on biomarkers of circadian regulation in the potto.

Although light intensities were matched between blue and red experimental conditions as a control measure, two animals in our study were serially exposed to blue light both in the dim (~ one lux) and moderate (~ 35 lux) range. The effects of light color revealed in analysis were for the most part due to changes observed in these pottos. A third animal, the gravid female at CZBG, also exhibited lower melatonin concentrations in the third condition (blue light) even though she was living in the exact same exhibit (and light environment) as in condition one. This change is not likely to be a result of the pregnancy; in humans, melatonin levels are generally elevated during the final stages of pregnancy (Wierrani et al., 1997). It seems that generally the effects documented here were a result of light intensity rather than wavelength.

Intensity thresholds for melatonin suppression are species-specific. In humans, half of maximal melatonin suppression is achieved by nocturnal exposure to light of 100 lux (Zeitzer et al., 2000); and exposure to ordinary levels of room light before bedtime suppresses melatonin production in human subjects (Gooley et al., 2010). In squirrel monkeys (Saimiri sciureus), 50% suppression of melatonin occurs at 200 lux (Hoban et al., 1990). However, melatonin is suppressed by lower

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intensities in nocturnal species, and light near one lux can suppress pineal melatonin production in Syrian hamsters (Brainard et al., 1982). Even so, it is possible that in the relatively dim lighting conditions occupied by many of our subjects (~ one lux), melatonin was not suppressed by either color of light.

An alternative explanation for our negative findings is that the pottos in our study were experiencing chronic melatonin suppression under their baseline lighting regimens. At all facilities, the pottos were exposed to uncontrolled light from visitors, keepers, and other sources. None of the facilities in this study consistently used red or dimmed light in keeper areas, and pottos were often exposed to white light as staff entered exhibits for animal care or saliva collection. In laboratory rats, exposure to light from hallways (as low as 0.2 lux) seeping into animal rooms underneath doors significantly decreased melatonin amplitude relative to control subjects housed in true darkness (Dauchy et al., 1997). This lack of experimental control—including the variable light phase for some animals at

CZBG—may have obscured subtle differences in melatonin expression between red and blue light at dim levels.

The effects of photic input on the circadian system are complicated by many factors, some of which were all but impossible to control in a zoo setting. The extent of melatonin suppression that occurs at a given intensity depends on the duration and timing of light exposure in experimental models (Kennaway and Rowe, 1994).

Furthermore, melatonin responses to light exposure can differ due to the nature of earlier lighting conditions (Smith et al., 2004). Although there were no obvious differences among our subjects based on the order of study conditions, prior light

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history still may have played a role in the negative results obtained. Alternatively, perhaps the two-week period given to adjust to each lighting condition, which was based on recommendations for managing jet lag in humans (Kolla and Auger, 2011), was inadequate.

Also likely obscuring any experimental results were individual differences in melatonin expression. Melatonin levels can be affected by sleep deprivation in humans, but this effect depends on age (Zeitzer et al., 2007); and melatonin levels are known to decline with age in humans (Arendt, 2005). Sensitivity of the circadian system to photoentrainment also decreases with age in nocturnal mouse lemurs

(Microcebus murinus) (Gomez et al., 2012). In our study, the highest melatonin levels were consistently measured in one of the youngest subjects (PP1), and the lowest were measured in the oldest (PP8). Furthermore, we had no measure of visual function in these subjects. Circadian dysfunction is common in blind humans (Sack and Lewy, 2001). If older pottos had experienced any retinal degeneration— perhaps resulting from years of constant light exposure (Bellhorn, 1980)—this may have obscured any effects of the experimental lighting.

Both cortisol and melatonin typically show a strong diurnal rhythm and as a result are considered biomarkers for circadian disruption. For the most part, the subjects in this study did not demonstrate a significant difference in concentrations of either hormone based on time of day. One explanation for this observation is that these animals were also experiencing chronic circadian dysregulation; however, logically this assumption cannot be made without first demonstrating that a normal circadian rhythm exists for these hormones in the potto. The ideal experiment to

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investigate this claim would be to house pottos in constant darkness and measure hormone levels to determine if an endogenous rhythm persists in the absence of photic input (Rietveld et al., 1993). Without these data, it is not possible to determine whether the subjects in this study were truly experiencing circadian dysregulation.

The role of cortisol in social stress is widely recognized (Tamashiro et al.,

2005), and it is likely that the cortisol data for several pottos in this study reflect changes in social rather than lighting conditions. The potto pair at CMZ (PP1 and

PP2) showed markedly elevated cortisol levels only after a galago was introduced into their exhibit. At CZBG, potto pair PP6 and PP7 both exhibited elevated cortisol when housed together in a small cage in a holding area but not in their exhibit.

However, neither the male PP6 nor the female PP3 exhibited cortisol elevations when he was introduced to her exhibit in condition three. There are many sources of stress in captivity and a great deal of variation in physiological response to stress in animals; as a result, many doubt the utility of cortisol as a proxy for stress or welfare

(Mormede et al., 2007; Rushen, 1991). It seems likely that the stress-cortisol relationship may have obscured any efforts to utilize cortisol as a biomarker for circadian regulation in this study.

In these experiments, we aimed to demonstrate an effect of nocturnal exhibit lighting color on endocrine markers of circadian function, melatonin and cortisol, in pottos. Our results suggest that some degree of melatonin suppression may occur in brighter exhibits, but differences based on light color were not evident in this study.

These results were likely hampered by a lack of experimental control over lighting

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and husbandry practices in the zoo setting and should not be taken as evidence that either red or blue light is preferable for illuminating nocturnal primate exhibits.

Future investigations examining the effects of light intensity, as well as controlled experiments establishing baseline hormone levels in true darkness, will likely clarify these issues.

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Chapter Nine

General Discussion

Summary of Findings and Conclusions

Together these studies provide a broad assessment of the health and care of lorisid primates living in North American zoos, while specifically addressing a deceptively simple question—which light is the best night light? As a whole, captive populations of lorises and pottos—like their wild counterparts—are struggling.

Lighting is only one of many husbandry issues needing assessment for these species.

This work represents a small step toward understanding the effects of lighting design on the behavior and welfare of captive nocturnal strepsirrhines, and there is much more work to be done.

Several trends in the captive care of lorisid primates were evident in the multi-institutional husbandry survey. Institutions currently housing lorises and pottos in North America are keeping animals in physical environments that meet current husbandry recommendations; however, larger cage sizes may be needed for some species and there is little consensus on lighting design. Second, a relatively small number of facilities engage in regular training with their lorisids, and more institutions could take advantage of the benefits of positive reinforcement training in providing animals with mental stimulation and reducing potential stressors associated with husbandry procedures. Third, there is much to learn about the social lives of lorisid primates, and more research is needed to understand how exhibit size and design can be modified to promote social housing and reproduction

195

in these species. Finally, improved husbandry knowledge has the potential to positively influence population sustainability, which is greatly needed given the tenuous status of aging populations of pottos, slender lorises, and slow lorises.

For the next study, medical records were reviewed to assess sources of mortality in captive lorises and pottos. This study identified poor neonate survivorship as a major impediment to population sustainability. Deaths due to trauma are common and often affect neonates and juveniles before they have the opportunity to reproduce. Major sources of mortality in adult animals are infection, neoplasia, renal disease, and degenerative changes. Improved diet and social management would both likely have beneficial effects for animal health.

Efforts to develop methods for understanding the role of light in health and behavior were complicated but promising in the zoo setting. Although actigraphy was not ultimately used for behavioral monitoring in this study, the pilot experiment reported here showed that this method could be used successfully and reliably in lorisid primates despite their slow-moving locomotor style. Circadian activity monitoring holds great promise for understanding health-behavior relationships in zoo animals, particularly species that are naturally inactive or that may hide overt signs of stress or illness.

Also promising is the use of salivary analysis for endocrine monitoring of zoo-housed primates. Although few facilities currently conduct training with their lorisids, we were able to train lorises, pottos, and an aye-aye for saliva collection with relative ease. In addition to biomarkers of circadian regulation, saliva can be used to non-invasively monitor stress and reproductive hormones, as well as insulin

196

and other health measures. The scientific possibilities of salivary hormone analysis in a zoo setting are seemingly endless.

Finally, the results of our experiments comparing endocrine and behavioral responses to red and blue exhibit lighting provide preliminary but compelling evidence that blue, but not red, nocturnal lighting may have detrimental effects on animal behavior and health. These effects were dramatically illustrated in a single aye-aye subject, which exhibited major behavioral changes under blue light accompanied by endocrine evidence of circadian dysregulation. Pottos and pygmy slow lorises were also more active under red light than blue, although blue light appears to have masked dark phase locomotor behavior in these subjects rather than altering circadian entrainment. There was also some evidence of melatonin suppression by bright blue light, but the effects of different wavelengths at the intensities tested were not clear. Ultimately, neither red nor blue lighting may be ideal, and future investigations into the effects of other wavelengths and intensity levels are needed.

Lighting Recommendations for Zoos

Despite rapidly accumulating evidence of the negative effects of blue light, this investigation did not reveal significant differences in markers of circadian disruption in pottos housed under red versus blue light. However, taking into account the wealth of laboratory and epidemiological evidence about the harms of exposure to (blue) light at night, the behavioral evidence from all the strepsirrhines studied here, and the hormonal responses observed in the aye-aye and some pottos,

197

a general recommendation against the use of blue light during the dark phase seems warranted.

This study confirmed that behavioral and hormonal responses to exhibit lighting design are likely to depend in part on the perceptional abilities of the species in question. Red light may be aversive for some species (including human zoo visitors). For this reason, neutral density filters are often recommended.

Perhaps more ideal would be filters that selectively block wavelengths that are known action spectra for melatonin suppression. Although the exact wavelengths that regulate the circadian system in nocturnal strepsirrhines are not known, as a rule light sources that emit wavelengths shorter than 540 nm should be avoided during the dark phase, as should white LED and metal halide lights (Falchi et al.,

2011). Lighting can be designed that appears to be full-spectrum while blocking out the wavelengths responsible for circadian disruption. This type of designer lighting may be quite useful for zoos, but its behavioral and endocrine effects should first be empirically evaluated.

Future Outlook

These studies show both the promise and challenges of understanding the factors shaping activity patterns of zoo-housed animals. Circadian patterns of activity are shaped by many factors, as are the physiological effects of exposure to light at night. These multitudinous variables interact and are difficult to control in an applied setting. For example, it is possible that our failure to biologically validate the melatonin assay for pygmy slow lorises occurred because our study subjects

198

were already experiencing chronic melatonin suppression. More controlled experiments using non-exhibited captive colonies could clarify these issues.

There are several experiments that can provide immediately useful recommendations for nocturnal lighting design in zoos using the methods developed here. First, the aye-aye findings should be replicated in additional study subjects. Next, experiments in a more photically controlled environment could be used to develop a dose-response curve for light exposure and melatonin suppression in the aye-aye and potto. This approach can be used to determine a threshold level for melatonin suppression under different intensity/wavelength combinations, which then could be used to derive recommendations for dark phase lighting intensity in these species.

While this research has touched on many applied issues for the maintenance of nocturnal primates in captivity, there are greater implications of these findings for human and animal health. All animals are increasingly affected by light pollution as new technologies spread across the globe while forested areas shrink. The line between captivity and “the wild” becomes increasingly blurred in this context. The long-term survival of nocturnal species may depend in part on their ability to adapt to this new reality. Understanding the physiological and behavioral effects of nocturnal lighting can facilitate the design of artificial lighting that minimizes its deleterious effects for humans their nocturnal relatives alike.

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Appendix I

Questions for Multi-Institutional Husbandry Survey

Question Response Type^ Basic Group Information How many lorisids are there in the group for open-ended (one or more animals) which you are completing this survey? Is this a breeding group, i.e. are efforts being made Yes/No to mate individuals within this group? Is this a mixed-species group? Yes/No Group Enclosures (Refer to methods text for definitions of primary and secondary enclosures included in survey instructions.) Where is the primary enclosure located? multiple choice* (single response permitted) Which best describes the secondary enclosure? multiple choice (single response permitted) How often is the group moved between multiple choice enclosures? (single response permitted) Is the primary enclosure temperature controlled? Yes/No - if so, provide temperature (or range) - open response What best describes the ventilation systems for multiple choice the primary enclosure? (single response permitted) Is relative humidity controlled in the primary Yes/No enclosure? - if so, at what relative humidity (& RH) is the -open response enclosure maintained? What are length, width, and height of the primary open response enclosure? What best describes the substrate in the primary multiple choice enclosure? (single response permitted) Does the primary enclosure contain any hiding or multiple choice sleeping sites? (multiple responses permitted) Lighting Conditions in the Primary Enclosure Which best describes the lighting conditions in the multiple choice enclosure? (single response permitted) For exhibits lit by artificial lighting, are day Yes/No lengths varied seasonally? What is the length of the dark phase (may be exact open response range or variable)? Is artificial lighting used to simulate twilight, i.e. Yes/No are there gradual transitions between the dark and light phases? What sources of light are used to illuminate the multiple choice enclosure during the light phase? (multiple responses permitted)

200

Question Response Type^ What sources of lights are used to illuminate the multiple choice enclosure during the dark phase? (multiple responses permitted) What color(s) of artificial light illuminate the multiple choice enclosure during the dark phase? (multiple responses permitted) Animal Care Practices How many keepers work directly with this group multiple choice during a given work week? (single response permitted) How often is the primary enclosure cleaned? multiple choice (single response permitted) In how many feedings per day is the main diet multiple choice presented? This total does not include (single response permitted) supplemental food used for training or other purposes. How often is enrichment presented for this group? multiple choice (single response permitted) How often are training sessions conducted with multiple choice this group? (single response permitted) Are specific members of the group generally open response removed during parturition events and/or while infants are being reared? Explain. How frequently do members of this group receive multiple choice physical exams by a veterinarian? (single response permitted) Individual Animal Questions Respondents were instructed to complete one set of the following questions for each group member. Enter studbook and local ID number. open response Select the individual’s species. multiple choice (single response permitted) Select the sex. multiple choice (single response permitted) How long has this individual been housed in the multiple choice physical conditions described for the group in this (single response permitted) survey? Please select from the following options that multiple choice which best represents your estimate of this (single response permitted) individual’s reproductive success. * See results tables in Chapter Two for response options for multiple choice questions. ^ For all multiple choice questions, survey respondents were always given the option to write in an alternative response or to explain further their response choice.

201

Appendix II

Detailed Tables for Cause of Death in Lorisid Primates by Species and Age Group

Primary cause of death or reason for euthanasia in Loris tardigradus nordicus housed in North American facilities. There were no juvenile deaths in this group.

Neonate Adult Geriatric All L.t. nordicus Age Class N=5 (2.2.1) N=4 (2.2) N=11 (6.5) N=20 (10.9.1)

Cause of Death M F U % of neonates M F % of adults M F % of geriatrics % of all animals

Gastrointestinal 1 9.1 5.0

202 Hepatic & Biliary 1 25.0 5.0

Ocular 1 9.1 5.0

Renal 3 2 45.5 25.0

Respiratory 1 25.0 1 9.1 10.0

Multisystemic 1 25.0 5.0

Trauma 1 20.0 5.0

Incomplete Record 1 25.0 2 1 27.3 20.0 Maternal Neglect 1 1 40.0 10.0 (neonates) Unexplained Neonate Death 1 1 40.0 10.0 (neonates)

Primary cause of death or reason for euthanasia in Loris tardigradus tardigradus housed in North American facilities.

All L.t. Neonate Juvenile Adult Geriatric tardigradus Age Class N=23 (7.5.11) N=4 (3.1) N=11 (6.4.1) N=34 (15.19) N=72 (31.29.12)

% of % of % of % of % of all Cause of Death M F U M F M F U M F neonates juveniles adults geriatrics animals

Cardiovascular & 2 1 8.8 4.2 Hemolymphatic CNS 2 5.9 2.8

Hepatic & Biliary 3 27.3 1 2.9 5.6

Integumentary 1 2.9 1.4 203

Musculoskeletal 2 5.9 2.8

Renal 1 9.1 4 8 35.3 18.1

Respiratory 1 2 8.8 4.2

Multisystemic 3 1 36.4 3 7 29.4 19.4

Trauma 4 1 1 26.1 1 1 50.0 2 18.2 13.9

Incomplete Record 2 50.0 1 9.1 4.2

Maternal Neglect (neonates) 1 4.3 1.4 Stillborn/Abortion 3 13.0 4.2 (neonates) Unexplained Neonate Death 3 3 7 56.5 18.1 (neonates)

Primary cause of death or reason for euthanasia in Nycticebus coucang housed in North American facilities.

All slow Neonate Juvenile Adult Geriatric lorises Age Class N=17 (7.3.7) N=4 (3.1) N=27 (13.14) N=61 (27.34) N= 109 (50.52.7) % of % of % of % of % of all Cause of Death M F U M F M F M F neonates juveniles adults geriatrics animals Cardiovascular & 1 3.7 3 4.9 3.7 Hemolymphatic Endocrine and Metabolic 1 2 4.9 2.8

Ear, Nose & Throat 1 3.7 2 3.3 2.8

Gastrointestinal 2 3 18.5 2 3.3 6.4

204 Hepatic & Biliary 1 1 7.4 4 6.6 5.5

Musculoskeletal 1 3.7 2 3.3 2.8 1 Renal 9 34.4 19.3 2 Reproductive 1 3.7 1 2 4.9 3.7

Respiratory 1 3.7 2 3.3 2.8

Multisystemic 1 25.0 5 3 29.6 9 7 26.2 22.9

Trauma 4 1 1 35.3 1 1 50.0 1 2 11.1 10.1

Unknown 1 25.0 1 1.6 1.8

Incomplete Record 3 1 14.8 1 1 3.3 5.5

Maternal Neglect (neonates) 1 5.9 0.9

All slow Neonate Juvenile Adult Geriatric lorises Age Class N=17 (7.3.7) N=4 (3.1) N=27 (13.14) N=61 (27.34) N= 109 (50.52.7) Stillborn/Abortion (neonates) 2 2 2 35.3 5.5 Unexplained Neonate Death 4 23.5 3.7 (neonates) 205

Primary cause of death or reason for euthanasia in Nycticebus pygmaeus housed in North American facilities.

All pygmy Neonate Juvenile Adult Geriatric lorises Age Class N=46 (17.12.17) N=3 (1.2) N=28 (10.18) N=56 (32.24) N=133 (59.57.17) Cause of Death % of % of % of % of % of all M F U M F M F M F neonates juveniles adults geriatrics animals Cardiovascular & 1 3.6 4 1 8.9 4.5 Hemolymphatic CNS 2 1 5.4 2.3 Endocrine & Metabolic 1 1.8 0.8 Ear, Nose, & Throat 1 1.8 0.8 Gastrointestinal 1 3.6 1 1.8 1.5 Hepatic & Biliary 2 7.1 2 3.6 3.0 Immunologic 1 3.6 0.8 206 Integumentary 1 1 3.6 1.5

Musculoskeletal 1 3.6 2 4 10.7 5.3 Ocular 1 1.8 0.8 Renal 5 2 25.0 7 6 23.2 15.0 Reproductive 1 1.8 0.8 Respiratory 1 2.2 1 1 7.1 4 7.1 5.3 Multisystemic 1 1 1 6.5 1 1 66.7 2 4 21.4 7 3 17.9 15.8 Trauma 3 3 5 23.9 1 33.3 1 3.6 1 1.8 10.5 Unknown 1 1.8 0.8 Incomplete Record 1 5 21.4 1 3 7.1 7.5 Maternal Neglect (neonates) 4 2 3 19.5 6.8

Stillborn/Abortion (neonates) 3 5 5 28.3 9.8 Unexplained Neonate Death 5 1 3 19.6 6.8 (neonates)

Primary cause of death or reason for euthanasia in Perodicticus potto housed in North American facilities. There were no juvenile deaths in this group.

Neonate Adult Geriatric All pottos Age Class N=20 (11.4.5) N=3 (1.2) N=10 (5.5) N=33 (17.11.5)

% of % of % of % of all Cause of Death M F U M F M F neonates adults geriatrics animals

Cardiovascular & Hemolymphatic 1 1 20.0 6.1

CNS 1 33.3 3.0

Renal 1 33.3 1 10.0 6.1

Reproductive 1 10.0 3.0 207 Respiratory 1 2 15.0 1 1 20.0 15.2

Multisystemic 1 1 10.0 2 1 30.0 15.2

Trauma 3 15.0 9.1

Incomplete Record 1 33.3 1 10.0 6.1

Maternal Neglect (neonates) 2 10.0 6.1

Stillborn/Abortion (neonates) 1 1 2 20.0 12.1 Unexplained Neonate Death 3 0 3 30.0 18.1 (neonates)

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