Behavioral, Physiological, and Neurological Influences of Pheromones and Interomones in Domestic Dogs

Home , Pregnanolone, Vandenbergh effect, Whitten effect

BEHAVIORAL, PHYSIOLOGICAL, AND NEUROLOGICAL INFLUENCES OF PHEROMONES AND INTEROMONES IN DOMESTIC DOGS

By Glenna Michelle Pirner, B.S., M.S.

A DISSERTATION in ANIMAL SCIENCE

Submitted to the Graduate Faculty Of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

John J. McGlone, Ph.D. Chairperson of the Committee Alexandra Protopopova, Ph.D. Nathaniel Hall, Ph.D. Arlene Garcia, Ph.D. Yehia Mechref, Ph.D.

Mark Sheridan, Ph.D. Dean of the Graduate School May 2018

Texas Tech University, Glenna M. Pirner, May 2018

ACKNOWLEDGEMENTS

When I accepted a staff position as a research aide at Texas Tech University, I never dreamed that work would culminate a Ph.D., and I would like to first express my gratitude to Dr. John McGlone for giving me this opportunity. Your patience and guidance have provided me with invaluable knowledge and skills that will remain with me throughout my career.

I would also like to thank Dr. Protopopova, Dr. Hall, Dr. Garcia, and Dr. Mechref for taking time to be a part of my committee and provide their feedback and advice. Your insight into each respective field has taught me to broaden my thinking and I look forward to future collaborations.

I am deeply appreciative of the undergraduate research assistants and my fellow graduate students both in our lab and the department for their encouragement and support during my years here, especially Guilherme, Matt, Edgar, Lingna, Alexis, Gizell, Adrian, and Garrett. The teamwork and friendship made even the toughest days more bearable, and I wish all of you the best in your future endeavors.

Last, but by no means least, I must express my love and gratitude for my parents,

Glen and Wanda Schmid, my husband Jack, and our daughter Mary, without whom this would not be possible. My parents have provided support in every aspect of my life, allowing me to choose my own path and encouraging that decision at every step along the way. Jack has been loving, patient, and understanding when I have to work long hours,

Texas Tech University, Glenna M. Pirner, May 2018 offering to watch Mary so I can study or cooking dinner because I will be home late.

Above all, Mary has been my strongest source of motivation. Her unconditional love is a constant reminder that no matter what happens, at the end of the day I am right where I am supposed to be.

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Texas Tech University, Glenna M. Pirner, May 2018

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

ABSTRACT ...... viii

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

LIST OF ABBREVIATIONS ...... xii

I. INTRODUCTION ...... 1

II. LITERATURE REVIEW ...... 3

Background ...... 3

Defining Pheromones ...... 3

Early and current literature ...... 3

Terminology ...... 4

Perception ...... 5

Characteristics ...... 11

Conservation of molecules across species ...... 12

Classes of Pheromones ...... 13

Alarm ...... 13

Maternal ...... 14

Social/aggregation ...... 14

Sexual/reproductive ...... 15

Sex Pheromones Identified in Different Species ...... 18

Humans ...... 18

Texas Tech University, Glenna M. Pirner, May 2018

Other Species ...... 20

Dog Specific Information ...... 23

Reproductive physiology ...... 23

Reproductive behavior ...... 24

Literature Cited ...... 27

III. IMPACT OF 2-METHYLBUT-2-ENAL ON ACUTE STRESS RESPONSES IN

CHRONICALLY STRESSED DOMESTIC DOGS ...... 39

Abstract ...... 39

Introduction ...... 40

Materials and Methods ...... 43

Results ...... 51

Discussion ...... 57

Conclusion ...... 62

Literature Cited ...... 63

IV. PHYSIOLOGICAL VARIABLES MAY BE USED IN PREDICTING STRESS

RESPONSE IN LABORATORY DOGS ...... 67

Abstract ...... 67

Introduction ...... 68

Materials and Methods ...... 71

Results ...... 77

Discussion ...... 82 v

Texas Tech University, Glenna M. Pirner, May 2018

Conclusion ...... 84

Literature Cited ...... 85

V. IDENTIFICATION OF PUTATIVE SEXUAL PHEROMONES IN MALE

DOGS BY SOLID-PHASE MICROEXTRACTION TECHNIQUE IN

COMBINATION WITH GAS CHROMATOGRAPHY-MASS

SPECTROMETRY ...... 88

Abstract ...... 88

Introduction ...... 89

Materials and Methods ...... 90

Results ...... 93

Discussion ...... 105

Conclusion ...... 109

Literature Cited ...... 110

VI. EVALUATION OF PHEROMONE-INDUCED ACTIVATION IN THE

HUMAN BRAIN USING FUNCTIONAL MAGNETIC RESONANCE

IMAGING (FMRI) ...... 114

Abstract ...... 114

Introduction ...... 115

Materials and Methods ...... 117

Results ...... 124

Discussion ...... 129 vi

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Conclusion ...... 131

Literature Cited ...... 132

VI. CONCLUSION ...... 135

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Texas Tech University, Glenna M. Pirner, May 2018

ABSTRACT

Pheromones provide a crucial method of information transfer for many species; however, this type of communication is not well understood in the domestic dog. Beyond intraspecific communication, evidence suggests that some molecules, known as interomones, may act as a pheromone in one species but elicit unpredictable effects in a different species. 2-methylbut-2-enal (2M2B), the rabbit maternal-neonatal pheromone, is one such molecule.

To determine the behavioral and physiological effects of 2M2B on chronically stressed domestic dogs two stress-induction models were used: a simulated thunderstorm and car travel. In both models, 2M2B elicited a faster return of elevated heart rate (HR) to baseline compared to control (CON). During the thunderstorm dogs treated with 2M2B spent 15.9% more time lying down compared to when treated with CON (p = 0.04). Heart rate variability measures, leukocyte differentials, and adrenocorticotropic hormone- stimulation tests were recorded from the dogs used in these studies to understand how acute stressors affect chronically stressed dogs, and how 2M2B might ameliorate this response. Average R-R interval was negatively, but not significantly, correlated with the magnitude of difference in heart rate between placebo and 2M2B ointment during the simulated thunderstorm.

There is limited research on domestic dog pheromones, and there are notable inconsistencies in the literature that does exist. To identify urinary volatiles that may act as pheromones, urine was collected from five individuals in each of six groups: juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact

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Texas Tech University, Glenna M. Pirner, May 2018 female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF).

Headspace sampling yielded six molecules with significantly different peak areas between groups. Octanal, 2-methyl-quinoline, methyl propyl sulfide, and 2-heptanone appear to be closely linked to male sex hormones as they had significantly higher peak areas in intact adult males. 3-ethylcyclopentanone appears to be associated with intact adult females and castrated or subordinate male animals. No sex or life stage patterns could be divulged from the data on 2-pentanone.

For a molecule to elicit a behavioral or physiological response, it stands to reason that regions of the brain corresponding to the effect should be activated. 5α-androst-16-en-

3-one elicits behavioral and physiological effects in women, especially during the time of ovulation. Functional magnetic resonance imaging (fMRI) and blood-oxygen-level dependent (BOLD) contrast were employed to determine which areas of the brain might be responsible for these effects. Ten women received an fMRI scan with ANDRO, 2M2B, rose odor (ROSE), and fresh air (CON) in a 15 s on / 45 s off block design, with three randomized repetitions. ANDRO activated the left insular region compared to CON (p =

0.04). 2M2B elicited activation in the somatosensory association cortex (p < 0.01), premotor cortex (p < 0.01), and Brodmann’s area 8 (p = 0.03) compared to CON. 2M2B also elicited activation in the posterior cingulate and angular gyri compared to ROSE (p <

0.01, both). The insula and amygdala are regions associated with olfactory processing and so were expected. 2M2B elicited activation mainly in motor processing regions, suggesting a motor response to the molecule. Evidence that either molecule activated the hypothalamus, as would be expected by a priming pheromone, was not observed.

Texas Tech University, Glenna M. Pirner, May 2018

LIST OF TABLES

Table 2.1. Characteristics of identified mammalian sexual pheromones ...... 38

Table 3.1. Description of dogs used in the thunderstorm simulation and

car travel models...... 44

Table 3.2. Ethogram from thunderstorm simulation and car travel studies ...... 47

Table 3.3. Dog behavior results in thunderstorm simulation model ...... 54

Table 3.4. Heart rate variability of dogs during car travel ...... 56

Table 3.5. Dog behavior results in car travel model ...... 57

Table 4.1. Comprehensive data from stepwise regression analysis ...... 76

Table 4.2. Comprehensive data from the stepwise regression analysis ...... 79

Table 5.1. Identity, retention time, and number of dogs having each

molecule in headspace of domestic dog urine ...... 94

Table 6.1. Regions of brain activation for functional magnetic resonance

imaging contrasts ...... 124

Texas Tech University, Glenna M. Pirner, May 2018

LIST OF FIGURES

Figure 2.1. Schematic of the olfactory system in the domestic dog...... 37

Figure 3.1. Thunderstorm simulation heart rate results...... 53

Figure 3.2. Heart rate of dogs during car travel...... 55

Figure 4.1. Diagnostic Protocol for Adrenocorticotropic Hormone

Stimulation Test...... 74

Figure 4.2. Correlation matrix of physiological variables in domestic dogs...... 78

Figure 4.3. Thunderstorm simulation heart rate results...... 80

Figure 5.1. Peak area of octanal in domestic dog urine...... 100

Figure 5.2. Peak area of 2-methyl-quinoline in domestic dog urine...... 101

Figure 5.3. Peak area of 3-ethylcyclopentanone in domestic dog urine...... 102

Figure 5.4. Peak area of 2-pentanone in domestic dog urine...... 103

Figure 5.5. Peak area of methyl propyl sulfide in domestic dog urine...... 104

Figure 5.6. Peak area of 2-heptanone in domestic dog urine...... 105

Figure 6.1. Diagram of functional magnetic resonance imaging

facility and olfactometer set up ...... 119

Figure 6.2. Olfactometer designed for this study...... 120

Figure 6.3. Block design representing baseline and odorant delivery schedule . . . . 122

Figure 6.4. Contrast map of androstenone – control condition ...... 126

Figure 6.5. Contrast map of 2-methylbut-2-enal – control condition ...... 127

Figure 6.6. Contrast map of 2-methylbut-2-enal – rose odor condition...... 128

Texas Tech University, Glenna M. Pirner, May 2018

LIST OF ABBREVIATIONS

2M2B 2-methylbut-2-enal

ACTH Adrenocorticotropic hormone

AOB Accessory olfactory bulb

AUC Area under the curve

BOLD Blood-oxygen-level dependent cAMP Cyclic adenosine monophosphate

CAR Carboxen

DAP Dog appeasing pheromone

DVB Divinylbenzene

EDTA Ethylenediaminetetraacetic acid fMRI Functional magnetic resonance imaging

FSH Follicle-stimulating hormone

GABA Gamma-aminobutyric acid

GC-D Guanylyl cyclase type D receptor

GC-MS Gas chromatography - mass spectrometry

GG Grueneberg ganglion

GnRH Gonadotropin-releasing hormone

HFP High frequency absolute power

HR Heart rate

HRV Heart rate variability

LFHFR Low frequency : high frequency ratio

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Texas Tech University, Glenna M. Pirner, May 2018

LFP Low frequency absolute power

LH Luteinizing Hormone

MOB Main olfactory bulb

MOE Main olfactory epithelium

NLR Neutrophil:lymphocyte ratio

PDMS Polydimethylsiloxane

PET Positron emission tomography

PTFE Polytetrafluoroethylene

RMSSD Root mean square of the successive differences

SO Septal organ of Masera

SPME Solid phase microextraction

TAAR Trace amine-associated receptor

VNO Vomeronasal organ of Jacobson

V1R Type I vomeronasal receptor

V2R Type II vomeronasal receptor

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CHAPTER I

INTRODUCTION

Pheromones, or chemicals used to communicate between individuals of a species, have been widely investigated in insects since the discovery and identification of bombykol in 1959 [1,2]. In contrast, the field of mammalian pheromones has not enjoyed so much success in identification and demonstration of such molecules [3]. Rodent pheromones are currently among the best described semiochemicals of the mammalian world, including the Whitten, Bruce, Lee-Boot, and Vandenbergh Effects [4 – 7].

Interomones, or chemical signals produced by an individual of one species which are detected by a second individual of a different species in which they elicit an unpredictable response, are a relatively new concept in the world of semiochemicals [8].

The primary difference between interomones and other allelochemicals, or molecules that convey information between species, is that allomones, kairomones, and synomones have a definitive benefit for the sender, the receiver, or both, whereas interomones do not have such obvious benefits [9 – 11].

Putative pheromone molecules have been identified and their behavioral influences demonstrated in pigs, rabbits, and some other species; however, behavioral effects alone do not, by definition, constitute a pheromone [12, 13]. To meet the accepted definition of “pheromone”, a molecule should be secreted to the outside of the body by a living individual and elicit specific, evolutionarily adaptive behavioral and physiological effects [1, 14].

Texas Tech University, Glenna M. Pirner, May 2018

A complete profile including identity, behavioral and physiological responses, as well as neurological response beyond the olfactory bulbs is not available for the vast majority of mammalian pheromones, and especially not for interomones. The objectives of the present studies were 1) to describe behavioral and physiological effects of 2- methylbut-2-enal in domestic dogs using models of acute stressors; 2) determine which physiological traits influence efficacy of 2M2B; 3) isolate and identify urinary volatiles unique to intact adult male and female dogs; and 4) determine regions of neural activation elicited by putative pheromones and interomones. The information obtained in this series of studies may provide new information pertinent to describing semiochemical effects within and across species.

Texas Tech University, Glenna M. Pirner, May 2018

CHAPTER II

LITERATURE REVIEW

Background

Defining Pheromones

Early & current literature. Chemical communication between animals conveys information such as a food source, danger, and reproductive status; essentially, this knowledge transfer is crucial for the overall species’ survival. While visual and auditory communication does play an important role in communication, spatial restrictions can vastly decrease the efficacy of these behaviors. On the other hand, olfactory communication depends on molecules transferred between animals, some of which may be patent for weeks after the signal has occurred. Alberts [15] found territorial scent marks are composed of molecules of higher molecular mass than those used in alarm signals. Odor from anal gland marks of hyenas and aardwolves can be detected up to 6 months after deposition [16, 17]. In 1959, Karlson & Luscher [1] coined the term

“pheromone” and defined these molecules as “substances secreted to the outside by an individual and received by a second individual of the same species in which they release a specific reaction.”

Most recently the definition of a pheromone has been modified to include four criteria: “(i) substances that are secreted to the outside by an individual and received by a second individual of the same species, in which they cause a specific reaction; (ii) substances that are effective in minute amounts; (iii) substances that are released from 3

Texas Tech University, Glenna M. Pirner, May 2018 living individuals; and (iv) substances that mediate communication for an evolutionarily adaptive function” [14]. Chemical communication in the animal kingdom comprises much more than just pheromones, however. As such, the need for better descriptions of types of chemical messengers led to new terminology and categorization of these molecules.

Terminology. Semiochemical is the overall term for any chemicals involved in communicatory interactions between organisms [18]. There are various subdivisions within semiochemicals, with pheromone being the primary term for same-species communication. There are two types of pheromones: releasers and primers. Releaser pheromones immediately elicit a behavioral response in the recipient. In contrast, primer pheromones have a longer latency period between detection and the elicited response because the response is generally modified by changes in the endocrine system [19].

Some chemical messengers have been demonstrated to convey information between species, deviating from the original definition of a pheromone; such molecules are known as allelochemicals [20]. Within the category of allelochemicals, there are allomones, kairomones, and synomones. The term “allomone” is defined as a chemical substance, produced or acquired by an animal, which elicits a behavioral or physiological reaction in the receiver which is adaptively favorable to the sender [10]. Relationships surrounding allomones can be mutualistic or antagonistic, with the primary effect of benefitting the sender. In contrast, a kairomone is a chemical signal which benefits the recipient rather than the sender. For example, a predator may detect an odor that is unique

Texas Tech University, Glenna M. Pirner, May 2018 to its prey species, alerting the predator that his next available meal may be nearby [21].

Synomones are substances produced or acquired by an organism which evokes a behavioral or physiological reaction in the receiver which benefits both the sender and the receiver [11]. A new class of interspecific chemical messengers, interomones, was introduced in 2012 [8]. Interomones are defined as a chemical that operates as a pheromone in a given species, but may have a different, unpredictable effect, on a receiver of a different species. This effect on the second species is not necessarily beneficial or harmful. The theoretical basis supporting intraspecific functionality of interomones is that some pheromone molecules are structurally similar across species

(See section Conservation of molecules across species).

Perception. The olfactory system in mammals is a complex system involving at least four different subsystems: the main olfactory epithelium (MOE), the vomeronasal organ

(VNO), the septal organ (SO) and the Grueneberg ganglion (GG) (Figure 2.1). Initially it was believed that the MOE functioned only in detection of general odorants while the

VNO functioned in detection of pheromone odors. Recent studies have demonstrated that all of these systems are involved in pheromone detection to some extent [22].

Texas Tech University, Glenna M. Pirner, May 2018

Figure 2.1. Schematic of the olfactory system in the domestic dog.

Approximate location of the main olfactory epithelium (MOE; light blue), main olfactory

bulb (MOB; dark blue), vomeronasal organ with V1R and V2R receptors (red and light

green), accessory olfactory bulb (AOB (dark red and dark green), Grueneberg ganglion

(GG; purple), and septal organ (SO; orange).

The main olfactory epithelium (MOE) lines the turbinates of the posterior nasal cavity; it is comprised of olfactory sensory neurons, basal cells, and support cells. The sensory neurons of the MOE are bipolar neurons with a single, ciliated dendrite protruding to the mucosa. These cilia are the primary mode of transduction for most odorants, as well as pheromones [22]. Axons of the sensory neurons conjoin after crossing the basal membrane to form olfactory nerves, which project to the main olfactory bulb. There are two small sub-regions within the main olfactory epithelium which are identified by the type of receptors they express. These are the trace amine- 6

Texas Tech University, Glenna M. Pirner, May 2018 associated receptors (TAARs) and the olfactory-specific guanylyl cyclase type D receptor

(GC-D)

The vomeronasal organ of Jacobson (VNO) is a blind, tubular cavity in the bone below the nasal septum of some animals [23]. Odors entering the nasal cavity via normal breathing cannot enter the VNO; therefore, these odorant molecules must be moved into the lumen of the VNO by behaviors such as the Flehmen response in ungulates and felines [24]. Similar to the MOE, the VNO lumen is lined by pseudostratified epithelium, with basal cells along the basal membrane of the sensory epithelium and supporting cells in the most superficial layer of the sensory epithelium. Two distinct populations of sensory neurons, apical and basal, overlap each other in the VNO, in the apical and basal regions as the names respectively suggest [25]. Sensory neurons on the apical side possess dendrites and apical microvilli; in contrast, basal sensory neurons have axons that cross the basal membrane, where they merge to form vomeronasal nerves that connect to the accessory olfactory bulb. These sensory neurons have the same origins as gonadotropin-releasing hormone (GnRH) neurons and γ-aminobutyric acid (GABA)- containing neurons; as such, the vomeronasal sensory neurons maintain a functional relationship with the hypothalamus in adult animals, resulting in pheromonal influence on the neuroendocrine system [26]. Based on the discovery that these two populations are characterized by two different G protein subunits, they have been separated into V1R and

V2R type receptors [27].

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V1R receptors represent the apical portion of the VNO sensory neurons; these neurons express the G protein subunit Gαi2 [28, 29]. V1R genes are scattered across chromosomes and expression is monoallelic and monogenic, which contribute to the expression of only one receptor type per cell [30]. V1R receptor genes vary greatly across species, but have been found in all species except chickens and chimpanzees, in which a functional VNO has not been discovered [22]. Rodents have the greatest expression of

V1R genes compared to other species; surprisingly, dogs have only eight functional V1R genes, compared to five in humans [31]. It is theorized that this low number of genes in the modern dog is due to domestication of this species and the reduced importance of olfaction [32]. There is some evidence that V1R homologs may be expressed in the

MOE, but currently no true, functional V1R genes have been discovered in any olfactory tissue except the apical region of the VNO [33, 34].

V2R receptors, representing the basal portion of the VNO sensory neurons, were discovered several years after V1R receptors; these belong to the group III G protein receptors and are co-expressed with the G protein subunit Gαo [35, 36]. The expression pattern of the V2R genes in mice suggest that these receptors are exclusively expressed in the VNO [37]. Interestingly, while this receptor group is expansive in rodents, it appears to be completely degenerated in primates, dogs, and cows. This, in combination with the findings regarding V1R receptors in domestic dogs, does not represent a reduction in pheromone relevance and function, but rather suggests a general decline of the vomeronasal system in conjunction with breeding and domestication [38, 39]. Another

Texas Tech University, Glenna M. Pirner, May 2018 characteristic of the V2R receptors besides region of expression, is that the basal neurons express two receptors, compared to only one V1R receptor in apical neurons [37].

Besides the MOE and VNO, a third, separate olfactory subsystem exists in mammals, known as the Organ of Masera or septal organ (SO). This small region of olfactory tissue is located at the ventral base of the nasal septum, near the entrance to the nasopharynx and is isolated from the MOE by respiratory epithelium [40]. Ma et al. [41] investigated the underlying signal transduction mechanisms in the SO and found that it closely resembled the MOE, except that the epithelium is slightly thinner. The major olfactory signal transduction mechanism in both systems is cyclic adenosine monophosphate (cAMP). Olfactory sensory neurons in both systems are very similar in physiological properties and in projection to the main olfactory bulb [42]. The physical position of the SO and its presence in a wide range of species suggests that it may be specialized for early detection of biologically active molecules, thus possibly playing a role in alarm detection [22].

The fourth olfactory subsystem, known as the Grueneberg ganglion (GG) was first described in 1973 as a small, bilateral, clustered group of neurons located in the rostral roof of the nasal cavity, on either side of the septum [43]. Unlike the other olfactory subsystems, neurons of the GG are clustered in small groups, and only glial cells and ciliated neurons have been identified in these clusters [44, 45]. A single axon from each neuron joins with others to form nerves projecting to a subpopulation of the necklace glomeruli in the main olfactory bulb, suggesting a function in detection of

Texas Tech University, Glenna M. Pirner, May 2018 maternal pheromones [46, 47]. More recently, this theory was rebutted by a study suggesting that volatile alarm pheromones conveying information about danger are the most likely molecules to be received by the GG [44].

Neurons from the MOE, SO, and GG project to the main olfactory bulb (MOB), which is separated from the MOE by the cribriform plate of the ethmoid bone. Axons from olfactory neurons that express the same receptors end in uniformly-sized glomeruli in the outermost layer of the MOB [48]. These glomeruli are organized into small clusters based on the type of odorant received by their corresponding olfactory receptors, providing a spatial map of odor identification. Mitral cells within the MOB project only one apical dendrite to a single glomerulus [49]. Lateral dendrites of the mitral cells synapse with granule cells, which form the deepest layer of the MOB [50].

The MOB sends signals to the cortical amygdala, the hypothalamus, the orbitofrontal cortex, the piriform cortex, and the hippocampus, where emotion, memory, and learning are the major types of processing [51 – 53]. The cortical amygdala signals aversion or attraction to the odor based on learned associations between the odor and memories and emotions [54]. The amygdala then passes the information to the hippocampus, which associates specific events with an odor. This is known as episodic memory (Rolls 2010). The piriform cortex contributes to this response by contributing information based on experiences [55]. The orbitofrontal cortex receives olfactory information from the amygdala and hippocampus, which stimulates the reward system.

For example, an odor associated with a favorite food greatly stimulates the reward

Texas Tech University, Glenna M. Pirner, May 2018 system, which in turn communicates with the anterior cingulate cortex to stimulate appetite [54, 56]. These regions have many interconnections via the primary olfactory cortex, suggesting the complex association between olfaction and emotion, memory, and behavior [57].

In contrast, the accessory olfactory bulb receives information only from the VNO neurons.V1R receptor neurons project axons to the anterior portion of the AOB; whereas the V2R neurons send axonal projections to the posterior AOB [58, 59]. As with the

MOB, the outermost layer of the AOB consists of a glomerular layer, but in the AOB these glomeruli are of highly variable size and not spatially organized. Additionally, these glomeruli may receive input from VNO neurons expressing different receptor types [60].

Mitral cells of the AOB can have up to six apical dendrites which each synapse with only one glomerulus [49].

The AOB communicates with the medial amygdala and posteromedial cortical nucleus; these two regions are collectively referred to as the vomeronasal amygdala.

State-dependent responses to pheromones appear to be mediated here [61]. Signals are then routed to the ventromedial hypothalamus. Subregions of the medial amygdala and ventromedial hypothalamus work together to respond to signals such as predator odors, reproductive pheromones, and juvenile odors appropriately. In many instances pathways between these regions are intermingled or even identical [62, 63].

Characteristics. Molecules with pheromonal properties have diverse chemical structures and thus are typically categorized according to biological function such as reproduction,

Texas Tech University, Glenna M. Pirner, May 2018 alarm, maternal, and others. Pheromones that are required to convey information across a considerable distance, such as alarm or attraction pheromones are typically small, volatile molecules. On the other hand, identifying molecules tend to be non-volatile compounds such as proteins or peptides, which will remain in the immediate environment [64]. In some instances, such as the major histocompatibility complex molecules of mice, larger molecules such as a protein binds smaller, volatile molecules, which together convey a great deal of information about the sending individual [65]. Steroid derived molecules such as androstenone, produced in male pig saliva, are derived from sex steroid hormones. As such, these molecules convey information about the sender’s internal state.

It has been postulated that sex pheromones are not produced by an animal until after puberty, as the pheromones are dependent on sex hormones; indeed, juvenile male mice do not produce sex pheromones in the urine, and concentrations of these molecules in the urine increase at the beginning of puberty and peak at full maturation [66]. Additionally, pheromones are secreted in sweat, urine, saliva, and exocrine glands, and can convey information that changes based on species, sex, age, genotype, and endocrine state [67].

Conservation of molecules across species. As there is little variation in common metabolic pathways leading to production of pheromone molecules, it stands to reason that the same molecules can act as pheromones in different species. Similar gene families are responsible for odor detection in many species, but the genes coding for the receptor types are what allows for variation [68]. Beyond the receptors, there are some strong similarities in the organization of the olfactory pathway between species. The mammalian olfactory bulb and the insect antennal lobe relays are nearly identical in organization, for 12

Texas Tech University, Glenna M. Pirner, May 2018 example [69]. The fact that this pathway has changed very little throughout evolution of species also reinforces the importance of chemical communication. Two hypotheses exist regarding the reasoning for this conservation: either the system evolved relatively early and was retained throughout evolution, or more likely, that different species each independently evolved an olfactory system, but with similar solutions. The latter argument is supported by observations that although there are significant variations in anatomy across species, the overall purpose and pathways are incredibly similar [68]. To alleviate confusion of signals between species, pheromones may be released in a blend of other molecules, or may not elicit a response at all if the appropriate social cues are not present [70, 71]. It is the combination of molecules and their designated receptors that allow for similar molecules detected by similar receptors to elicit completely different responses across species [72].

Classes of pheromones

Alarm. Alarm pheromones in insects began to be recognized and described as early as the 1950’s. Within a decade nearly 20 different insect alarm pheromones were identified and confirmed to influence conspecific behavior [73]. Researchers discovered secretions found in exocrine glands on insects’ bodies, which when presented to other insects of the same species, elicited a reaction common to all individuals in the target population. These reactions included increased rate of locomotion or flight characterized by zig-zag or circular motions [74]. Increased aggression and defensive behavior are also characteristic responses of worker insects [9]. There is evidence that alarm pheromones may also

Texas Tech University, Glenna M. Pirner, May 2018 function as aggregation or attraction pheromones to attract more members of the species to the site of disturbance [75]. Existence of alarm pheromones in mice [76], rats [77], and cattle [78] has been demonstrated, but the identification of these putative pheromones has not been confirmed as there are far fewer studies in non-insect alarm pheromones.

Maternal. The vast majority of mammalian neonates are altricial; therefore, females have evolved a method of advertising the location of the mammary glands in a way that the blind young can easily interpret. Maternal pheromones help the offspring locate the mother and make nursing bouts more efficient. The faster a neonate begins suckling after birth, the sooner it receives hydration, nutrients, and passive immunization that help to determine the viability of the offspring. Maternal pheromones are secreted both in the colostrum and milk as well as by glands in the skin surrounding the teat; these glands represent eccrine, apocrine, and sebaceous glands, as well as specialized glands in some species (e.g. Montgomery’s glands in humans; [79, 80]). Production of maternal pheromones appears to be regulated by hormones associated with lactation; for instance, rabbit maternal pheromones produced later in lactation is less effective in attracting the pups to the nipple than pheromones produced early in lactation [13].

Social/aggregation. Aggregation pheromones in insects function to attract conspecifics to the area in which the signal was released. As defined by Shorey [81], these pheromones are compounds released by an emitter that cause aggregative behavior in conspecifics of both sexes or of the same sex as the emitter. This definition was provided to distinguish aggregation pheromones from sex pheromones, which can cause gathering

Texas Tech University, Glenna M. Pirner, May 2018 of large numbers of conspecifics of the opposite sex of the emitter, with the intention of copulation. The ratio of compounds and the quantity released vary greatly even within species, and is often dependent on life stage and geography [82, 83]. Purposes for aggregation include feeding, aggregated oviposition (multiple females depositing eggs in a given location), mate location, and defense. At high concentrations, some aggregation pheromones can act as alarm pheromones.

Sexual/Reproductive. Sex pheromones are found in both insects and mammals, with the function of these and aggregation pheromones often overlapping in insects. In mammals, males and females of each species produce unique odors to signal the individual’s current reproductive status. Females may use chemical odors to signal to a male that she is in estrus and is approaching ovulation. The frequency of urine marking increases as a female approaches estrus compared to anestrus [84]. Comparatively, males will often countermark the female’s urine and increase scent marking himself during courtship.

Recently, evidence that sex pheromone production is influenced by the life stage of the emitter has been demonstrated [85]. Many sex pheromones are derivatives of sex hormones such as testosterone, estradiol, and pregnanolone [86]. It is evolutionarily beneficial for signals to honestly represent the fitness of the emitter; as such these hormone-based signals reliably indicate reproductive state and dominance status.

Conversely, sex hormones appear to modulate responses to pheromone signals in the receiver. Neural pathways of the olfactory system relay to the brain only signals that are relevant to the receiver’s current status. For instance, an anestrus female will be behaviorally indifferent to male pheromones; on the other hand, these same molecules 15

Texas Tech University, Glenna M. Pirner, May 2018 elicit strong mating responses in the same female when she is in estrus. Similar differences in response to juvenile pheromones occur in virgin and experienced parent mice [87]. This mechanism appears to be due to sex-hormone mediated silencing of VNO neurons, which may function to filter out irrelevant signals [88,89]. The responses elicited by sex pheromones are incredibly diverse; however, there are four primer effects which occur in mice and are strong examples of the kind of responses elicited by sex pheromones.

The Lee-Boot effect is the phenomenon in which group-housed (i.e. more than 2) female mice show lengthened estrous cycles in absence of male mouse odor [6, 90]. The length of the cycle is positively correlated with group size, and in groups of 12 or more, some females stopped cycling altogether [91]. This effect was originally believed to be a result of overcrowding; however, if male odor is introduced to the group, cycling can be reinstated, suggesting that it is rather an energy preservation function: if there is no male present, there is no need to waste energy on reproduction. In 1998, two pheromones were found to be responsible for this phenomenon: 2,5-dimethylpyrazine is shown to be secreted in significantly higher concentration in group-housed females, and α/β Farnesene

[92]. A third compound, n-pentyl acetate, is also suggested to play a role in this phenomenon [93].

The Whitten effect describes the influence of male mouse urine on the length of estrous cycles in female mice. When females with suppressed estrous cycles (as in the

Lee-Boot effect) are exposed to the odor of a male mouse, they will come into estrus 2 to

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3 days after exposure; considerably shorter than the normal 4 to 5 day cycle. Also, the resumed estrous cycles will become synchronized throughout the group [4, 94]. Two androgen-dependent pheromones produced in the male’s urine are thought to work in combination to produce this effect: 2-sec-butyl-4,5-dihydrothiazole and 2,3-dehydro-exo- brevicomin [95].

The Vandenbergh effect is a phenomenon in which immature female mice experience accelerated puberty when exposed to urine of an adult male [7, 96]. This effect, as well as the Whitten effect, is only associated with exposure to urine from a sexually mature male mouse, suggesting that the involved pheromones are androgen dependent. There are 5 compounds suggested to play a role in this phenomenon: 2-sec- butyl-4,5-dihydrothiazole, 2,3-dehydro-exo-brevicomin, α/β Farnesene, 6-hydroxyl-6- methyl-3-heptanone, and 3-cyclohexene-1-methanol [97 – 99].

The Bruce effect occurs when a recently mated female mouse aborts her pregnancy when exposed to the urine odor of a novel (i.e. not the father) male mouse [5].

This effect occurs up until approximately 4 days after mating, when the fertilized embryo implants in the uterine wall. Prior to implantation, the female’s response to the unfamiliar male involves increased estrogen and thus decreased prolactin and progesterone [100].

This effect has evolutionary advantages for both the male and the female. If a male is able to interrupt a pregnancy, the female will subsequently return to estrus, and he will be able to mate with her, thus passing on his own genetic material. In females that are mated with a subordinate male, she may actively seek out a higher-quality male, thus

Texas Tech University, Glenna M. Pirner, May 2018 terminating the pregnancy and increasing the viability of her offspring by mating with the new male [101, 102].

Sex pheromones identified in different species

Humans. The importance of pheromones in human social interactions is a highly debated subject. Humans possess two types of sweat glands, eccrine and apocrine. Eccrine glands secrete large volumes of dilute, non-odorous sweat; on the other hand, apocrine glands secrete very small amounts of material, but the secretions are important for odor production. Apocrine glands typical produce secretions at times of emotion, stress, or sexual arousal. Additionally, these glands do not fully develop until the sex hormones of puberty are present. Many of the characteristic odors associated with different areas of the body are a result of bacteria and/or yeast associated with the glandular secretions. The axillary region in humans is the focus of putative pheromone research for several reasons: gland activity in this region is one of the most conspicuous indicators of puberty, axillary glands are unique to humans and great apes, males and females have distinctly different odors, and the odors can be detected from some distance. The primary compounds found to be responsible for male axillary odor are 3α-androstenol, 5α-androstenone, and other steroids [103], along with branched chained volatiles and unsaturated aliphatic acids [104

– 106].

Putative pheromones in humans include 5α-16-androsten-3α-ol (androstenol) and

5-α-androst-16en-3α-on (androstenone). These compounds are sexual pheromones produced in boar saliva and are determined as the compounds responsible for boar taint

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[107]. These compounds are produced from apocrine glands in the axillary region of both adult men and women, but are approximately 20x higher in concentration in axillary sweat of men [108]. When exposed to androstenol, both men and women rated photos of other people as “warmer” and “more friendly” compared to the control group. Those exposed to androstenol also had higher levels of skin conductance, in accordance with

Monti-Bloch & Grosser [109, 110]. As such, androstenone and androstenol have been suggested as putative human male pheromones that are attractive to females. As expected, there are differences in the responses of males and females to this proposed male pheromone.

A blinded study conducted by Cowley & Brooksbank [111] found that men experiencing androstenol reported no difference in number, depth, or duration of social exchanges with either men or women compared to control. In contrast, women experiencing androstenol did not report differences in social exchanges with other females, but did report increases in number, depth, and duration of interactions with males. Additionally, in the control situation, men were more socially active than females; in the pheromone group, this pattern was reversed. Savic et al., [112] examined neural activation of both men and women in response to androstenone using positron emission tomography (PET) and found that women had activation of the hypothalamus, preoptic, and ventromedial nuclei in response to the androgen. At a lower threshold, androstenone resulted in activation of these regions in addition to the piriform cortex, anterior cingulate, right amygdala, and right lingual gyrus in females. Men did not show any

Texas Tech University, Glenna M. Pirner, May 2018 activation of these regions in response to androstenone, but did show similar activation when exposed to an estrogen-like substance.

Positron emission tomography (PET) is a commonly used imaging technique in which a radioactive tracer is introduced into the body, then areas of interest can be isolated by detection of the positrons emitted from the tracer. In the case of neuroimaging, the tracer is typically oxygen-based, and so can be associated with increased blood flow to regions of activation in the brain [113]. While PET scans are beneficial in observing regions of activation in the brain, there are several aspects of the procedure that can be undesirable. The radioactive tracers are typically injected into the body intravenously, followed by approximately a one hour wait to allow the tracers to distribute throughout the body. Additionally, PET scans record images over 60 to 90 second scans. This time frame is not compatible with activation of brain regions in response to odor exposure [114].

Other Species. To date, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptonic acid) and four derivatives of 3-mercapto-3-methyl-1-butanol have been described in the urine spray of intact male cats [115]. No behavioral evidence of these compounds as sex pheromones has been published. Similarly, 2-methylquinoline and 2-butanone are proposed male sex pheromones isolated from red fox urine and lion urine, respectively, but no sexual behavior studies have been conducted [116, 117]. Putative sex pheromones in white-tailed deer, black-tailed deer, and pampas deer have been suggested based on seasonal changes in identity and concentration of some compounds from various

Texas Tech University, Glenna M. Pirner, May 2018 cutaneous glands [118 – 120]. Unfortunately, this incomplete information obtained in these studies does not provide a strong enough argument to classify the identified molecules as pheromones. Table 2.1 lists identified and biologically assayed sex pheromones in various species.

Table 2.1. Characteristics of identified mammalian sexual pheromones. Table adapted

from Tirindelli et al., [22].

Molecule Site of Site of Behavior Authors production reception Response Mouse 2,5-dimethyl pyrazine Female urine V1R Suppresses estrus [92,93] neurons cycle (Lee-Boot) 2-sec-butyl-4,5- Male urine V1R Estrus induction; [97, 98, dihydrothiazole; neurons estrus 121, 122] 2,3-dehydro-exo- synchronization brevicomin (Whitten); puberty acceleration (Vandenbergh); Female attraction α- and β-Farnesene Preputial V1R Puberty [92, 97, glands neurons acceleration 98, 122] (Vandenbergh); Female attraction; estrus cycle suppression (Lee- Boot) 6-hydroxyl-6-methyl-3- Male urine V1R Puberty [97, 98] heptanone; neurons acceleration Major Urinary Proteins (Vandenbergh) (MUP) 2-heptanone Urine V1R Estrus extension [122] neurons n-pentyl acetate Urine V1R Suppression of [93] neurons estrus cycle (Lee- Boot)

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Molecule Site of Site of Behavior Authors production reception Response Isobutylamine Male urine V1R Estrus [123, neurons acceleration and 124] vaginal opening 3-amino-s-triazole; 4- Male urine ??? Female attraction [125] ethyl phenol; 3-ethyl-2,7-dimethyl octane; 3-cyclohexene-1- methanol SYFPEITHE; Urine MOE Pregnancy block [126] AAPDNRETF (Bruce) Other Rodents Aphrodisin (Hamster) Vaginal VNO Stimulates male [127] glands copulatory behavior Squalene (rat) Preputial ??? Female attraction [128] gland 2-heptanone; Male urine ??? Female attraction [66, 128] 4-ethyl phenol; 4-heptanone; 3-ethyl-2-heptanone; 2-octanone; 2-nonanone; 4-nonanone; (rat) Elephant (Z)-7-dodecen-1-yl Female urine ??? Sexual behavior in [129] acetate; males

(+)(-)1,5-dimethyl-6,8- Male temporal ??? Sexual behavior [130] dioxabicycle[3,2,1]octane gland, urine, (Frontalin) breath Swine Androstenone; Male saliva MOE Stimulates sexual [12, 131, Androstenol; behavior in 132] Quinoline females

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Molecule Site of Site of Behavior Authors production reception Response

Goat/Sheep 4-ethyloctanal Glands on MOE Elicits GnRH [133, 4-ethyloctanoic acid head primary; pulses in female 134] (goat) VNO secondary 1,2-hexadecanediol Ante orbital MOE Induces LH peak [134, 1,2-octadecanediol gland; fleece primary; and ovulation in 135] Fatty acids VNO anestrus ewes secondary Dog Methyl p- Vaginal VNO Stimulates sexual [136] hydroxybenzoate secretions behavior in males (peak estrus) Horse p-cresol; Urine (estrus) ??? Elicits sexual [137] m-cresol excitation in stallions Cattle Viz. acetic acid; Estrus feces ??? Stimulates sexual [138] Propionic acid; behavior in males 1-iodo undecane Rabbit 2-methylbut-2-enal Milk MOE Stimulates nipple [13, 139] search and grasp behavior of pups

Dog specific information

Reproductive physiology. Bitches typically have two estrous cycles per year, but some can experience anestrus intervals as short as four months or as long as thirteen months.

The anestrus phase is characterized by inactive ovaries and strict resistance to mounting attempts by males. Toward the end of the anestrus phase, luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations begin to increase. The rising hormone 23

Texas Tech University, Glenna M. Pirner, May 2018 levels mark the beginning of the proestrus phase. This phase is characterized by swelling of the vulva and a blood-tinged vaginal discharge. Males will show increased interest in the bitch, but mounting attempts are still resisted. Estrogen levels rise during proestrus.

This phase lasts an average of nine days but can vary greatly between individuals and even between cycles.

As estrogen levels peak the bitch enters estrus, which correlates to ovulation. At this point in the cycle the female is receptive to the male. Throughout estrus estrogen levels decline and progesterone levels increase. The length of this phase also varies greatly between three day and three weeks, with an average of nine days. As estrus wanes, the bitch will begin resisting breeding attempts again. If fertilization has occurred, the female’s body will adapt accordingly to maintain the pregnancy. If breeding has not occurred or was unsuccessful, the bitch will enter the fourth estrous phase known as diestrus, which lasts approximately three months, at which time the cycle begins again with anestrus [140].

Reproductive behavior. Courtship and mating in the dog are characterized by a predictable series of behaviors and events, beginning with olfactory detection of the bitch’s estrous phase and concluding with a copulatory tie and ejaculation. When the dog encounters a urine mark produced by an estrus female, he often counter-marks the scent and then attempts to locate the bitch. Dogs do not display Flehmen behavior, but rather respond to the urine mark by “tongueing”, which involves a rapid flick of the tongue over the tip of the nose, thought to facilitate introduction of pheromones to the VNO duct,

Texas Tech University, Glenna M. Pirner, May 2018 located behind the front upper incisors [141]. Upon locating the estrus bitch, the male may sniff her head and body, gradually paying more attention to her anogenital region.

Identification of the molecule or combination of molecules responsible for attraction of the male to the estrus female has yet to be confirmed. Early studies found that dogs are more attracted to urine than to vaginal or anal gland secretions [142].

Goodwin [136] analyzed vaginal secretions obtained at different phases of the estrous cycle and found that methyl-p hydroxybenzoate, or methyl paraben, was only present during peak estrus, and that males were significantly more attracted to females with methyl paraben applied to the vulva. Several years later this finding was refuted by Kruse

& Howard [143]. This group applied methyl paraben or saline to the vulva of anestrus bitches and found no difference in interest levels of the male dogs between treatments.

The methodology of this study may explain the disagreement in results: the putative pheromone applied in the Kruse & Howard study was in powder form. The delivery method of the test molecule may have prevented detection of the methyl paraben in the

VNO. More recently, Dzieciol et al., [144] further argued against methyl paraben as the dog sex pheromone after analyzing urine from female dogs during proestrus and estrus.

These researchers did not detect methyl paraben in any concentration in these samples and proposed that it may have appeared in Goodwin’s analysis as a contaminant. This study also has a major methodological issue, however. Goodwin did collect urine from the females, but it was not analyzed; rather, methyl paraben was detected in vaginal secretions obtained on swabs. This disagreement in the literature need to be addressed to determine which molecule or molecules may act as canine sex pheromones. 25

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The female’s response is highly correlated with her stage of estrus. During proestrus, she may run around or engage in play with the male, but is generally intolerant of anogenital investigation or mounting attempts [145]. As peak estrus approaches, running and playing behavior wanes and the female will stand still for longer periods of time to permit the male’s investigation. The bitch will accept the male toward the end of the estrus phase, corresponding to the time of ovulation, promoting successful fertilization [146]. A receptive female will stand motionless with her hindquarters elevated and tail “flagged” or diverted to one side. These behavioral changes represent changes in hormonal status, and are likely also correlated with changes in pheromone production.

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[113] Nilsson, L.G., Markowitsch, H.J. 1999. Cognitive Neuroscience of Memory. Hogrefe & Huber Publishers, Boston, Massachusetts.

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[125] Achiraman, S., Archunan, G. 2002. Characterization of urinary volatiles in Swiss male mice (Mus musculus): bioassay of identified compounds. J Biosci 27: 679- 686.

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CHAPTER III

IMPACT OF 2-METHYLBUT-2-ENAL ON ACUTE STRESS RESPONSES IN

CHRONICALLY STRESSED DOMESTIC DOGS

Abstract

Thunderstorms and car travel are stressors that domestic dogs are likely to experience during their lifetime. For dogs living in a chronic state of stress, such as with disease, these acute stressors have a more detrimental influence than in non-stressed dogs. Pheromone therapy in stress alleviation has gained interest in recent years.

Interomones, or molecules that act as pheromones in one species but that elicit an unrelated effect in a second species, may also impact behavioral and physiological responses to stressors. 2-methylbut-2-enal (2M2B), a rabbit maternal pheromone, is one such molecule. This study examined the effects of 2M2B on behavior and physiology of dogs exposed to a simulated thunderstorm and car travel. Dogs in this study were shown to be chronically stressed by an adrenocorticotropic hormone stimulation test. In the thunderstorm simulation model heart rate (HR) of dogs in both groups increased during the simulated thunderstorm, but 2M2B elicited a faster return of HR to baseline compared to control (CON). Dogs treated with 2M2B also spent 15.9% more time lying down during the simulation compared to CON (p = 0.04). In the car travel model, dogs treated with 2M2B had an average HR of 110 bpm, compared to 131 bpm in the CON group (p =

0.01). High frequency absolute power (HFP) and the root mean square of the successive differences (RMSSD) increased from basal values in the “pre” and “drive” periods (p <

Texas Tech University, Glenna M. Pirner, May 2018

0.01 and p < 0.01, respectively). Use of 2M2B to reduce the impact of an acute stressor during periods of chronic stress, such as in illness, may have important implications in canine health and welfare.

Introduction

In 2012 there were approximately 70 million pet dogs in the United States [1].

With so many dogs living alongside humans, inevitably some owners will express concern regarding situations eliciting fear or stress in their pet. Seventy-four percent of

U.S. dog owners report noise phobia in their pet, with most claiming thunderstorms, fireworks, and gunshots are of greatest concern [2]. Fear is a natural, adaptive response intended to help the animal survive real or perceived potential threats, such as a thunderstorm [3]. Some dogs, however, may develop a phobic response to these situations. Thunderstorm phobia results in maladaptive responses to a storm, such as self- mutilation or environmental destruction [3]. Car travel is another common situation which may induce anxiety or fear in dogs. A survey revealed that approximately 75% of owners traveled with their dog by vehicle more than ten times per year for veterinary care and leisure [4]. For dogs unaccustomed to car travel, fear or discomfort may occur because of unfamiliar noises, trouble balancing, confinement, and motion sickness [5, 6].

In the survey by Mariti et al., [4], 23.8% of the total number of dogs surveyed showed problematic behaviors during car travel such as restlessness, vomiting, panting, and vocalizing. Veterinarians often recommend sedative-type drugs or behavior therapy to treat anxiety-related behavior problems in dogs [6, 7].

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Mariti el al., [4] found that 96.3% of owners with dogs experiencing aversion to car travel did not seek treatment or administer any intervention to alleviate the negative effects of transport in their pet. Some owners find medication undesirable due to prolonged sedation and difficulty administering the medication [8, 9]. Behavioral modification training can be time-consuming and expensive, making this type of intervention undesirable for some owners as well [8]. Owners who are unable to cope with or prevent problem behaviors in their pets may become frustrated, leading to deterioration of the human-animal bond and, potentially, relinquishment of the pet to an animal shelter [10]. Of the owners whose dog displayed problem behaviors during car travel, 14.5% of owners refused to transport their pet by car again, restricting the potential for leisure activities and reduced veterinary care [4]. Recently, pheromone- based products have gained popularity with pet owners seeking alternative interventions to alleviate anxiety in their pets.

Pheromones are chemical signals produced by one individual and received by a second individual of the same species in which they elicit specific responses [11].

Throughout mammalian evolution metabolic pathways responsible for producing these molecules and olfactory receptor gene families have been relatively conserved; thus, a pheromone molecule in one species may elicit a different response if received by an individual of a second species [12]. Such molecules are referred to as interomones [13].

Rabbit maternal pheromone (2-methylbut-2-enal; 2M2B) is secreted in milk from the dam to attract pups to the nipple and calm them during nursing [14, 15]. Dog Appeasing

Pheromone (DAP, Ceva Santé Animale) is a maternal pheromone produced by the bitch 41

Texas Tech University, Glenna M. Pirner, May 2018 during lactation that is marketed for alleviation of anxiety in dogs in various situations such as thunderstorms and car travel [16 – 18]. These studies largely rely on behavioral observations to determine the efficacy of the intervention; however, elicitation of physiological effects as well as behavioral effects is an important criterion for pheromone molecules [11]. Heart rate variability (HRV) is a non-invasive method used to monitor and predict the outcome of both physical and psychological illness in humans [19]. More recently, the ability to use these measurements in other species has allowed researchers to study emotional state and stress response in domestic dogs. The balance between the sympathetic and parasympathetic nervous system is constantly changing, allowing the animal to adapt to its surroundings and thus maintain homeostasis. HRV measures the variation in time between consecutive heart beats, or the R-R interval [19]. During acute stress the sympathetic nervous system exerts the most control over the sinoatrial node, increasing HR and decreasing HRV [20]. The root mean square of the successive differences (RMSSD) in HRV allows for determination of the amount of influence exerted by the vagus nerve on the heart while considering respiratory sinus arrhythmia.

RMSSD is also sensitive to fluctuations in low frequency ranges which reflect sympathetic nervous system inputs [19, 21]. High-frequency activity decreases during acute stress and elevated anxiety levels [22, 23]. In contrast, low-frequency activity increases under states of stress [24, 25]. Often, these two parameters are combined into a ratio of low to high frequency, and an increase in this ratio is associated with activation of the sympathetic nervous system [26].

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Based on the knowledge regarding physiological conservation and the calming effects of 2M2B in rabbits, it was hypothesized that 2M2B may act as an interomone in domestic dogs, potentially having a calming effect during periods of acute stress. The objective of this study was to determine if 2M2B, acting as an interomone, modifies physiology and behavior in dogs using models of a simulated thunderstorm and car travel.

Methods and Materials

General

All research was conducted after approval of the Texas Tech University

Institutional Animal Care and Use Committee (Protocol 14009-01). Space, management, and care of dogs were consistent with the US Animal Welfare Act. Food was provided once daily, and water was available ad libitum. Research was conducted both at Texas

Tech University and at a contract research facility in Texas.

Thunderstorm Simulation Model

Twenty (10 intact males and 10 intact females) mixed breed dogs between three and eight years of age and weighing 19.14 ± 3.60 (SEM) kg were tested in five groups of four dogs each. Detailed information on dogs is provided in Table 3.1. One male was removed from the study due to health concerns. Thirteen of these dogs were available approximately six months after the conclusion of the study; these dogs were given adrenocorticotropic hormone (ACTH) stimulation tests to evaluate basal HPA axis activity. Twelve dogs had results consistent with hyperadrenocorticism based on 43

Texas Tech University, Glenna M. Pirner, May 2018 observation that plasma cortisol concentrations increased significantly with administration of exogenous ACTH. Routine physical examination suggests this finding is due to chronic stress rather than Cushing’s disease. Dogs were individually housed at

Texas Tech University in 1.22 m x 1.83 m kennels, with two kennels per room. Rooms were maintained at positive air pressure with 100% fresh air circulation. A three-day environmental acclimation period was allowed prior to testing, followed by a three-day heart rate monitor acclimation period.

Table 3.1. Description of dogs used in the thunderstorm simulation and car travel models.

Age Weight Car Dog ID Sex Breed Thunderstorm (y) (kg) travel 533 F 4 Border collie 14.4 X X 527 M 9 Mixed 10.8 X X 536 M 4 Mixed 17.7 X 538 M 4 Labrador ret. 25.8 X 525 F 7 Labrador ret. 20.5 X 587 M 5 Labrador ret. 22.1 X X 545 F 10 Border collie 18.0 X X 515 F 7 Mixed 16.7 X X 549 M 6 Labrador ret. 30.4 X X 483 M 9 Mixed 16.1 X X 569 F 6 Labrador ret. 16.2 X 520 F 6 Mixed 13.5 X 620 M 5 Pit bull 22.3 X X 470 F 9 Mixed 10.6 X 625 M 2 Mixed 9.7 X X 44

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Age Weight Car Dog ID Sex Breed Thunderstorm (y) (kg) travel 510 F 7 Cattle dog 17.0 X X 467 M 9 Jack Russell Ter. 6.3 X X 626 F 1 Mixed 8.8 X X 528 M 4 Mixed 10 X X 624 F 2 Mixed 11.0 X 591 F 4 Mixed 13.3 X 629 F 4 Mixed 12.6 X

Treatments. Researchers were blind to treatments. Petroleum-based ointments were formulated by Perrigo Animal Health (Omaha, NE) and labeled as FORM1 and FORM2.

Unblinding revealed FORM1 contained no pheromone (CON) and FORM2 contained 1

µg/mL 2-methylbut-2-enal (2M2B).

Experimental Design. This study was a randomized crossover design with two rooms and two treatments, with each dog receiving each treatment in random order. Trials began at 11:00am, with 48 h rest between trials. Trials consisted of a 15 min “before” period to collect baseline data. The designated ointment was massaged onto the dorsal aspect of the dog’s nose at the end of the “before” period and immediately prior to the simulated thunderstorm. In the “during” period, a 15 min simulated thunderstorm soundtrack

(Sounds Scary!, Sound Therapy 4 Pets Ltd., Chester, England) was played on a Fender

Passport Event stereo system (Fender Musical Instruments Corp., Scottsdale, AZ). Sound levels ranged from 69 db to 96 db. This was immediately followed by a 15-min recovery

(“after”) period. 45

Texas Tech University, Glenna M. Pirner, May 2018

Car Travel Model

Sixteen (n = 8 intact males and 8 intact females) mixed breed dogs, estimated to be between two and ten years of age, and weighing 15.0 ± 1.4 kg at the beginning of the study were used in this study. Detailed information on dogs is provided in Table 4.1. The thirteen dogs with known ACTH stimulation test results were included in this study.

Dogs were housed in 3.05 m x 1.22 m kennels at the contract research facility. Transport occurred during daylight hours along local roads in Texas.

Treatments. Researchers were blind to treatments. Aerosol sprays formulated by Perrigo

Animal Health (Omaha, NE) were utilized in this model. Spray was used in place of ointment for car travel due to ease of administration when the dogs were in kennels. The control was a placebo spray with no pheromone odor (CON). The pheromone spray contained 1 µg/mL of 2-methylbut-2-enal (2M2B).

Travel. Vehicles used in this study were three full-size commercial vans. Each vehicle was designated as CON or 2M2B to prevent cross-contamination of the treatments; two vehicles served as both, in which case the CON trials took place prior to the 2M2B trials.

A plastic carrier was secured in the second row of seats, with the long axis perpendicular to the vehicle. Carriers were lined with cotton towels; between drives, carriers were washed and towels were replaced. Travel consisted of a drive along a pre-determined route on an interstate highway for 56.3 km in one direction, a brief turnaround, and 56.3 miles back to the facility. Highway speed was maintained at 112 km/h, air conditioning

Texas Tech University, Glenna M. Pirner, May 2018 was maintained at the same setting in each vehicle, and no music or food was permitted inside the vehicles at any time.

Experimental Design. This study used a parallel design in which dogs were randomly assigned to receive either CON or 2M2B spray, with eight dogs per treatment, balanced by sex and weight, and then assigned to an appropriately designated vehicle. Dogs were given a ten-minute HR belt acclimation period, and then were walked via leash to the running vans, where they were loaded into the carriers. Once inside the van, treatments were administered by spraying the towel for three consecutive seconds, and then spraying the air in front of the animal for one second. Next, HR and behavior data were collected for ten minutes in the stationary vehicles (“pre” period), followed by the transport period

(“drive” period). Upon return to the facility, HR monitors were removed and dogs were returned to their home kennels. Transport carriers were cleaned and a new towel was placed inside the carrier.

Data Collection

Behavior. Behavior in both studies was recorded in real time using a Panasonic HDC-

TM90 video camera (Osaka, Japan). Behaviors of interest in the Thunderstorm

Simulation Model were sitting, standing, lying down, locomotion, and vocalization.

Behaviors of interest in the Car Travel Model were sitting, standing, lying down, and locomotion (postural behaviors), as well as panting, vocalizing, and lip-licking.

Vocalization and all behaviors are defined in the ethogram in Table 3.2.

Texas Tech University, Glenna M. Pirner, May 2018

Table 3.2. Ethogram with description of dogs’ behaviors observed in the simulated thunderstorm and car travel models.

Behavior Definition Sitting Caudal body touching table, supported by front limbs Lying Animal is recumbent, either laterally or sternally Standing Animal is upright with body supported by all limbs Locomotion Movement in any pattern, includes jumping Vocalization Any whine, bark, yelp, growl, or other audible sound produced by the animal Lip-Lick Tongue is extended and moved along any part of the upper lip

All videos were viewed at Texas Tech University by observers blind to the treatments. Observers were trained and validated, with the correlation between observers required to be > 90 % and no difference in scoring means (P > 0.05). Behavior was recorded using a one-minute scan sample technique, with the dog’s postural behavior recorded as well as whether the dog was panting (Car Travel Model). Vocalization and lip-licking are typically brief occurrences and so these were recorded as they occurred.

Heart Rate and Heart Rate Variability. HR and HRV measures were obtained using a

Polar Pro RS800CX telemetry monitor (Polar Electro, Lake Success, NY). Belts were placed around the thorax, caudal to the shoulders, with the sensor centered on the sternum. To maximize conduction fur was shaved from the sensor contact area and conduction gel was applied. Basal HR and HRV parameters were measured while dogs were in their home kennels during a quiet period of thirty minutes. In the Thunderstorm

Simulation Model, dogs wore the same monitor for each trial. HR was averaged every

Texas Tech University, Glenna M. Pirner, May 2018 one min, and the treatment application period was omitted from the calculations. HRV measures are not available for this model due to equipment error. In the Car Travel

Model, HR was averaged every five min and the time during turnaround was omitted from the calculations. Heart rate variability was processed using Kubios Heart Rate

Variability Analysis software 3.0 (Kubios Oy, 2017). The root mean square of the successive differences (RMSSD; ms), high frequency absolute power (HFP; ms2), low frequency absolute power (LFP; ms2), and the low frequency:high frequency ratio

(LFHFR) were obtained after processing. Frequency-domain variables were calculated using Fast Fourier Transform (FFT). The low frequency range was fixed between 0.04 and 0.15 Hz, and high frequency was considered above 0.15 Hz [27].

Data Analyses

Thunderstorm Simulation Model. Heart rate data were checked for normality using

Shapiro-Wilks test and failed to meet this assumption (W1495 = 0.98, P < 0.01); after log transformation, data met the normality assumption using Kolmogorov-Smirnov (D1495 =

0.01, P > 0.15). Transformed data were analyzed using PROC MIXED and repeated measures of SAS 9.4 (SAS Inst., Inc., Cary, NC). Data were analyzed to determine if sex had an effect; it did not and therefore was omitted from the model. HR was first analyzed by minute for examination, and then by period. The HR by minute model included effects of TREATMENT, TIME, and the interaction of TREATMENT*TIME, using

DOG*TREATMENT as an error term. The HR by period model included effects of

PERIOD, TREATMENT, and the interactions of TREATMENT*PERIOD and

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DOG*TREATMENT*PERIOD. Least Squares means and standard errors were back- transformed and calculated using raw data for clarity of reporting. Post-hoc tests to determine differences were conducted where appropriate. Basal HR values compared to

HR during the simulated thunderstorm when treated with 2M2B ointment were used to categorize dogs as having a reverse response (HR higher with 2M2B compared to CON during simulation, p < 0.05), no response (HR not different between 2M2B and CON during simulation, p > 0.05), or expected response (HR lower with 2M2B compared to

CON during simulation, p < 0.05).

Behavior data were initially examined using PROCGLM; however, normality assumptions were not met and data were also analyzed using Wilcoxon signed rank tests where deemed appropriate based on significance in the GLM output. The number of instances of each behavior was compared between treatments in each period. Data are reported as average percent of time dogs spent engaged in each behavior during each period. Vocalizations are presented as the average number of occurrences during each period.

Car Travel Model. HR data met the assumption of normality using Shapiro-Wilks test

(W123 = 0.99, P = 0.48) in SAS 9.4. HRV parameters were also checked for normality.

RMSSD and HFP were normally distributed (W47 = 0.99, p = 0.93; W47 = 0.98, p = 0.52, respectively). First, HR, RMSSD, and HFP data in the “drive” period were analyzed by five-minute periods compared to the “pre” and “basal” period using PROC MIXED and repeated measures procedures of SAS 9.4, with main effects of SEX, TREATMENT,

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TIME, and interactions of SEX*TREATMENT and TREATMENT*TIME, using DOG within TIME as the error term. SEX had no effect and so was removed from the model.

The final model included TREATMENT, TIME, and TREATMENT*TIME. HR,

RMSSD, and HFP were also compared across periods using main effects of

TREATMENT, PERIOD, and TREATMENT*PERIOD. Post hoc comparisons were made using Least Squares means where appropriate. LFP and LFHFR were not normally distributed and so were compared between PERIOD and TREATMENT using the

Wilcoxon Signed Rank test.

Behavior data were not normally distributed and so were analyzed using the

Wilcoxon Signed Rank test to compare number of instances of each behavior between treatments during the study. For ease of understanding, behavior data are presented as percent of time dogs spent engaged in sitting, lying, standing, panting, and moving behavior immediately before and during transport. Vocalizations and lip-licking are presented as the average number of occurrences of each behavior during the study.

Results

Thunderstorm Simulation Model

Heart Rate. Average HR of dogs when treated with CON or 2M2B ointments before, during, and after the simulated thunderstorm is presented in Figure 3.1. There was no effect of TREATMENT*TIME interaction on HR. There was an overall effect of

PERIOD (p = 0.03), with HR in the “during” period being higher than in the “before”

Texas Tech University, Glenna M. Pirner, May 2018 period (p = 0.03) as well as in the “after” period (p = 0.01). Similarly, there was an overall effect of TIME (p < 0.01). Post-hoc tests revealed no differences at any minute in the “before” and “after” periods. At the onset of the simulation there was a rise in HR from an average of 109 bpm in minute 15 to an average of 125 bpm in minute 16 (p <

0.01). Average overall HR began to decrease at minute 20, returning to average baseline by minute 30. When data were examined by period, there was no effect of interaction of

TREATMENT*PERIOD. As with the HR by minute analysis, there was no overall effect of TREATMENT in the “before” or “after” periods, but there was a TREATMENT effect in the “during” period (p = 0.02). When treated with CON ointment dogs’ average HR during the simulated thunderstorm was 126 bpm. When treated with 2M2B ointment dogs’ average HR during the simulated thunderstorm was 109 bpm. There was a significant TREATMENT*DOG*PER effect, with 11 dogs experiencing significantly different HRs between treatments during the simulated thunderstorm (p < 0.01).

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160 Before During After a b a 140

120 HR, bpm HR, 100

80 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Time, min 2M2B CON

Figure 3.1. Thunderstorm simulation heart rate results. Least Squares means of dogs’ heart rate per minute before, during, and after a simulated thunderstorm. Dogs received either control ointment (CON) or 1 µg/mL 2-methylbut-2-enal ointment (2M2B). Black vertical lines indicate periods. n = 19 dogs/treatment. SEpooled = 4.76.

Behavior. In the PROCGLM analysis, no differences in sitting, standing, moving, or vocalization were observed between treatments in any period (Table 3.3). There was a

TREATMENT*PERIOD effect on lying behavior (p = 0.04). When dogs were treated with 2M2B ointment they spent 66.8% of the “during” period lying down, compared to

50.9% when treated with CON ointment (p = 0.04). Wilcoxon Rank Sign tests showed no differences in lying, standing, moving, or vocalization were observed between treatments in any period. Dogs in the 2M2B group spent 27.0% of the “before” period sitting, compared to 17.5% for dogs in the CON group (p = 0.02).

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Table 3.3. Dog behavior results in thunderstorm simulation model. Average percent of time dogs spent engaged in sitting, lying, standing, or locomotion behaviors before, during, and after a simulated thunderstorm when treated with control ointment (CON) or 1µg/mL rabbit maternal pheromone ointment (2M2B). Vocalizations are given as average number per period. n = 19 dogs/treatment; each period = 15 minutes.

Treatment Period Sit (%) Lay (%) Stand (%) Locomotion (%) Vocal (#) CON Before 17.5y 58.6 8.8 15.1 0.05 CON During 41.1 50.9a 1.7 6.3 0.58 CON After 29.1 56.5 7.4 7.0 0.11 2M2B Before 27.0z 47.7 12.3 13.0 0.68 2M2B During 28.4 66.8b 1.7 3.1 0.00 2M2B After 19.7 67.0 4.5 8.8 0.21 SE 6.30 5.30 1.97 2.18 0.30 Treatment P value 0.42 0.24 0.89 0.52 0.83 TRT*PER P value 0.18 0.04 0.29 0.50 0.14 a,b LS Means differ, p < 0.05. y,z Wilcoxon Rank Sign results differ, p < 0.05. Car Travel Model

Heart Rate. BHR was not different between the two treatment groups (p = 0.29). The interaction of TREATMENT*TIME on HR was not significant. There was a significant effect of TREATMENT on HR (p = 0.01), as shown in Figure 3.2. Dogs in the CON group had an average overall HR of 131 bpm, while those in the 2M2B group had an overall average HR of 110 bpm. There was also a main effect of TIME (p < 0.01). In general, HR was higher than BHR in the “pre” period and during the first thirty minutes of the “drive” period, after which it returned to BHR levels.

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150

140

130

120 CON HR, HR, BPM 2M2B 110

100

90 BHR PRE 0 5 10 15 20 30 35 40 45 50 TIME

Figure 3.2. Heart rate of dogs during car travel. Least squares means of dogs’ basal heart rate (BHR) and heart rate before (PRE) and during (DRIVE) car travel. Dogs received either control spray (CON) or 1 µg/mL 2-methylbut-2-enal spray (2M2B). n = 8 dogs / treatment. SEpooled = 7.44.

Heart Rate Variability. HRV results are shown in Table 3.4. No basal HRV parameters were different between the two treatment groups. There were no significant differences in

LFP or LFHFR in this study. There was a significant main effect of TIME on RMSSD and HFP (p < 0.01, both).

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Table 3.4. Least Squares means of the root mean square of successive differences (RMSSD), low frequency absolute power (LFP), high frequency absolute power (HFP), and the low frequency : high frequency absolute power ratio (LFHFR) of dogs in a basal condition (BHRV), immediately prior to car travel (PRE) and during car travel (DRIVE). Dogs received either control spray (CON) or 1 µg/mL 2-methylbut-2-enal spray (2M2B). n = 8 dogs / treatment.

Treatment Time RMSSD LFP (ms2) HFP (ms2) LFHFR (ms) CON BHRV 19.35a 340.47 290.16a 2.04 CON PRE 39.14b 1575.66 1051.37a 1.56 CON DRIVE 38.15b 931.85 905.07 1.07 2M2B BHRV 25.12a 385.64 287.84b 1.48 2M2B PRE 38.31b 1055.27 783.22b 1.18 2M2B DRIVE 48.54b 1229.19 1023.09 1.17 SE 3.60 99.53 143.22 0.40 TRT p-value 0.12 - 0.69 - TIME p-value < 0.01 - < 0.01 - TRT*TIME p-value 0.27 - 0.36 - a,b Among treatments, LS Means differ between periods, p < 0.05.

Behavior. Behavior data are shown in Table 3.5; reported data are derived from GLM output for ease of understanding. Overall there were no differences in sitting, lying, standing, moving, or panting behavior between dogs receiving CON spray and those receiving 2M2B spray at any time during the study. Dogs in the CON group vocalized an average of 2.20 times during the study; in contrast, dogs in the 2M2B group vocalized an average of 0.11 times during the study (S = 26.5, P < 0.01). Whining was the most common type of vocalization noted, with only two instances of barking observed during

Texas Tech University, Glenna M. Pirner, May 2018 the entire study. Dogs in the CON group exhibited an average of 5.53 lip-licks during the

“drive” period, compared to an average of 3.53 lip-licks in dogs in the 2M2B group (S =

25.5, P = 0.02).

Table 3.5. Average percent of time dogs spent sitting, lying down, standing, moving, or panting, and average number of vocalizations and lip-licks during ground transport when treated with a placebo spray (CON) or Sentry Calming Spray (CALM). n = 8 dogs/treatment

Sitting Lying Standing Moving Panting Vocalizations Lip-Licks Treatment Period (%) (%) (%) (%) (%) (#) (#) CON Pre 21.30 18.80 18.80 20.00 31.20 7.50 9.44 CALM Pre 17.20 30.00 32.80 20.00 71.40 1.14 6.50 SE Pre 0.01 0.03 0.05 0.02 0.01 0.01 0.01 P-value Pre 0.50 0.50 0.50 1.00 0.50 0.50 0.50 CON Drive 37.50 45.60 6.00 11.00 44.58 2.20a 5.53a CALM Drive 34.80 44.00 12.60 8.80 39.97 0.11b 3.53b SE Drive 0.06 0.05 0.02 0.03 0.31 0.44 0.62 P-value Drive 0.63 1.00 0.06 0.36 0.34 <0.01 0.02

a,b Within period, LS Means differ between treatments, p < 0.05.

Discussion

All dogs experienced a rise in HR at the beginning of the simulated thunderstorm as well as when loaded into the vehicle prior to travel, indicating these dogs were good representatives of dogs experiencing minor reactions to both stressors. In the thunderstorm simulation study, the HR of dogs treated with 2M2B ointment returned to

Texas Tech University, Glenna M. Pirner, May 2018 baseline twice as quickly as when they were treated with CON ointment. The fact that

HR of CON dogs did return to baseline during the simulation suggests dogs do acclimate to stressors; thus, it appears that the first ten minutes of a thunderstorm are the most stressful to the animal. Similar studies evaluating Dog Appeasing Pheromone (DAP,

Adaptil, Ceva Santé Animale) have not examined heart rate data, and so the generality of this observation cannot be commented upon [17, 28]. Similarly, HR of CON dogs in the car travel model increased, and then decreased but remained above BHR. In contrast, HR of dogs treated with 2M2B increased, but at no time was significantly higher than BHR.

This suggests that 2M2B does modify HR of dogs exposed to an acute stressor.

In the simulated thunderstorm model, significant behavior results between GLM and Wilcoxon signed-rank were conflicting. Although Wilcoxon tests are more appropriate for the non-normally distributed data, GLM provides a more comprehensive view of the data without compromising error rates. With this consideration, behavior can only be appropriately superficially discussed for this study. The observation that dogs treated with 2M2B ointment spent more time lying during the simulation is consistent with the findings of Levine et al., [28] in that dogs treated with DAP showed less pacing and running behaviors during a CD-based fireworks program. One major difference is that dogs evaluated in Levine’s study also received eight weeks of desensitization and counter-conditioning training, whereas dogs in the current study did not receive such therapy. Pairing of 2M2B with such training procedures would likely be of great value to owners with noise-phobic dogs.

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An obvious limitation of this study is that although this study was designed to be highly controlled in order to evaluate the effects of the pheromone, some environmental changes associated with a true thunderstorm could not be replicated. It was not possible to incorporate factors such as barometric pressure changes, atmospheric electromagnetic disturbances, and wind into this study. Some studies show that although these variables may not directly be the cause of the phobia, the dogs may become classically conditioned to associate these stimuli with an impending storm [9]. The Sounds Scary! Thunderstorm track is an accurate representation of the sounds of thunder and rain, with volume, duration, and intensity of thunderclaps varying throughout the track. Playing the track on stereo system in which bass and treble could be adjusted enhanced the quality of the simulation.

In the car travel model it was anticipated that moving, vocalization, and lip- licking would be reduced in dogs receiving 2M2B spray. While moving behavior was not significantly different, it was reduced in the 2M2B group. Lip-licking can be interpreted as either a sign of distress or a sign of nausea, both of which can be attributed to riding in a vehicle [29, 30]. Only two dogs in the 2M2B group and one dog in the CON group vomited, so nausea could not be statistically assessed in this study. The higher frequency of lip-licking in CON dogs may be associated with a higher level of stress during transport, which is supported by the higher HR in the CON group. As anticipated the number of vocalizations, an indicator of stress in domestic dogs, was higher in dogs in the CON group, with whining being the most commonly noted type of vocalization [31].

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Most of the vocalizations occurred in two of the eight dogs in the CON group and so could not be statistically assessed.

Until recently HRV was used primarily in humans to assess disease state and mortality risk. It has been shown to be useful in assessing welfare and stress of several livestock species including sheep, cattle, and swine [32 – 34]. HRV has been measured in domestic dogs but mainly for emotional state determination [35, 36]. Stubsjøen et al.,

[32] and Kovács et al., [33] used HRV to evaluate chronic stress in lambs and cows due to lameness. Both species had a high RMSSD, consistent with the findings of the present study. Interestingly, RMSSD increased during acute stress in the current study, rather than decreasing with acute stress as anticipated [37]. In dogs treated with CON spray,

RMSSD increased in the “pre” period compared to baseline, then decreased slightly, but not significantly in the “drive” period. In dogs treated with 2M2B spray, RMSSD increased significantly from baseline to “pre” and from “pre” to “drive”. 2M2B may have prevented the decrease in RMSSD associated with acute stress that was somewhat present in CON dogs. Stubsjøen and Kovács reported conflicting findings regarding frequency- domain measurements. Lambs with foot rot were reported to have a lower HFP and higher LFP, and thus a higher LFHFR compared to non-lame counterparts; this is consistent with the general finding of acute stress and dominance of the sympathetic nervous system in HR control [22, 24, 26, 32]. In cattle, only HFP and LFHFR were evaluated, and it was observed that HFP increased and LFHFR decreased [33]. In the current study, HFP elevated from baseline in both treatment groups, but decreased

Texas Tech University, Glenna M. Pirner, May 2018 slightly in the CON group in the “drive” period compared to the “pre” period. In contrast,

HFP increased in each period in dogs treated with 2M2B. Each of these studies uses a model of pain-induced chronic stress; it is possible that endogenous opioids modify the physiological response to long term stress, explaining differences observed between these studies and the present study [38].

One important distinction of dogs in this study is that twelve of the dogs reported in this study have been determined to be experiencing a chronic state of stress as evidenced by results of an adrenocorticotropic hormone (ACTH) stimulation test. In the aforementioned studies the animals were assumed to be experiencing chronic stress based on previous knowledge of dairy cow and lamb husbandry [32, 33]. In humans with chronic life stress, such as being a primary caregiver for a loved one, introduction of an acute stressor resulted in an exaggerated sympathomedullary response, releasing high levels of epinephrine from the adrenal medulla. It also resulted in blunted natural killer cell activity, which is linked to susceptibility to viral infections and neoplasia [39, 40]. It appears that 2M2B may modify HR and some HRV parameters by acting upon the sympathomedullary system and suppressing the exaggerated stress response. Future work should investigate the effects of 2M2B on stress response parameters such as epinephrine release and NK cell activity.

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Conclusions

For dogs that are chronically stressed, exposure to an acute stressor such as car travel or thunderstorm may induce an exaggerated stress response, including increased

HR and altered HRV parameters. 2M2B appears to reduce the magnitude of physiological effects of this exaggerated stress response, possibly by modification of the sympathomedullary system. Understanding the mechanism by which 2M2B alleviates the effects of acute stress in chronically stressed dogs may have important implications in improving the health and overall welfare of domestic dogs.

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[13] J. McGlone, The Pheromone Site. http://www.depts.ttu.edu/animalwelfare/Research/Pheromones/index.php, 2011 (accessed 2 June 2017). 63

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[14] B. Schaal, G. Coureaud, D. Langlois, C. Ginies, E. Semon, G. Perrier, Chemical and behavioural characterization of the rabbit mammary pheromone. Nature 424 (2003) 68-72. [15] G. Coureaud, D. Langlois, G. Sicard, B. Schaal, Newborn rabbit responsiveness to the mammary pheromone is concentration dependent. Chem. Senses 29 (2003) 341-350. [16] P. Pageat, E. Gaultier, Current research in canine and feline pheromones. Vet. Clin. N. Am. - Small 33 (2003) 187–211. [17] G. M. Landsberg, A. Beck, A. Lopez, M. Deniaud, J.A. Araugo, N.W. Milgram, Dog-appeasing pheromone collars reduce sound-induced fear and anxiety in beagle dogs: a placebo-controlled study. Vet. Rec. 177 (2015) 260. [18] M. G. Estellés, D.S. Mills, Signs of travel-related problems in dogs and their response to treatment with dog-appeasing pheromone. Vet. Rec. 159 (2006) 143- 147. [19] A.J. Camm, M. Malik, J.T. Bigger, G. Breithardt, S. Cerutti, R.J. Cohen, F. Lombardi, Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 93 (1996) 1043-1065. [20] J.B. Clapp, S. Croarkin, C. Dolphin, S.K. Lyons, Heart rate variability: a biomarker of dairy calf welfare. Anim. Prod. Sci. 55 (2015) 1289-1294. [21] G.G. Berntson, D.L. Lozano, Y.J. Chen, Filter properties of root mean square successive difference (RMSSD) for heart rate. Psychophysiology, 42 (2005) 246- 252. [22] P. Nickel, F. Nachreiner, Sensitivity and Diagnostics of the 0.1-Hz Component of Heart Rate Variability as an Indicator of Mental Workload. Hum. Factors 45 (2003) 575–590. [23] P. Jönsson, Respiratory sinus arrhythmia as a function of state anxiety in healthy individuals. Int. J. Psychophysiol. 63 (2007) 48–54. [24] A. Malliani, M. Pagani, F. Lombardi, S. Cerutti, Cardiovascular neural regulation explored in the frequency domain. Circulation 84 (1991) 1482-1492.

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[26] R.P. Sloan, P.A. Shapiro, E. Bagiella, M.M. Myers, J.T. Bigger, R.C. Steinman, J.M. Gorman, Brief interval heart period variability by different methods of analysis correlates highly with 24 h analyses in normals. Biol. Psych. 38 (1994) 133-142.

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[28] E. D. Levine, D. Ramos, D.S. Mills, A prospective study of two self-help CD based desensitization and counter-conditioning programmes with the use of Dog Appeasing Pheromone for the treatment of firework fears in dogs (Canis familiaris). Appl. Anim. Behav. Sci. 105 (2007) 311-329. [29] D. Schwizgebel, Zusammenhänge zwischen dem Verhalten des Tierlehrers und dem Verhalten des Deutschen Schäferhundes im Hinblick auf tiergerechte Ausbildung. Aktuelle Arbeiten zur artgemassen Tierhaltung, (1982) 138-148.

[30] D.C. Twedt DC, Vomiting, in: S.J. Ettinger, E.C. Feldman (Eds.), Textbook of Veterinary Internal Medicine Vol. 1 (5th edn) WB Saunders Co., Philadelphia, PA, USA, 2000, pp. 117–121.

[31] B. Beerda, M.B. Schilder, J.A. van Hooff, H.W. de Vries, Manifestations of chronic and acute stress in dogs. Appl. Anim. Beh. Sci. 52 (1997) 307-319.

[32] S.M. Stubsjøen, M. Knappe-Poindecker, J. Langbein, T. Fjeldaas, J. Bohlin, Assessment of chronic stress in sheep (part II): Exploring heart rate variability as a non-invasive measure to evaluate cardiac regulation. Small Ruminant Res. 133 (2015) 30-35.

[33] L. Kovács, F.L. Kézér, V. Jurkovich, M. Kulcsár-Huszenicza, J. Tőzsér, Heart rate variability as an indicator of chronic stress caused by lameness in dairy cows. PloS one, 10(8) (2015), e0134792.

[34] I.C. de Jong, A. Sgoifo, E. Lambooij, S.M. Korte, H.J. Blokhuis, J.M. Koolhaas JM, Effects of social stress on heart rate and heart rate variability in growing pigs. Can. J. Anim. Sci. 80 (2000) 273-280. [35] T. Romero, A. Konno, T. Hasegawa, Familiarity bias and physiological responses in contagious yawning by dogs support link to empathy. PloS ONE 8(8) (2013) e71365. [36] M. Zupan, J. Buskas, J. Altimiras, L.J. Keeling, Assessing positive emotional states in dogs using heart rate and heartrate variability. Physiol. Behav. 155 (2016) 102- 111.

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[37] B. Verkuil, J.F. Brosschot, J. F. Thayer, Cardiac reactivity to and recovery from acute stress: Temporal associations with implicit anxiety. Int. J. Psychophysiol. 92 (2014) 85-91. [38] C.J. Loijens, J.G. Janssensb, W.G.P. Schoutena, V.M. Wiegant, Opioid activity in behavioral and heart rate responses of tethered pigs to acute stress. Physiol. Behav. 75 (2002) 621–626. [39] J.L. Pike, T.L. Smith, R.L. Hauger, P.M. Nicassio, T.L. Patterson, J. McClintick, M.R. Irwin, Chronic life stress alters sympathetic, neuroendocrine, and immune responsivity to an acute psychological stressor in humans. Psychosom. Med. 59 (1997) 447-457. [40] G. Trinchieri, Biology of natural killer cells. Adv. Immunol. 47 (1989) 187-376.

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CHAPTER IV RELATIONSHIPS AMONG PHYSIOLOGICAL MEASURES OF STRESS AND IMPACT OF 2-METHYLBUT-2-ENAL IN STRESS AMELIORATION Abstract:

Domestic dogs are susceptible to both acute and chronic stress. The physiological response to stress involves a balance of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic-adrenal-medullary (SAM) axis, resulting in distinct changes in plasma cortisol and heart rate variability (HRV) measures. Evidence suggests 2- methylbut-2-enal (2M2B) may modify the acute stress response of chronically stressed dogs. Thirteen dogs previously exposed to a simulated thunderstorm in a crossover design comparing placebo or 2M2B ointment underwent adrenocorticotropic hormone

(ACTH) stimulation testing, measurement of HRV parameters, and leukocyte differentials. Correlations and stepwise regression modeling were used to confirm that dogs were chronically stressed and to predict which dogs might benefit from 2M2B intervention. Dogs exhibited physiological parameters consistent with chronic stress, including elevated neutrophil:lymphocyte ratios, basal heart rates, and select time- and frequency- domain measures of HRV. Cortisol was within normal limits at baseline but increased markedly one hour after administration of exogenous ACTH, suggesting chronic HPA axis activation. Average R-R interval was negatively, but not significantly, correlated with the magnitude of difference in heart rate between placebo and 2M2B ointment during the simulated thunderstorm. With the reliability and ease of acquiring

HRV measurements, the relationship between HRV and 2M2B responsiveness should be

Texas Tech University, Glenna M. Pirner, May 2018 further investigated. Understanding the effects of 2M2B on the HPA axis during a stress response has important implications in reducing animal stress and promoting welfare.

Introduction

Chronic stress in domestic dogs occurs due to disease, poor environmental conditions, and inappropriate handling, among other reasons [1]. Dogs in shelters and laboratory environments are susceptible to chronic stress [2]. Responding to an acute stressor during a period of chronic stress involves interaction of the hypothalamic- pituitary-adrenal (HPA) axis and the sympathetic-adrenal-medullary (SAM) axis.

Behavior tests in this context are not standardized and may not accurately reflect the underlying physiological processes responsible for the stress response [3]; therefore, certain physiological measures provide a reliable method for determining an animal’s state of stress.

Cortisol concentrations in hair, nails, and feces can be a reliable indicator of chronic stress-related activation of the HPA axis with the added benefit of being non- invasive [4 – 6]. However, these matrices may provide inconsistent results. Bennet and

Hayssen [7] found differences in cortisol concentrations in different colored hair within the same subject. It is also possible that hair may reflect localized production of cortisol as well as systemic levels [8]. Adrenocorticotropic hormone (ACTH) stimulation testing is used in veterinary medicine to diagnose disfunction of the HPA axis, typically in

Addison’s or Cushing’s diseases [9]. In the absence of disease, administration of exogenous ACTH mimics the body’s stress response by stimulating release of

Texas Tech University, Glenna M. Pirner, May 2018 glucocorticoids from the adrenal glands and increasing the neutrophil:lymphocyte ratio

[10, 11]. Heart rate variability (HRV) is a common, non-invasive method used extensively to monitor and predict the outcome of both physical and psychological illness in humans [12]. More recently, the ability to use these same measurements to investigate the activity of the autonomic nervous system in other species has allowed researchers to study emotional state and stress response in domestic dogs

Evaluation of heart rate alone is insufficient to determine which branch of the autonomic nervous system is responsible for a change in heart rate [13]. The balance between the sympathetic and parasympathetic nervous system is a dynamic relationship, constantly changing to allow the animal to adapt to its surroundings and thus maintain homeostasis. HRV measures the variation in time between consecutive heart beats, or the

R-R interval [12]. During a stress response the sympathetic nervous system exerts a slower effect on heart rate than during rest, and so HRV will be lower as a result [14].

The root mean square of the successive differences (RMSSD) in HRV allows for determination of the amount of influence exerted by the vagus nerve on the heart. This measurement allows for consideration of respiratory sinus arrhythmia but also can detect fluctuations in low frequency ranges which reflect sympathetic nervous system inputs

[12, 15]. Changes in the frequency-domain parameters are typically measured using high- and low-frequency absolute power, and the ratio of these powers. High-frequency activity decreases during acute stress and elevated anxiety levels [16, 17]. In contrast, low- frequency activity increases under states of stress [18, 19]. Often, these two parameters

Texas Tech University, Glenna M. Pirner, May 2018 are combined into a ratio of low to high frequency, and an increase in this ratio is associated with activation of the sympathetic nervous system [20].

Pheromone-based products have gained popularity as a method of stress relief.

Pheromones are chemical signals produced by one individual and received by a second individual of the same species in which they elicit specific responses [21]. Throughout mammalian evolution metabolic pathways responsible for producing these molecules and olfactory receptor gene families have been relatively conserved; thus, a pheromone molecule in one species may elicit a different response if received by an individual of a second species [22]. Such molecules are referred to as interomones [23]. Rabbit maternal pheromone (2-methylbut-2-enal; 2M2B) is secreted in milk from the dam to attract pups to the nipple and calm them during nursing [24, 25]. Previous work in this laboratory has shown that 2M2B ointment modifies heart rate of laboratory dogs during a simulated thunderstorm and during car travel [26]. Results of these studies suggest an individualized response not only to the stressor, but also to the interomone treatment.

The objectives of this study were to determine the average stress level of the dogs used in acute stressor models, and to use that information to determine if correlations exist between a range of physiological variables and the heart rate response of dogs experiencing a simulated thunderstorm in the presence or absence of 2M2B. The overall goal of this study was to determine which physiological parameters influence the effectiveness of 2M2B in domestic dogs.

Texas Tech University, Glenna M. Pirner, May 2018

Methods and Materials.

General

Research was approved by the Texas Tech University Institutional Animal Care and Use Committee (Protocol 14009-01). Husbandry and housing of dogs were consistent with the U.S. Animal Welfare Act. Dogs were individually housed in 1.22 m x 1.83 m kennels with a food dish, water dish, toys, blanket, and an elevated shelf for sleeping.

Thirteen (nine males and four females) mixed breed dogs between one and ten years of age and weighing 16.88 ± 1.95 (SEM) kg were treated with either a placebo ointment

(CON) or an ointment containing 1 µg/mL 2-methylbut-2-enal (2M2B) and then exposed to a simulated thunderstorm soundtrack (Sounds Scary! Sound Therapy 4 Pets Ltd.,

Chester, England).

Data Collection.

Heart Rate and Heart Rate Variability. Basal heart rate (BHR) and heart rate variability measures were collected during a thirty min quiet period when dogs were in their home kennels. These were obtained using a Polar Pro RS800CX telemetry monitor

(Polar Electro, Lake Success, NY). Belts were placed around the thorax, caudal to the shoulders, with the sensor centered on the sternum. To maximize conduction fur was shaved at the sensor contact area and conduction gel was applied. Heart rate was recorded every 1 s and averaged per min. For heart rate, recordings below 60 bpm or above 180 bpm were considered inaccurate and were omitted from data. Heart rate variability was processed using Kubios Heart Rate Variability Analysis software 3.0 (Kubios Oy, 2017). 71

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The root mean square of the successive differences (RMSSD; ms), high frequency absolute power (HFP; ms2), low frequency absolute power (LFP; ms2), and the low frequency:high frequency ratio (LFHFR) were obtained after processing. Frequency- domain variables were calculated using Fast Fourier Transform (FFT). The low frequency range was fixed between 0.04 and 0.15 Hz, and high frequency was considered above 0.15 Hz.

Heart rate was recorded before, during, and after the thunderstorm simulation.

Dogs’ heart rate during the simulation when treated with CON ointment (CHR) and

2M2B ointment (MBHR) was averaged for the fifteen min period. The difference in HR between BHR and HR during the thunderstorm with CON ointment (CBDIFF), the difference between BHR and HR during the thunderstorm with 2M2B ointment

(MBBDIFF), and the difference between CHR and MBHR (MBCDIFF) were calculated.

Heart rate variability measures are not available from this study due to equipment error.

Adrenocorticotropic Hormone Stimulation Testing. Adrenocorticotropic hormone

(ACTH) stimulation testing took place approximately six months after the simulated thunderstorm trials. Fur was shaved from the neck and skin cleaned using diluted

Betadine solution (Spectrum Chemical Mfg. Corp., New Brunswick, NJ) and 70% isopropyl alcohol. At each time point, 3 mL of blood was drawn via jugular venipuncture into a 5 mL EDTA Vacutainer tube (BD Vacutainers®, Becton, Dickinson, and Co.,

Franklin Lakes, NJ). Whole blood was centrifuged at 1500 rpm for 10 minutes, and plasma was separated into two aliquots and placed on ice to be transported. Time 0 was

Texas Tech University, Glenna M. Pirner, May 2018 considered baseline. Immediately following the Time 0 draw, dogs received an intravenous injection of Cortrosyn® (Amphastar Pharmaceuticals, Inc., Rancho

Cucamonga, CA) at 5 µg/kg in the cephalic vein. Blood was collected 60 (Time 1), 90

(Time 2), 120 (Time 3), and 150 (Time 4) min after injection. Dogs were returned to their home kennels between blood draws.

Plasma was thawed to room temperature and cortisol concentrations were measured in duplicate using an enzyme-linked immunosorbent assay kit (Enzo Life

Sciences, Inc., Farmingdale, NY, USA) with a sensitivity of 56.72 pg/mL. Results were plotted against a standard curve and cortisol concentrations calculated in µg/dL. Time 0 cortisol (BCORT), Time 1 cortisol (HRCORT) and area under the curve for all time points (AUC) are used in the predictive model. The Diagnostic Protocol for Cases of

Suspected Cushing’s Syndrome or Addison’s disease provided by Idexx Laboratories was used to determine the adrenal function of each dog ([9]; Figure 4.1). Dogs were categorized as consistent with hypoadrenocorticism, normal, or consistent with hyperadrenocorticism.

Texas Tech University, Glenna M. Pirner, May 2018

Figure 4.1. Diagnostic Protocol for Adrenocorticotropic Hormone Stimulation Test.

Leukocyte Quantification. A peripheral blood smear was prepared using whole blood obtained at the Time 0 blood draw and stained using a three-step method. Neutrophils and lymphocytes were quantified per 100 cells at 100x magnification and neutrophil:lymphocyte ratio was calculated (NLR).

Texas Tech University, Glenna M. Pirner, May 2018

Data Analyses

Pearson Correlation Coefficients between variables were calculated using the

PROC CORR function of SAS 9.4 to determine direction and magnitude of relationships between variables. Predictive variables for each dependent variable were determined using the PROC STEPWISE function of SAS 9.4. The overall model R2 and significance of each independent variable in the model were computed. Related variables were assessed and removed from each computation as necessary. For example, HFP and LFP were not included in the model for LFHFR. Correlations and relationships were considered significant at an alpha level of ≤ 0.05. Table 4.1 gives the comprehensive results used in relationship calculations.

Table 4.1. Results used in relationship calculations.

MBH HRCOR AG CHR CBDIF R MBBDI MBCDI BCORT T E WT BHR RMSS LFP HFP LFHF (BPM F (BPM FF FF (UG/DL (UG/DL NL DOG (Y) (KG) (BPM) D (ms) (ms2) (ms2) R ) (BPM) ) (BPM) (BPM) AUC ) ) R 483 9 16.1 78 26.91 462.68 964.65 0.48 71 7 86 -8 15 120.79 4.35 24.58 1.44 545 10 18 81 13.42 467.48 248.65 1.88 66 15 65 16 -1 60.66 1.63 24.90 2.96 536 4 17.7 81 32.28 473.06 181.19 2.61 99 -18 113 -32 14 118.64 21.69 49.44 3.13 533 4 14.4 86 21.91 497.78 190.24 2.62 89 -3 93 -7 4 153.09 6.36 74.02 2.24 626 1 8.8 98 39.28 752.16 524.36 1.43 164 -66 150 -52 -14 243.64 5.47 76.38 7.00 549 6 30.4 105 22.04 402.77 418.40 0.96 155 -50 99 6 -56 97.94 1.46 42.07 3.14 467 9 6.3 108 45.34 149.33 353.66 0.42 128 -20 115 -7 -13 185.18 2.08 52.52 5.71 515 7 16.7 109 14.41 330.00 149.83 2.20 124 -15 99 10 -25 39.26 2.32 22.74 3.10 587 5 22.1 110 35.68 595.27 536.76 1.11 124 -14 72 38 -52 193.88 1.66 54.46 4.87 538 4 25.8 120 17.03 1035.40 261.71 3.95 126 -6 118 2 -8 78.03 11.47 55.21 4.75 527 9 10.8 128 27.10 653.18 352.63 1.85 150 -22 133 -5 -17 111.60 1.31 17.56 8.00 620 5 22.3 136 4.59 26.986 7.7764 3.47 131 5 157 -21 26 153.15 3.51 46.42 3.14 528 4 10 147 2.62 3.2565 1.1228 2.90 144 3 111 36 -33 137.43 5.73 54.44 3.89

Results.

ACTH Stimulation Testing

Ten of the 13 dogs exhibited normal cortisol concentrations at Time 0 (range 1.31 to 5.73) µg/dL. The remaining three dogs had basal cortisol levels > 6.00 µg/dL. After

Cortrosyn® administration 12 of the 13 dogs displayed a cortisol response consistent with hyperadrenocorticism; only one dog displayed a normal cortisol response to Cortrosyn® administration.

Correlations

The correlation matrix is shown in Figure 4.2. Body weight, LFP, and BCORT were not significantly correlated with any other single variables. Age was negatively correlated w HRCORT (r = -0.82, P < 0.01). BHR was positively correlated with CHR (r

= 0.68, P = 0.01). RMSSD was positively correlated with HFP (r = 0.58, P = 0.04) and

AUC (r = 0.55, P = 0.05). RMSSD was negatively correlated with LFHFR (r = -0.67, P =

0.01) and CBDIFF (r = -0.55, P = 0.05). CHR was positively correlated NLR (r = 0.68, P

= 0.01).

Texas Tech University, Glenna M. Pirner, May 2018

Figure 4.2. Correlation matrix of physiological variables in domestic dogs. Orange- red indicates negative correlations; cyan-blue indicates positive correlations. n = 13 dogs.

Predictive Models

Comprehensive results for all stepwise regression analyses can be found in Table

4.2.

Texas Tech University, Glenna M. Pirner, May 2018

Table 4.2. Comprehensive data from the stepwise regression analysis.

DEPENDENT INDEPENDENT P- MODEL MODEL P- R VARIABLE VARIABLE VALUE R2 VALUE NEUT:LYMPH RATIO 0.41 0.17 RMSSD -0.45 0.12 BASAL HR 0.82 < 0.01 LOW FREQ POWER -0.31 0.30 AVERAGE R-R 0.15 0.63 AVERAGE R-R N/A N/A N/A N/A N/A CHR - BHR DIFF -0.55 0.05 BASAL HR -0.45 0.12 RMSSD 0.77 < 0.01 NEUT:LYMPH RATIO 0.44 0.13 CORT AUC 0.55 0.05 LOW FREQ N/A N/A N/A N/A N/A POWER HIGH FREQ BASAL HR -0.51 0.08 0.25 0.08 POWER RMSSD -0.67 0.01 LF:HF RATIO BASAL CORTISOL 0.46 0.12 0.82 < 0.01 NEUT:LYMPH RATIO -0.10 0.74 CON HR NEUT:LYMPH RATIO 0.68 0.01 0.47 0.01 CON HR - RMSSD -0.55 0.05 0.31 0.05 BASAL HR NEUT:LYMPH RATIO 0.50 0.08 2M2B HR 0.41 0.07 LF:HF RATIO 0.35 0.234 AVERAGE R-R 0.56 0.05 2M2B - BASAL CORT AUC -0.36 0.23 0.82 0.04 HR BASAL CORTISOL -0.40 0.18 1-HR CORTISOL -0.28 0.35 2M2B - CON HR AVERAGE R-R 0.46 0.11 0.21 0.11 1-HR CORTISOL 0.70 < 0.01 CORT AUC LF:HF RATIO -0.30 0.33 0.77 < 0.01 NEUT:LYMPH RATIO 0.41 0.16 LF:HF RATIO 0.46 0.12 BASAL RMSSD 0.08 0.80 0.67 0.01 CORTISOL NEUT:LYMPH RATIO -0.17 0.58 1-HR CORTISOL N/A N/A N/A N/A N/A NEUT:LYMPH CON HR 0.6821 0.0102 0.61 < 0.01 RATIO RMSSD 0.444 0.1285

Texas Tech University, Glenna M. Pirner, May 2018

No significant predictive models were found for RR, LFP, HFP, HRCORT, MBCDIFF, or MBHR.

The effect of 2M2B on CON HR and 2M2B HR during the simulated thunderstorm is presented in Figure 4.3. Dogs experienced a higher HR with CON ointment compared to 2M2B ointment during the simulation. When dogs were treated with 2M2B ointment, HR returned to baseline twice as quickly as when they were treated with CON ointment.

160 Before During After a b a 140

120 HR, bpm HR, 100

80 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Time, min 2M2B CON

Figure 4.3. Thunderstorm simulation heart rate results. Least Squares means of dogs’ heart rate per minute before, during, and after a simulated thunderstorm. Dogs received either control ointment (CON) or 1 µg/mL 2-methylbut-2-enal ointment (2M2B). Black vertical lines indicate periods. n = 19 dogs/treatment.

BHR had no significant predictive parameters. NLR (r = 0.41), RMSSD (r = -

0.45), LFP (r = -0.31), and RR (r = 0.15) cumulatively account for approximately 81.9% of the variability in BHR (P < 0.01). NLR was positively correlated with CHR (r = 0.68), accounting for 46.5% of the variability in CON HR during the simulation (P = 0.01).

RMSSD was negatively correlated with the difference between CON HR and BHR (r = - 80

Texas Tech University, Glenna M. Pirner, May 2018

0.55), accounting for 30.8% of the variability in this difference (P = 0.05). The difference in HR between BHR and 2M2B HR during the simulation was positively correlated with

RR (r = 0.56, P = 0.05). AUC (r = -0.36), BCORT (r = -0.40), and HRCORT (r = -0.28) were negatively, but not significantly, correlated with the difference in HR between BHR and 2M2B HR during the simulation. Cumulatively, these variables account for 82.3% of the variability in the difference in HR between BHR and 2M2B HR during the simulation

(P = 0.04).

BCORT had no significant predictive parameters. LFHFR (r = 0.46), RMSSD (r =

0.08), and NLR (r = -0.28) cumulatively account for approximately 67.2% of the variability in BCORT (P = 0.01). AUC was positively correlated with HRCORT (r =

0.70, P < 0.01). LFHFR (r = -0.30) and NLR (r = 0.41) were included in the AUC model but were not significant. Cumulatively, these variables account for 76.5% of the variability in AUC (P < 0.01). NLR was positively correlated with CHR (r = 0.68, P =

0.01). RMSSD (r = 0.44) was included in the NLR model but was not significant.

Cumulatively, these variables account for 61.3% of the variability in NLR (P < 0.01).

RMSSD was negatively correlated with CBDIFF (r = -0.55, P = 0.05) and positively correlated with AUC (r = 0.55, P = 0.05). BHR and NLR were included in the

RMSSD model but were not significant. Cumulatively, these variables account for 77.3% of the variability in RMSSD (P < 0.01). LFHFR was negatively correlated with RMSSD

(r = 0.67, P = 0.01). BCORT (r = 0.71) and NLR (r = -0.10) were also included in the

LFHFR model but were not significant. Cumulatively, these variables account for 82.3% of the variability in LFHFR (P < 0.01).

Texas Tech University, Glenna M. Pirner, May 2018

Discussion

Age was negatively correlated with the concentration of cortisol 1 h after ACTH stimulation. The influence of age of healthy dogs (i.e. non-Cushing’s or non-Addison’s disease) on ACTH stimulation test results has not been directly investigated. The results of the current study contrast those of Reul et al., [27], which demonstrate that older dogs

(> 11 yr) had a significantly higher basal cortisol level compared to younger dogs.

Currently, limited literature exists which uses HRV measures to evaluate stress in domestic dogs; rather, HRV is used to determine emotional state or monitor disease progression. In one study by Katayama et al., [28], RMSSD was reduced in dogs in a negative emotional state. While stress and emotions are linked, it is possible to have negative emotions and not experience a state of stress, and vice versa [29]. RMSSD and

HFP were both found to be higher in dairy cows with chronic lameness compared to non- lame cows, which is consistent with the findings of the current study [30]. This finding is also strengthened by the observed positive correlation between AUC after the ACTH stimulation test and RMSSD. Animals in a state of chronic stress exhibit hypersensitivity of the HPA axis to acute stressors, resulting in an elevated level of plasma cortisol; this is mimicked by the administration of exogenous ACTH [9, 31].

Dogs with higher HR during the simulated thunderstorm in the CON condition also had higher monocyte numbers and NLR. This is consistent with the general stress response of increased NLR [32 – 34].

Texas Tech University, Glenna M. Pirner, May 2018

In chronically stressed rats the increased sympathetic nervous system tone, rather than the HPA axis, mediates the glucocorticoid response [35]. Sympathetic nervous system activity increases LFHFR in humans, which is consistent with the findings of the current study [12]. CBDIFF describes the elevation of the dogs’ HR from BHR during the simulated thunderstorm without interomone intervention. Given that these dogs are chronically stressed and thus experience a high, rapid rate of cortisol release during an acute stressor, it is logical that activation of the sympathetic nervous system during the simulation would result in an elevated HR and reduced RMSSD.

Predictive Modeling

Dogs in this study exhibit physiological parameters consistent with a chronic state of stress, including elevated NLR, BHR, LFHFR, and RMSSD [12, 30, 32].

Regarding predicting the effectiveness of 2M2B on dogs’ response to a chronic stressor, the stepwise regression model was non-significant, with only average RR correlating negatively to MBCDIFF. In the simulated thunderstorm study, dogs were considered to “respond” to 2M2B if the difference between their heart rate with the placebo ointment was not significantly different than with the 2M2B ointment during the simulation. Thus, as MBCDIFF increased, average RR decreased, consistent with the findings of de Jong et al., [36] in that socially stressed pigs exhibited an elevated heart rate and decreased R-R interval. Four dogs in the current study had an RR between 300 and 400 ms; of these, only one was a “responder”. Seven of the remaining dogs had an

RR between 401 and 500 ms, and two had an RR > 501 ms. Six of these nine dogs were

“responders”. 83

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One limitation of this study is the small sample size as a result of availability of all data points on the original twenty dogs chosen for the simulated thunderstorm study.

A larger sample size will be beneficial in narrowing down the ranges of average RR interval so that a more definite conclusion regarding the relationship between HRV and

2M2B responsiveness can be made.

Conclusions

Based on the observed physiological parameters, the dogs used in the current study are valid representatives of domestic dogs experiencing a state of chronic stress. Of these dogs, those that appear to benefit the most from 2M2B intervention during an acute stressor such as a thunderstorm are those that have an average R-R interval greater than

401 ms. The ease and non-invasiveness of obtaining HRV measures will be beneficial in further exploring this relationship in the future.

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[2] Beerda B, Schilder MB, Bernadina W, Van Hooff JA, De Vries HW, Mol JA. 1999. Chronic stress in dogs subjected to social and spatial restriction. II. Hormonal and immunological responses. Physiol Behav 66:243-254.

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[3] Döring D, Haberland BE, Ossig A, Küchenhoff H, Dobenecker B, Hack R, Erhard MH. 2016. Behavior of laboratory beagles: Assessment in a standardized behavior test using novel stimuli and situations. J Vet 11:18-25.

[4] Mesarcova L, Kottferova J, Skurkova L, Leskova L, Kmecova N. 2017. Analysis of cortisol in dog hair-a potential biomarker of chronic stress: a review. Vet Med- Czech 62:363-376.

[5] Accorsi PA, Carloni E, Valsecchi P, Viggiani R, Garnberoni M, Tarnanini C, Seren E. 2008. Cortisol determination in hair and faeces from domestic cats and dogs. Gen Comp Endocr 155:398–402.

[6] Veronesi MC, Comin A, Meloni T, Faustini M, Rota A, Prandi A. 2015. Coat and claws as new matrices for noninvasive long-term cortisol assessment in dogs from birth up to 30 days of age. Theriogenology 84:791-796.

[7] Bennett A, Hayssen V. 2010. Measuring cortisol in hair and saliva from dogs: coat color and pigment differences. Domest Anim Endocrin 39:171-180.

[8] Ito N, Ito T, Kromminga A, Bettermann A, Takigawa M, Kees F, Straub RH, Paus R. 2005. Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol. FASEB J 19:1332— 1334.

[9] Feldman EC, Nelson RW .2004. Canine and Feline Endocrinology and Reproduction, 3rd ed. St Louis, MO: Saunders.

[10] Davis AK, Maney DL, Maerz JC. 2008 The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22:760-772.

[11] Bilandžić N, Žurić M, Lojkić M, Šimić B, Milić D, Barač I. 2006. Cortisol and immune measures in boars exposed to three-day administration of exogenous adrenocorticotropic hormone. Vet Res Commun 30:433-444. [12] Camm AJ, Malik M, Bigger JT, Breithardt G, Cerutti S, Cohen RJ, Coumel P, Fallen EL, Kennedy HL, Kleiger RE, Lombardi F. 1996. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 93:1043-1065.

[13] Hainsworth, R. 1995. The control and physiological importance of heart rate. Heart rate variability. 3-19.

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[14] Clapp JB, Croarkin BS, Dolphin C, Lyons SK. 2015. Heart Rate Variability: A biomarker of dairy calf welfare. Anim Prod Sci 55:1289–1294.

[15] Berntson GG, Lozano DL, Chen YJ. 2005. Filter properties of root mean square successive difference (RMSSD) for heart rate. Psychophysiology 42:246-252.

[16] Nickel P, Nachreiner F. 2003. Sensitivity and Diagnostics of the 0.1-Hz Component of Heart Rate Variability as an Indicator of Mental Workload. Hum Factors 45:575–590.

[17] Jönsson P. 2007. Respiratory sinus arrhythmia as a function of state anxiety in healthy individuals. Int J Psychophysiol 63: 48–54.

[18] Malliani A, Pagani M, Lombardi F, Cerutti S. 1991. Cardiovascular neural regulation explored in the frequency domain. Circulation. 84:1482-1492.

[19] Rimoldi O, Pierini S, Ferrari A, Cerutti S, Pagani M, Malliani A. 1990. Analysis of short-term oscillations of R-R and arterial pressure in conscious dogs. Am J Physiol 258:967-976.

[20] Sloan RP, Shapiro PA, Bagiella E, Myers MM, Bigger JT, Steinman RC, Gorman JM. 1994. Brief interval heart period variability by different methods of analysis correlates highly with 24 h analyses in normals. Biol Psych 38:133-142.

[21] Karlson P, Luscher M. 1959. Pheromones: a new term for a class of biologically active substances. Nature 183:55–56.

[22] Ache BW, Young JM. 2005. Olfaction: diverse species, conserved principles. Neuron 48:471-430.

[23] McGlone J. 2011. The Pheromone Site. http://www.depts.ttu.edu/animalwelfare/Research/Pheromones/index.php (accessed 2 June 2017). [24] Schaal B, Coureaud G, Langlois D, Ginies C, Semon E, Perrier G. 2003. Chemical and behavioural characterization of the rabbit mammary pheromone. Nature 424:68-72.

[25] Coureaud G, Langlois D, Sicard G, Schaal B. 2003. Newborn rabbit responsiveness to the mammary pheromone is concentration dependent. Chem Senses 29:341-350.

[26] G. Pirner, A. Garcia, J. McGlone. Impact of 2-methylbut-2-enal on acute stress responses in chronically stressed domestic dogs. Submitted for publication 2018.

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[27] Reul JM, Rothuizen J, de Kloet ER. 1991. Age-related changes in the dog hypothalamic-pituitary-adrenocortical system: neuroendocrine activity and corticosteroid receptors. J Steroid Biochem Mol Biol 40:63-69.

[28] Katayama M, Kubo T, Mogi K, Ikeda K, Nagasawa M, Kikusui T. 2016. Heart rate variability predicts the emotional state in dogs. Behav Process 128:108-112.

[29] Spielberger CD, Sarason IG, Kulcsar Z, Van Heck GL. 2015. Stress and emotion. Taylor & Francis.

[30] Kovács L, Kézér FL, Jurkovich V, Kulcsár-Huszenicza M, Tőzsér J. 2015. Heart rate variability as an indicator of chronic stress caused by lameness in dairy cows. PloS one, 10:e0134792.

[31] Franco AJ, Chen C, Scullen T, Zsombok A, Salahudeen AA, Di S, Herman JP, Tasker JG. 2016. Sensitization of the hypothalamic-pituitary-adrenal axis in a male rat chronic stress model. Endocrinology 157:2346-2355.

[32] Maes M, Scharpé S, Meltzer HY Cosyns P. 1993. Relationships between increased haptoglobin plasma levels and activation of cell-mediated immunity in depression. Biol Psych 34:690-701.

[33] Landmann RM, Müller FB, Perini CH, Wesp M, Erne P, Bühler FR. 1984. Changes of immunoregulatory cells induced by psychological and physical stress: relationship to plasma catecholamines. Clin Exp Immunol 58:127.

[34] Fauci AS. 1975. Mechanisms of corticosteroid action on lymphocyte subpopulations 1. Redistribution of circulating T-lymphocytes and B-lymphocytes to bone marrow. J Immunol 28:669–680.

[35] Lowrance SA, Ionadi A, McKay E, Douglas X, Johnson JD. 2016. Sympathetic nervous system contributes to enhanced corticosterone levels following chronic stress. Psychoneuroendocrino 68:163-170. [36] de Jong IC, Sgoifo A, Lambooij E, Korte SM, Blokhuis HJ, Koolhaas JM (2000) Effects of social stress on heart rate and heart rate variability in growing pigs. Can J Anim Sci 80:273-280.

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CHAPTER V

IDENTIFICATION OF PUTATIVE SEXUAL PHEROMONES IN MALE DOGS

BY SOLID-PHASE MICROEXTRACTION TECHNIQUE IN COMBINATION

WITH GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Abstract

Pheromones are essential for effective communication in insects and mammals.

Many pheromones that convey information about reproductive status and age are dependent on the sex hormones which increase in the body after puberty. This study used solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) to isolate and identify volatile molecules in the headspace of domestic dog urine. Urine was collected via free-catch from five individuals in each of six groups: juvenile intact male (JIM), adult intact male (AIM), adult castrated male

(AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF). Headspace sampling yielded 100 volatile molecules across all samples, of which six had significantly different peak areas between groups. Octanal, 2-methyl-quinoline, methyl propyl sulfide, and 2-heptanone appear to be closely linked to male sex hormones as they had higher peak areas in intact adult males.

3-ethylcyclopentanone appears to be associated with intact adult females and castrated or subordinate male animals. No sex or life stage patterns could be divulged from the data on 2-pentanone. Future research should investigate changes in the female across the phases of the estrous cycle and, most importantly, determine behavioral and physiological effects of these molecules on conspecifics. 88

Texas Tech University, Glenna M. Pirner, May 2018

Introduction

Pheromones play significant roles in mammalian communication, including identity, reproduction, alarm, and maternal recognition, among others. Pheromones signaling the identity of an individual do not vary throughout the animal’s lifespan; however, many studies have documented drastic changes in pheromone production occurring with puberty [1]. At puberty the body begins producing higher concentrations of steroid-derived sex hormones such as testosterone and estradiol, which serve as the precursor for many sex pheromones [2].

Takacs et al., [3] hypothesized that sex pheromones in the brown rat would appear only as juveniles reached maturity. Indeed, collection of urine from the same rats from four weeks of age through puberty found that five ketone molecules (3-ethyl-2- pentanone, 2-heptanone, 4-heptanone, 3-ethyl-2-heptanone, and 4-nonanone) steadily increased in concentration from weeks 5 to 11, with 3-ethyl-2-pentanone, 4-heptanone, and 4-nonanone appearing as the males progressed from week 5 to week 6. Booth [4] measured androstenone and androstenol concentration in testes of boars at varying ages and found that these two 16-androstene compounds were not abundantly produced until between 18 and 24 weeks, which correlates with the beginning of puberty and spermatogenesis [5].

An abundant body of information exists regarding the effects of male odors on females in rodents and ungulates; however, this effect is not as thoroughly documented in canines [6 – 12]. While sex pheromones have been studied in some canid species, those of the domestic dog remain unidentified. 89

Texas Tech University, Glenna M. Pirner, May 2018

Sex pheromones are anticipated to be volatile as they should be able to carry information to potential mates some distance away; therefore, solid phase microextraction (SPME) is an ideal method to detect these potential pheromone molecules.

SPME provides a reagent-free method of collecting volatile molecules that involves less sample cleanup and preparation compared to previously used methods. In maned wolves, hemiterpenoids, hemiterpenoid alcohols, and pyrazines have been identified as molecules that may be unique to canids but that may differ slightly across species [13, 14]. Volatile organic compounds such as these are of the most interest as potential pheromones.

The objectives of this study are to isolate and identify volatile molecules unique to adult and juvenile male and female dogs, and then to determine if the molecule(s) potentially have pheromonal properties using behavioral and physiological measures in bioassays. We hypothesize that there will be molecules which are dependent on sex hormones that are unique to or in greater concentration in adult males and females compared to juveniles and altered adults.

Methods and Materials

General

Research was approved by Texas Tech University’s Institutional Animal Care and

Use Committee (Protocol 14009-01). Space, management, and husbandry of dogs were consistent with the US Animal Welfare Act.

Animals

Urine samples were collected from owner-surrendered dogs at a municipal shelter in Lubbock, Texas. Owner-surrendered dogs were chosen because the approximate age

Texas Tech University, Glenna M. Pirner, May 2018 and sex status were able to be documented. Urine was collected from five dogs in each of six life stages: juvenile intact male (JIM), adult intact male (AIM), adult castrated male

(AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF). Juvenile was defined as a dog < 6 m of age, with deciduous incisors and non-palpable testicles in males. Adult was defined as between 1 and 8 yr of age, with palpable testicles in males. AIFs were confirmed to be in anestrous by visual observation (no vulvar edema, sanguinous vaginal discharge, or breeding stance observed; [15]) as well as by vaginal smears. Vaginal smears were collected from the caudodorsal aspect of the vagina using a sterile cotton swab moistened with 0.9% sterile saline. The swab was rolled across a glass microscope slide and allowed to air dry, followed by a Wright-Giemsa Three-Step Staining procedure (Fisher Scientific

Company, LLC, Hampton, NH) and finally rinsed with distilled water. Smears were examined using light microscopy at 40x. Cells were identified and ratio between cell types of 100 cells was determined. Anestrus was defined as > 90% parabasal and intermediate cells [16, 17]. Castration and ovariohysterectomy were confirmed by owner records as occurring at least one year prior to date of urine collection.

Urine Collection

Urine was collected via free-catch method into a sterile glass container, transferred into sterile glass tubes with PTFE caps with effort made to minimize air space in the tube, and placed on ice for transport to the laboratory. Samples were stored at -4°C until analysis.

Gas chromatography – mass spectrometry

Texas Tech University, Glenna M. Pirner, May 2018

The procedure used in this study was derived from Mozuraitis et al., [18]. All samples were analyzed within seven days of collection. Urine was thawed to room temperature and vortexed to ensure thorough mixing of contents. 2.0 mL of urine was transferred to a 4 mL sterile headspace vial with a Teflon septum (Supelco, Bellefonte,

PA). The sample was saturated with NaCl and gradually heated to 37°C with constant stirring. A 2 cm 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane

(DVB/CAR/PDMS) SPME fiber was exposed to the headspace of the warmed sample for

30 minutes. Prior to sampling the SPME fiber was conditioned at 270°C for 10 minutes in the GC injector.

The fiber was injected into a splitless injector of a Trace GC Ultra chromatograph fitted with a Restek Rtx-5 column (30 m x 0.25 mm i.d. x 0.25 µm df; Restek, Bellefonte,

PA). The injector temperature was held at 225°C. Helium was used as the carrier gas with column flow at 2.4 mL/min. The temperature program of the oven was maintained at

40°C for 2 min, then increased by 5°C / min to 230°C, and then maintained at 230°C for 10 min. Mass spectra were recorded in electron-impact (EI) mode at 70 eV with a mass range from 40 u to 450 u. Blank runs of the DVB/CAR/PDMS fiber were conducted with the same program and subtracted from the urine sample chromatograms to reduce noise.

Data Analyses

Peak area and identity were determined for all peaks exceeding 15% relative abundance. Peak area was averaged across each group, and the following averaged total ion chromatographs (TIC) were subtracted: 1) AIM – AXM, 2) AIM – JIM, 3) AIM –

AIF, 4) AIF – AXF, 5) AIF – JIF, 6) JIM – JIF.

Texas Tech University, Glenna M. Pirner, May 2018

Significant peaks unique to each life stage were identified using Qual Browser within Xcalibur (Thermo Fisher Scientific Inc., Waltham, MA). To be included in the final analysis, the molecule was required to be present in all individuals in the group for which it was unique.

Data were tested for normality using Shapiro-Wilks in SAS 9.4 (SAS Inst., Inc.,

Cary, NC) and failed to meet this assumption. Data for each individual molecule was ranked and analyzed using Wilcoxon Rank-Sign and Kruskal-Wallis tests in SAS 9.4.

Post-hoc tests for differences between groups were conducted where appropriate.

Differences were considered significant at α = 0.05.

Results

One hundred molecules were isolated and identified across all six groups of dogs.

Of these 100 molecules, six had significant differences between some of the groups.

Nineteen molecules were found in all six groups. Table 1 gives the information for all molecules isolated from the headspace of the urine samples.

Table 5.1. Identity, retention time, and number of dogs having each molecule in headspace of domestic dog urine. Groups are juvenile intact male (JIM), adult intact male

(AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female

(AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

Texas Tech University, Glenna M. Pirner, May 2018

AVG MOLECULE RT AXF AIF JIF AIM AMX JIM Acetaldehyde 2.67 1 1 0 0 0 0

4-(2-amino-1-hydroxyethyl)-1,2-

benzenediol 2.82 1 0 2 0 0 0

1-methylethyl-hydroperoxide 2.95 1 4 3 0 0 0

1,2,4-butanetriol 3.16 0 0 1 0 0 0

5,6-dimethyl-6-hydroxytetrahydro-

1,3-thiazin-2-thione 3.63 1 3 2 0 0 0

2-pentanone 4.62 4 5 3 5 5 1

1-(methylthio)-propane 5.00 5 5 0 5 5 0

3-methyl-1-butanethiol 6.50 0 1 0 5 3 0

Hexanal 6.87 5 5 5 5 5 5

N-ethyl-1,3-dithioisoindoline 7.28 5 5 5 4 4 1

1-methylthio-butane 7.32 0 2 0 1 4 0

1,3-octadiene 7.58 2 1 1 1 4 0

3-methylcyclopentanone 8.22 1 5 1 4 5 0

2-mercapto-3-butanol 8.36 0 0 1 0 0 0 4-heptanone 8.97 1 1 0 5 4 1

Methylthiofuran 9.00 0 0 1 1 0 0

9-oxabicyclo-nonane 9.10 1 1 0 0 0 0

3-methyl-1-(methylthio)-butane 9.16 1 2 0 3 5 0

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MOLECULE AVG AXF AIF JIF AIM AMX JIM RT 2-methyl-6-phenyl-5,6-dihydro-4H-

1,3-oxazine 9.21 0 1 2 0 0 0

2,4-Dithiapentane 9.24 0 1 1 0 0 0

2-heptanone 9.46 3 2 0 5 5 3

5,6-bis(methylidene)-2-norbornen-7-

one 9.47 3 2 0 0 1 3

8-hydroxy-2-octanone 9.67 1 1 0 5 0 0

Heptanal 9.88 0 0 0 4 2 5

3(2)-mercaptopentanal 9.95 0 0 0 1 0 0

Methoxy-phenyl-oxime 9.96 1 0 0 0 3 0

2-bromo-octane 10.79 1 2 3 0 0 0

1,5-dimethyl-6,8-dioxabicyclo-

octane 10.89 0 0 1 0 0 0

3-ethylcyclopentanone 11.42 5 5 3 5 5 2

N-acetylpyrrole 11.55 2 0 0 4 5 2

Benzaldehyde 11.60 2 3 4 4 5 5

1-octen-3-ol 12.16 4 3 2 5 5 4

2-octanone 12.37 2 2 0 0 0 2

2-pentyl-furan 12.56 1 0 0 2 3 1

3-octen-1-ol 12.60 1 0 0 3 5 1

Texas Tech University, Glenna M. Pirner, May 2018

AVG MOLECULE RT AXF AIF JIF AIM AMX JIM Octanal 12.82 3 4 2 5 5 5

2-methyl-propanoic acid, butyl ester 13.16 0 0 0 2 2 5

2-cyano-2,5-dimethylpyrrole 13.28 0 1 0 0 0 0

2-octen-1-ol 13.45 4 4 3 3 4 3

4-cyanocyclohexene 13.48 0 0 0 2 2 0

2-ethyl-1-hexanol 13.57 3 1 3 3 2 5

5,9-dimethyl-1-decanol 13.71 0 2 2 2 4 2

3,5-octadien-2-ol 14.03 1 0 0 4 5 0

1-nitro-hexane 14.35 1 0 0 0 0 0

11-chloro-1-undecene 14.55 5 3 3 3 4 0

2-octenal 14.63 0 1 0 5 5 4

1-phenyl-ethanone 14.77 3 5 2 4 4 2

1-chloro-octane 14.81 1 0 0 0 0 0

2,3-dimethyl-cyclohexanol 15.16 4 3 4 2 1 2

4-methyl-phenol 15.21 0 0 0 0 1 0

3-methyl-phenol 15.22 0 0 0 0 1 0

2,6-dimethyl-cyclohexanol 15.37 2 2 1 5 5 3

2-[2-(p-chlorophenyl)-1-

methylethenyl]-benzimidazole 15.42 2 1 0 0 0 0

3,5-octadien-2-one 15.69 0 0 0 0 2 0

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AVG MOLECULE RT AXF AIF JIF AIM AMX JIM Nonanal 15.79 5 5 5 5 5 5

2,6-dimethyl-7-octen-2-ol 17.40 0 0 0 2 0 2

2-nonenal 17.62 0 0 0 2 1 1

2-Methyl-7-endo-vinylbicyclo-oct-1-

ene 17.62 2 0 1 2 3 0

3-butyl-cyclopentanone 17.69 0 0 0 1 1 0

6-(acetoxy)-4-methyl-4-heptenal 17.85 0 0 2 0 0 0

2-isopropoxyphenol 17.91 0 1 0 0 0 0

1,3,5-undecatriene 18.17 1 0 0 2 2 1

2-methyl-4-decanone 18.20 0 0 0 1 4 0

Decanal 18.69 3 0 1 5 5 5

6-chloro-N-ethyl-1,3,5-triazine-2,4-

diamine 18.74 1 1 0 0 0 0

1-(1,2,2,3-tetramethylcyclopentyl)- 18.86 0 2 3 4 3 5 ethanone

1,1,1,4,4-pentabromobut-3-en-2-ol 18.91 5 5 5 5 5 4

4-t-butyl-3-cyano-6-methyl-2(1H)-

pyridinone 19.29 1 1 2 0 0 0

2,3-dihydro-benzofuran 19.40 0 0 0 3 5 0

(+)-tricycloekasantatal 19.43 0 0 0 1 1 0

Texas Tech University, Glenna M. Pirner, May 2018

AVG MOLECULE RT AXF AIF JIF AIM AMX JIM 2-decanol, methyl ether 19.60 3 0 0 4 2 5

2-Butyl-2,7-octadien-1-ol 19.85 4 4 3 4 3 4

2,2-dimethyl-1-hexanol 20.00 3 0 0 4 2 4

2-methyl-5-(1-methylethenyl)-2-

cyclohexen-1-one 20.11 1 0 0 0 0 0

Nonanoic Acid 20.65 1 0 0 3 4 0

Dihydroedulan II 21.16 1 1 0 3 4 3

2,6,10,10-tetramethyl-1-

oxaspiro[4,5]dec-6-ene 21.48 2 2 3 3 5 3

2-methyl-quinoline 21.78 2 1 0 4 3 1

2,4,4-trimethyl-3-(3-

methylbutyl)cyclohex-2-enone 21.79 1 1 3 0 0 0

2-methoxy-4-vinylphenol 21.89 0 0 0 1 3 0

Decanoic acid, methyl ester 22.05 1 0 0 0 0 0

2,4,4-trimethylcyclohexa-5-en-1-ol 22.60 0 0 0 3 5 0

2,4,4-trimethyl-2-pentene 22.77 0 0 0 1 0 0

2-methyl-propanoic acid, 3-hydroxy-

2,4,4-trimethylpentyl ester 23.27 2 1 2 5 5 4

Menthoxyacetyl chloride 23.29 0 3 1 0 0 0

A-damascenone 23.55 1 1 0 2 5 0

Texas Tech University, Glenna M. Pirner, May 2018

AVG MOLECULE RT AXF AIF JIF AIM AMX JIM 2,4,4,6,6,8,8-heptamethyl-1-nonene 23.72 0 0 0 3 3 0

cis-Jasmone 23.95 1 0 0 5 5 1

3,3-dimethyl-cyclopentanecarboxylic

acid 24.33 0 0 0 5 3 4

4-(2,6,6-trimethyl-1,3-

cyclohexadien-1-yl)-2-butanone 24.52 0 0 0 1 1 1

Isopentyl n-pentyl disulfide 24.63 0 0 0 1 0 0

Ethyl(3-methylbutyl)-propanedioic

acid, diethyl ester 24.75 3 2 4 0 0 0

3-tetradecyn-1-ol 25.08 0 0 0 1 1 2

6,10-dimethyl-5,9-undecadien-2-one 25.27 0 0 0 2 3 3

2-methyl-8-quinolinol 25.31 0 0 0 3 4 0

1-dodecanol 25.90 3 0 0 0 0 3

2-tert-butyl-4-isopropyl-5-

methylphenol 26.56 4 2 2 5 5 3

Dodecanoic acid, methyl ester 26.94 1 0 0 0 0 0

3-(1-methylethenyl)-1-

phenylcyclobuten-1-ol 27.10 0 1 0 3 5 0

2-methyl-propanoic acid, 1-(1,1-

dimethylethyl)-2-methyl-1,3-

propanediyl ester 28.35 4 5 4 5 5 5

Texas Tech University, Glenna M. Pirner, May 2018

Octanal (RT 12.82 min) was present every male dog in all groups but had the greatest peak area in AIM (4.68 x 108 counts; Figure 5.1). Octanal was found only in four

AIF dogs, three AXF dogs, and two JIF dogs. The peak area of octanal in AIM was greater than in AXF and JIF (p = 0.01 and p < 0.01, respectively). Likewise, the peak area of octanal in JIM was greater than in AXF and JIF (p = 0.04 and p = 0.03, respectively).

8.00E+08 a

7.00E+08

6.00E+08

5.00E+08

4.00E+08

3.00E+08

ab a ac 2.00E+08 bc bc 1.00E+08

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05 Figure 5.1. Peak area of octanal in domestic dog urine. Peak area of octanal obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

2-methyl-quinoline (RT 21.88 min) was present in all groups except JIF (Figure

5.2). 2-methyl-quinoline was found in four AIM dogs, 3 AXM dogs, one JIM and one

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AIF dog, and two AXF dogs. The greatest peak areas of 2-methyl-quinoline occurred in

AIM and AXM (4.30 x 108 counts and 3.31 x 108 counts, respectively).

7.00E+08 a ab 6.00E+08

5.00E+08

4.00E+08

3.00E+08

2.00E+08 b b

1.00E+08 b bc

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05 Figure 5.2. Peak area of 2-methyl-quinoline in domestic dog urine. Peak area of 2- methyl-quinoline obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

3-ethylcyclopentanone (RT was present in all groups but had the highest peak area in adult intact and castrated males and females (Figure 5.3). 3-ethylcyclopentanone was present in all individuals in AIM, AXM, and AIF, as well as two JIM dogs, four

AXF dogs, and three JIF dogs. The greatest peak area of 3-ethylcyclopentanone was 1.13 x 109 in AIF dogs.

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1.60E+09 a 1.40E+09 a

1.20E+09 a

1.00E+09 ab

8.00E+08

6.00E+08 ab 4.00E+08 b 2.00E+08

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05

Figure 5.3. Peak area of 3-ethylcyclopentanone in domestic dog urine. Peak area of 3- ethylcyclopentanone obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

2-pentanone was present in all groups with the greatest peak area occurring in

AXF dogs; however, only four of the five AXF dogs’ urine contained 2-pentanone

(Figure 5.4). 2-pentanone was present in all individuals in AIM, AXM, and AIF, as well as one JIM dog, and three JIF dogs.

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1.20E+09 ab 1.00E+09

8.00E+08

a 6.00E+08

PEAKAREA a 4.00E+08 a ab 2.00E+08 b

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05

Figure 5.4. Peak area of 2-pentanone in domestic dog urine. Peak area of 2-pentanone obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

Methyl propyl sulfide was present only in every individual in AIM, AXM, AIF, and AXF groups, but not present in any JIF or JIM individuals (Figure 5.5). Peak area was highest in AIM dogs (3.07 x 109 counts), but this peak area was not different from any of the other adult groups.

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5.00E+09 a 4.50E+09

4.00E+09

3.50E+09 a 3.00E+09 a 2.50E+09 a

2.00E+09

1.50E+09

1.00E+09 b b 5.00E+08

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05

Figure 5.5. Peak area of methyl propyl sulfide in domestic dog urine. Peak area of methyl propyl sulfide obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

2-heptanone was present in all groups except JIF, with the highest peak area in

AIM dogs (9.76 x 108 counts; Figure 5.6). All five individuals in the AIM and AXM groups had 2-pentanone in the urine, as well as three JIM dogs, two AIF dogs, and three

AXF dogs.

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1.40E+09

1.20E+09 a

1.00E+09

8.00E+08

6.00E+08

4.00E+08 ab a a ab 2.00E+08 b

0.00E+00 AIM AXM JIM AIF AXF JIF

a,b,c Least Squares means differ, p < 0.05

Figure 5.6. Peak area of 2-heptanone in domestic dog urine. Peak area of 2-heptanone obtained via solid-phase microextraction (SPME) in juvenile intact male (JIM), adult intact male (AIM), adult castrated male (AXM), juvenile intact female (JIF), adult intact female (AIF), and adult ovariohysterectomized female (AXF) domestic dogs. n = 5 dogs per group.

Discussion

Octanal has previously been identified in both male and female African Wild Dog urine and feces, and in greater abundance in male urine than in female urine [19, 20].

These findings are consistent with the findings of the present study, in which urine of both sexes of all life stages contained octanal. In female coyotes, octanal has been observed to increase in concentration in female urine during the estrous season, suggesting a potential female sex attractant pheromone role [21]. Females in the present

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Texas Tech University, Glenna M. Pirner, May 2018 study were confirmed to be in anestrus, which may explain the contradictory results with those of Schultz et al., [21]. Goodwin et al., [22] proposed that methyl-p hydroxy benzoate, which was present only in vaginal secretions of bitches in estrus, was a female sex attractant pheromone. This finding has since been refuted, possibly due to methodology inconsistencies [23, 24]. Methyl paraben was not found in any of the anestrus female urine samples in the present study, which is consistent with the findings of Dzięcioł et al., [24]. Octanal has also been described as an aggregation pheromone in several species of insects and is present in the interdigital secretions of antelopes [25 -

27].

Wolfram [28] identified 2-methylquinoline in the urine of male and female red wolves, gray wolves, wolf-dog hybrids, and domestic dogs; however, the abundance of the molecule in each sex or species was not provided. In the present study, 2- methylquinoline is significantly higher in adult males compared to juvenile males and all female groups. Similarly, 2-methylquinoline is unique to male ferret urine [29]. 2- methylquinoline has also been identified in deermouse urine, in which it appears to be unique to adult mice and completely absent in juveniles; it also has a higher concentration in males than in females [30]. The presence of this molecule across species and its prevalence in adult males suggests 2-methylquinoline is a sex hormone-related molecule which possibly plays a role in male-specific behavior or mate attraction.

In the present study, 3-ethylcyclopentanone appears to be associated with post- pubertal males and females. Interestingly, the peak area of the molecule increases in

AXM compared to AIM, but decreases in AXF compared to AIF. 3-ethylcyclopentanone

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Texas Tech University, Glenna M. Pirner, May 2018 has been identified in the urine of female wolves and male domestic dogs. In wolves, 3- ethylcyclopentanone is elevated in castrated males, consistent with the findings of the present study [28, 31]. 3-ethylcyclopentanone is also found in the urine of subordinate male white tail deer during the breeding season [32]. Initially, it appears that 3- ethylcyclopentanone is sex-hormone dependent; additionally, the observed higher concentrations in females, subordinate males, and castrated males would suggest it is an estrogen-dependent molecule.

2-pentanone acts as an alarm pheromone in ants and an aggregation pheromone in grain borers [33, 34]. 2-pentanone is also found in relatively equal concentrations in male, female, castrated, and juvenile deermice [30]. Similarly, 2-pentanone does not appear to be strongly associated with either sex or any life stage in domestic dogs. It does not appear that 2-pentanone is sex hormone-dependent.

Methyl propyl sulfide, also referred to as methyl propyl sulfide, was reported as a major constituent of female Beagle urine which increased during the time of estrus [35].

In wolves, methyl propyl sulfide increased in abundance in intact and castrated male and ovariohysterectomized female urine after treatment with exogenous testosterone [31].

Methyl propyl sulfide is likely dependent on testosterone, which is consistent with the absence of the molecule in juvenile domestic dogs and the patterns observed in adult males and females.

2-heptanone is found in a wide array of mammals and insects, including female

African elephants, pine voles, antelopes, Bengal tigers, mice, ants, and honey bees [27,

36 – 41]. 2-heptanone is also a common constituent of canid urine, including gray and red

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Texas Tech University, Glenna M. Pirner, May 2018 wolves, domestic dogs, and African Wild dogs [20, 28]. When wolves were treated with exogenous testosterone, 2-heptanone increased in abundance in the urine of intact adults; however, the same effect was not observed in ovariohysterectomized females or castrated males [31]. Likewise, 2-heptanone is found in increased concentration in male white tail deer during breeding season compared to non-breeding season [32]. In combination with other volatile ketones, 2-heptanone is one of the primary pheromones responsible for puberty acceleration and estrus extension in female mice [39]. Combined evidence suggests that this molecule is responsible for male reproductive behavior and influences on the female during breeding.

Of the six molecules found to have different peak areas between sex and life stage, only 2-pentanone did not support the hypothesis that unique molecules would be sex hormone-dependent. This is especially true of 2-heptanone and methyl propyl sulfide, which have been found to increase in concentration after administration of exogenous testosterone. Interestingly, no molecules had significantly higher peak areas in intact adult females; this may be related to the fact that females in this study were in anestrus.

Schultz et al., [35] found methyl propyl sulfide increased during the estrus phase, but two compounds in this analysis remained unidentified. Advances in headspace sampling and

GC-MS techniques over the past thirty years warrant a new look at urine and vaginal secretions from females in each phase of the estrous cycle. Additionally, future research should address the behavioral and physiological effects of unique molecules on conspecifics in order to determine the biological activity of such molecules.

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Conclusions

Of the 100 molecules found in the headspace of the urine samples, only six molecules met the criteria of having higher peak areas in one or more groups, and being present in every individual in the group for which the significant difference existed.

Octanal, 2-methyl-quinoline, methyl propyl sulfide, and 2-heptanone appear to be closely linked to male sex hormones as they had higher peak areas in intact adult males. 3- ethylcyclopentanone appears to be associated with intact adult females and castrated or subordinate male animals. No sex or life stage patterns could be divulged from the data on 2-pentanone; therefore, it is likely that if this molecule has biological activity in dogs, it is not related to reproduction. Future research should address changes in the female across the phases of the estrous cycle, as well as determining physiological and behavioral effects of putative pheromones on conspecific animals.

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[4] Booth, W. D. 1975. Changes with age in the occurrence of C19 steroids in the testis and submaxillary gland of the boar. J. Reprod. Fert. 42: 459-472.

[5] Merck Veterinary Manual. 2015. Boar Management. Available: https://www.merckvetmanual.com/management-and-nutrition/management-of- reproduction-pigs/boar-management. Accessed 1 February 2018.

[6] Lee, S., van der Boot, L.M. 1955. Spontaneous pseudopregnancy in mice. Acta Phys Pharmacol. Neerl 4: 442-443.

[7] Whitten, W.K. 1956. Modification of the oestrus cycle of the mouse by external stimuli associated with the male. J. Endocrinol. 13: 399-404.

[8] Bruce, H.M. 1959. An exteroceptive block to pregnancy in the mouse. Nature 184: 105.

[9] Vandenbergh, J.G. 1967. Effect of the presence of a male on the sexual maturation of female mice. Endocrinology 81: 345-349.

[10] Cohen-Tannoudji, J., Lavenet, C., Locatelli, A., Tillet, Y., & Signoret, J. P. 1989. Non-involvement of the accessory olfactory system in the LH response of anoestrous ewes to male odour. J. Reprod. Fert. 86: 135-144.

[11] Gelez, H., & Fabre-Nys, C. 2004. The “male effect” in sheep and goats: a review of the respective roles of the two olfactory systems. Horm. Behav. 46: 257-271.

[12] Murata, K., Wakabayashi, Y., Kitago, M., Ohara, H., Watanabe, H., Tamogami, S., Okamura, H. 2009. Modulation of Gonadotrophin‐Releasing Hormone Pulse Generator Activity by the Pheromone in Small Ruminants. J. Neuroendocrin. 21: 346-350.

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[13] Kester, M. E., Freeman, E. W., Songsasen, N., & Huff, T. B. 2016. Automated headspace solid-phase microextraction of urinary VOCs from eleven maned wolves (Chrysocyon brachyurus): a recursive workflow for GC–MS analysis. In Chemical Signals in Vertebrates 13 (pp. 477-498). Springer, Cham.

[14] Goodwin, T.E., Songsasen, N., Broederdorf, L.J., Burkert, B.A., Chen, C.J., Jackson, S.R., Keplinger, K.B., Rountree, M.E., Waldrip, Z.J., Weddell, M.E. and Desrochers, L.P., 2013. Hemiterpenoids and pyrazines in the odoriferous urine of the maned wolf (Chrysocyon brachyurus). In Chemical Signals in Vertebrates 12 (pp. 171-184). Springer, New York, NY.

[15] Jöchle, Wolfgang, and Allen C. Andersen. 1977. The estrous cycle in the dog: a review. Theriogenology 7: 113-140.

[16] Aydin, I., Sur, E., Ozaydin, T., Dinc, D.A. 2011. Determination of the Stages of the Sexual Cycle of the Bitch by Direct Examination. Journal of Animal and Veterinary Advances 10: 1962-1967.

[17] Feldman, E.C., Nelson, R.W. 1996. Canine and Feline Endocrinology and Reproduction. 2nd Edition, W.B. Sounders Company, Philadelphia, USA. Pp. 526- 546.

[18] Mozūraitis, R., Būda, V., & Borg-Karlson, A. K. 2010. Optimization of solid- phase microextraction sampling for analysis of volatile compounds emitted from oestrous urine of mares. Zeitschrift für Naturforschung C, 65: 127-133.

[19] Apps, P., Mmualefe, L., McNutt, J.W. 2013. A reverse-engineering approach to identifying which compounds to bioassay for signaling activity in the scent marks of African wild dogs (Lycaon pictus). In: Chemical Signals in Vertebrates 12. Springer, New York, N.Y. pp 417-432.

[20] Parker, M. N. 2010. Territoriality and scent marking behavior of African wild dogs in northern Botswana. Master’s Thesis. University of Montana.

[21] Schultz, T. H., Flath, R. A., Stern, D. J., Mon, T. R., Teranishi, R., Kruse, S. M., & Howard, W. E. 1988. Coyote estrous urine volatiles. Journal of Chemical Ecology, 14: 701-712.

[22] Goodwin, M., Gooding, K.M., Regnier, F. 1979. Sex pheromone in the dog. Science 203: 559-561.

[23] Kruse, S.M., Howard, W.E. 1983. Canid sex attractant studies. Journal of Chemical Ecology 9: 1503-1510. 111

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[24] Dzięcioł, M., Politowicz, J., Szumny, A., Nizanski, W. 2015. Methyl paraben as a sex pheromone in canine urine – is the question still open? Polish Journal of Veterinary Sciences 17: 601-605.

[25] Torto, B., Obeng-Ofori, D., Njagi, P. G., Hassanali, A., & Amiani, H. 1994. Aggregation pheromone system of adult gregarious desert locust Schistocerca gregaria (Forskal). Journal of Chemical Ecology, 20: 1749-1762.

[26] Siljander, E., Gries, R., Khaskin, G., & Gries, G. 2008. Identification of the airborne aggregation pheromone of the common bed bug, Cimex lectularius. Journal of Chemical Ecology, 34: 708.

[27] Jumean, Z., Gries, R., Unruh, T., Rowland, E., & Gries, G. 2005. Identification of the larval aggregation pheromone of codling moth, Cydia pomonella. Journal of Chemical Ecology, 31: 911-924.

[28] Wolfram, W. 2013. Scent-marking: Investigating chemosensory signals in wolf urine. University of Exeter. Masters Thesis.

[29] Zhang, J. X., Soini, H. A., Bruce, K. E., Wiesler, D., Woodley, S. K., Baum, M. J., & Novotny, M. V. 2005) Putative chemosignals of the ferret (Mustela furo) associated with individual and gender recognition. Chemical Senses, 30: 727-737.

[30] Ma, W., Miao, Z., Novotny, M.V. 1998. Role of the adrenal gland and adrenal- mediated chemosignals in suppression of estrus in the house mouse: the Lee-Boot effect revisited. Biol. Reprod. 59: 1317-1320.

[31] Raymer, J., Wiesler, D., Novotny, M., Asa, C., Seal, U. S., & Mech, L. D. 1986. Chemical scent constituents in urine of wolf (Canis lupus) and their dependence on reproductive hormones. Journal of Chemical Ecology, 12: 297-314.

[32] Miller, K. V., Jemiolo, B., Gassett, J. W., Jelinek, I., Wiesler, D., & Novotny, M. 1998. Putative chemical signals from white-tailed deer (Odocoileus virginianus): social and seasonal effects on urinary volatile excretion in males. Journal of Chemical Ecology, 24: 673-683.

[33] Moser, J. C., Brownlee, R. C., & Silverstein, R. 1968. Alarm pheromones of the ant Atta texana. Journal of Insect Physiology, 14: 529-535.

[34] Williams, H. J., Silverstein, R. M., Burkholder, W. E., & Khorramshahi, A. 1981. Dominicalure 1 and 2: Components of aggregation pheromone from male lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). Journal of Chemical Ecology, 7: 759-780.

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[35] Schultz, T. H., Kruse, S. M., & Flath, R. A. 1985. Some volatile constituents of female dog urine. Journal of chemical ecology, 11: 169-175.

[36] Goodwin, T. E., Rasmussen, L. E. L., Schulte, B. A., Brown, P. A., Davis, B. L., Dill, W. M., Loizi, H. (2005). Chemical analysis of preovulatory female African elephant urine: a search for putative pheromones. In Chemical Signals in Vertebrates 10 (pp. 128-139). Springer, Boston, MA.

[37] Boyer, M. L., Jemiolo, B., Andreolini, F., Wiesler, D., & Novotny, M. 1989. Urinary volatile profiles of pine vole, Microtus pinetorum, and their endocrine dependency. Journal of chemical ecology, 15: 649-662.

[38] Soso, S. B., & Koziel, J. A. 2016. Analysis of odorants in marking fluid of Siberian tiger (Panthera tigris altaica) using simultaneous sensory and chemical analysis with headspace solid-phase microextraction and multidimensional gas chromatography-mass spectrometry-olfactometry. Molecules, 21: 834.

[39] Jemiolo, B., Gubernick, D. J., Yoder, M. C., & Novotny, M. 1994. Chemical characterization of urinary volatile compounds of Peromyscus californicus, a monogamous biparental rodent. Journal of Chemical Ecology, 20: 2489-2500.

[40] Riley, R. G., Silverstein, R. M., & Moser, J. C. 1974. Biological responses of Atta texana to its alarm pheromone and the enantiomer of the pheromone. Science, 183: 760-762.

[41] Shearer, D. A., & Boch, R. 1965) 2-Heptanone in the mandibular gland secretion of the honey-bee. Nature, 206: 530.

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CHAPTER VI

EVALUATION OF PHEROMONE-INDUCED ACTIVATION IN THE HUMAN

BRAIN USING FUNCTIONAL MAGNETIC RESONANCE IMAGING (FMRI)

Abstract

Human pheromones have been the subject of debate for decades. Although humans lack a functional vomeronasal organ there is evidence that some androgenic compounds, namely 5α-androst-16-en-3-one and androsta-4,16-dien-3-one, elicit behavioral and physiological effects in women, especially during the time of ovulation.

This study utilized functional magnetic resonance imaging (fMRI) and blood-oxygen- level dependent (BOLD) contrast to determine which regions of the brain were activated by the putative pheromones 5α-androst-16-en-3-one (ANDRO) and 2-methylbut-2-enal

(2M2B). Ten women between the ages of 18 and 30 received an fMRI scan in a 3 T machine with ANDRO, 2M2B, rose odor (ROSE), and fresh air (CON) in a 15 s on / 45 s off block design, with three randomized repetitions. No significant activations were noted with ROSE. ANDRO activated the left insular region compared to CON (p = 0.04).

2M2B elicited activation in the somatosensory association cortex (p < 0.01), premotor cortex (p < 0.01), and Brodmann’s area 8 (p = 0.03) compared to CON. 2M2B also elicited activation in the posterior cingulate and angular gyri compared to ROSE (p <

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Introduction

Chemical communication in the form of pheromones has proven to be essential for survival of nearly all terrestrial vertebrates; however, there is much debate over the importance of chemical communication in humans. Humans consciously rely on auditory and visual communication in social encounters, but there may be an olfactory component to these interactions, especially in close proximity.

The vomeronasal organ (VNO) is a structure located at the base of the nasal septum that is physically separated from the nasal cavity and main olfactory epithelium.

The VNO has been determined to be the primary region of pheromone detection in vertebrates. When a molecule binds the chemoreceptors in the lumen of the VNO, neural signals are sent to the accessory olfactory bulb in the brain, which communicates appropriately with the amygdala, orbitofrontal cortex, and the hippocampus [1, 2].

The presence of a functional VNO in humans is a subject of much debate. Early studies suggest that the human VNO is functional only in infants and then becomes vestigial as the human advances into adulthood [3, 4]. More recently, studies have indicated that adults do possess a VNO that is capable of detecting picogram quantities of putative pheromones, with potentials recorded in the VNO epithelium that have similar properties to summated receptor potentials in other organs [5]. Additional support of the existence of this specialized olfactory organ in humans is the discovery of a pheromone receptor gene (V1RL1) in the olfactory mucosa of humans [6].

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Putative pheromones in humans include 5α-16-androsten-3α-ol (androstenol) and

5-α-androst-16en-3α-one (androstenone). These compounds are sexual pheromones produced in boar saliva but are also produced from apocrine glands in the axillary region of both adult men and women; concentrations in male sweat are approximately 20x higher than in female sweat [7]. While androstenol does not grossly affect social interactions between men, women exposed to androstenol reported an increase in number, duration, and depth of conversations with men [8]. Savic et al., [9] examined neural activation of women in response to androstenone using positron emission tomography (PET) and found that women had activation of the hypothalamus, preoptic, and ventromedial nuclei in response to the androgen.

PET scans are not desirable for this type of study because they employ radioactive isotopes and collect data using 60 – 90 second scans. This large time frame is not useful for studies examining discrete odor exposure [10]. Functional magnetic resonance imaging (fMRI) measures blood oxygen level dependent (BOLD) changes in regions of the brain based on the paradigm that blood flow increases to a region when neural activity increases [11]. These scans can be taken at a much faster rate than is available with PET.

The objective of this study was to use fMRI to quantify differences in regional brain activation elicited by a putative pheromone, androstenone, in adult women.

Investigation of brain activation by 2M2B is a novel concept, and so no hypotheses could be formulated for this odorant. The authors hypothesized that ANDRO would elicit

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Methods and Materials

General

All research was conducted after approval of the Texas Tech University Human

Research Protection Program. Research was conducted at the Texas Tech Neuroimaging

Institute.

Participants

Ten women between the ages of 18 and 40 years were recruited from the student population at Texas Tech University. Participants were self-reported as healthy, having no upper respiratory disease or disorder, not using any type of contraceptive (oral, implant, etc.) and had regular, predictable menstrual cycles.

Olfactometer

A custom-built olfactometer was used to deliver odors to the participants (Figures

6.1 and 6.2). The olfactometer consisted of a three-sided box (35.6 cm H x 40.6 cm L x

55.9 cm W) constructed of 0.64 cm thick particle board. Air intake was facilitated by an air pump (EcoPlus 3000 LPH Commercial Air Pump). Air passed through a drying filter, a particulate filter, a charcoal filter, and a micro-particulate filter via 6.35 mm O.D. x

4.77 mm I.D. polyethylene tubing (Industrial Specialties Mfg. Co., Englewood, CO).

After filtering, the tubing was split into two lines and passed through flow meters (Dwyer 117

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Instruments, Michigan City, IN). A carrier line held at 1.5 L/min air flow was used to maintain a constant air pressure delivery in order to reduce participant awareness of condition changes. The second line, maintained at 250 mL/min air flow was connected to a three-way solenoid valve. In the baseline condition, air passed directly to the odorant manifold. In the odorant conditions, air was directed through one of four two-way solenoid valves, each connected to a respective valve on the odorant manifold. These valves were controlled by a computer program (described in Experimental Design).

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Figure 6.1. Diagram of functional magnetic resonance imaging facility and

olfactometer set up.

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Figure 6.2. Olfactometer designed for this study.

From the olfactometer, the four odorant air supply lines and the carrier line extended 7.92 m into the MRI room. Each of the four odorant air supply lines were connected via nylon fittings to the odorant jars. Jars were 500 mL glass PTFE-lined, with

PTFE-lined lids (Thomas Sci., Swedesboro, NJ). Nylon fittings were made air-tight in the lid by PTFE O-rings and Teflon tape. An 11.43 cm piece of tubing extended into the odorant jar, ending approximately 3.81 cm above the odorant solution (described in

Odorants). This served to pass air across the solution. Odorized air then passed out of the jar through a second fitting connected to 6.35 mm O.D. x 4.77 mm I.D. FEP Wall

VersilonTM DualityTM Tubing (US Plastics, Lima, OH). From the jar, odorized air passed

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12.70 cm to the odorant manifold and then 0.91 m to the MRI table. A one-way valve was placed at the exit end of each junction to prevent retrograde air flow.

A Nylon fitting was used to connect the FEP tubing to 1.22 m of soft PVC tubing, with a nasal cannula in the center. This setup allowed the tubing to be placed behind the participants’ ears with the cannula firmly held in the nostrils, as in an oxygen supply line.

A new nasal cannula was used for each participant. On the opposite side of the participant from the odorant supply, the soft PVC tubing was connected to a 7.92 m length of FEP tubing, which returned to the olfactometer box. Respirations were monitored by a pressure sensor (Digi-Key Electronics, Thief River Falls, MN) to ensure participants were not actively sniffing during odor delivery.

Odorants

Three odorant conditions and a negative control were used in this study. Each odorant was mixed with 10 mL of mineral oil, which also served as the negative control

(CON). 2-phenylethanol (rose odor; ROSE) was prepared at 0.5% concentration and served as a positive control. The two putative pheromones in this study were 5-α-androst-

16en-3α-one (ANDRO), prepared at 20 mM, and 2-methylbut-2-enal (2M2B), prepared at 0.1 % concentration. Concentrations were determined based upon literature review and preliminary testing.

Experimental Design

Odorants were delivered to each participant using a block design (Figure 6.3).

Delivery times were determined based on review of previous block design trials

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45 s of baseline (i.e. air from carrier line only), followed by 15 s of a randomly selected odorant, and followed again by 45 s of baseline. This pattern repeated until all four odorants were delivered, and then the program was repeated twice more. Essentially, each participant experienced each odor three times, with each set of four odorants delivered in random order.

Figure 6.3. Block design representing baseline and odorant delivery schedule.

Scanning Parameters

Data were collected on a Siemens 3 Tesla SKYRA Magnetic Resonance Imaging machine (Siemens, Germany) with a 20-channel head coil. High-resolution MPRAGE anatomical scans were acquired in the sagittal plane using the following parameter settings: repetition time (TR) = 1900 ms, echo time (TE) = 2.49 ms, flip angle (FA) =

90°, field of view (FOV) = 256°, matrix = 256 x 256, slice thickness = 1 mm, number of slices = 192. Functional runs used Siemen’s product echo planar imaging (EPI) sequence with the following parameter settings: TR = 2040 ms, TE = 25 ms, FA = 70°, FOV =

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192°, matrix = 64 x 64, number of slices = 41, slice thickness = 2.5 mm with 0.5 mm gap.

Slices were acquired in ascending order in the axial plane and oriented approximately 30° off of the anterior commissure/posterior commissure line to reduce orbital dropout [13].

Data Analysis

FMRIB’s Software Library (FSL; Oxford University, UK) and Freesurfer were used for preprocessing. Functional images were converted from DICOM to NIFTI.

Motion correction was applied to all functional images with a 6-DOF rigid body alignment of each functional volume to the center volume of the respective run. Skull stripping was accomplished using the Brain Extraction tool in the FSL software package.

All images were spatially smoothed with a six mm Gaussian kernel as well as a 100 s cut- off high pass filter.

Processing of fMRI data was conducted using the fMRI Expert Analysis Tool

(FEAT) Version 6.0 (FSL; Oxford University, UK). Higher-level analyses were carried out using FMRIB’s Local Analysis of Mixed Effects (FLAME) stages 1 and 2 [14 – 16].

The following contrasts were made: ANDRO – CON, 2M2B – CON, ROSE – CON,

ANDRO – 2M2B, ANDRO – ROSE, and 2M2B – ROSE. Z (Gaussianised T/F) statistic images were thresholded using clusters determined by Z > 2.3 and a corrected cluster significance threshold of p ≤ 0.05 [17].

MNI coordinates provided in the FLAME analyses were entered into the Yale

BioImage Suite Package (BioImage Suite, Xenios Papademetris, Yale University, 2014).

Brodmann’s area definitions in this package are derived from [18].

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Results

No significant differences in activation were found for the contrasts of ROSE –

CON, ANDRO – ROSE, or ANDRO – 2M2B. Areas of activation for remaining contrasts are shown in Table 6.1.

Table 6.1. Regions of brain activation for functional magnetic resonance imaging contrasts. Contrasts are androstenone – control (ANDRO – CON), 2-methylbut-2-enal –

control (2M2B – CON), and 2-methylbut-2-enal – rose odor (2M2B – ROSE). If no p-

value is given, p > 0.05. n = 10 participants.

MNI Voxels Z- P-value Region Left/Right coordinates (#) value (corrected) ANDRO - CON

Insula L -40 10 -2 494 3.95 0.04

Amygdala L -24 -2 -14 ___ 3.43 ___

Inf. Frontal Gyrus L -26 6 -16 ___ 3.19 ___

Temporal Lobe L -40 12 -12 ___ 3.03 ___

2M2B – CON

Cerebellum --- 28 -82 -14 4361 4.23 < 0.01

Somatosensory Assoc. L -34 -50 48 4262 4.16 < 0.01

Cortex

Premotor/Supplementary L -20 -10 64 696 3.34 < 0.01

Motor Cortex

BA 8 L -46 18 36 472 3.48 0.03

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MNI Voxels Z- P-value Region Left/Right coordinates (#) value (corrected) Visual Assoc. R 12 -92 -6 ___ 4.16 ___

Somatosensory Assoc. R 36 -54 54 ___ 3.78 ___

Cortex

Premotor/Supplementary R 26 6 62 ___ 2.99 ___

Motor Cortex

Sup. Frontal Gyrus R 38 32 38 ___ 3.18 ___

Sup. Frontal Gyrus L -30 34 42 ___ 2.79 ___

BA 44 L -52 14 24 ___ 2.75 ___

2M2B - ROSE

Post. Cingulate Gyrus R 4 -32 42 2099 4.40 < 0.01

Angular Gyrus L -32 -54 44 1007 4.38 < 0.01

Cerebellum ___ 10 -78 -18 619 4.09 0.03

In the ANDRO – CON contrast 494 voxels in the left insula (MNI X = -40, Y =

10, Z = -2) showed greater activation in the ANDRO condition (p = 0.04). Other near- significant activations included the left amygdala, left inferior frontal gyrus (Brodmann’s area 47), and a section of the temporal lobe (Brodmann’s area 38). The contrast map for

ANDRO – CON is shown in Figure 6.4.

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Figure 6.4. Contrast map for ANDRO – CON. Areas of activation elicited by ANDRO

are shown in red/yellow.

Four regions showed significant activation in the 2M2B condition compared to the CON condition (Figure 6.5). These included 4361 voxels in the cerebellum (X = 28,

Y = -82, Z = -14; p < 0.01), 4262 voxels in the left somatosensory association cortex (X =

-34, Y = -50, Z = 48; p < 0.01), 696 voxels in the left premotor/supplementary motor cortex (X = -20, Y = -10, Z = 64, p < 0.01), and 472 voxels in the left Brodmann’s area 8

(X = -46, Y = 18, Z = 36, p = 0.03). There were a number of near-significant activations 126

Texas Tech University, Glenna M. Pirner, May 2018 associated with each cluster, including the right visual association cortex, and the left and right superior frontal gyrus.

Figure 6.5. Contrast map for 2M2B – CON. Areas of activation elicited by 2M2B are

shown in red/yellow.

Additionally, three regions showed significant activation in the 2M2B condition compared to the ROSE condition (Figure 6.6). These included 2099 voxels in the posterior cingulate gyrus (X = 4, Y = -32, Z = 42; p < 0.01), 1007 voxels in the angular

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Texas Tech University, Glenna M. Pirner, May 2018 gyrus (X = -32, Y = -54, Z = 44; p < 0.01), and 619 voxels in the cerebellum (X = 1-, Y =

-78, Z = -18; p = 0.03).

Figure 6.6. Contrast map for 2M2B – ROSE. Areas of activation elicited by 2M2B are

shown in red/yellow.

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Discussion

Sniffing behavior was not noted by any of the participants during this study. The left insula has been implicated in several studies examining the effects of various odorants on the brain [19, 20]. Savic et al., [21] concluded that the insula appears to be a crucial part of the olfactory input processing sequence. Given this conclusion, it is interesting that the insula did not show significant activation in the 2M2B – CON condition. It is currently unknown if 2M2B has biological activity in humans; however, regardless of biological activity the molecule does have a perceivable odor and should, according to the above conclusion, activate the insula. The brain is a vastly complex organ and the function of every region is not completely understood. Thus, it is possible that some undiscovered aspect of the insula results in its activation with ANDRO and not

2M2B.

Activation of the amygdala by ANDRO is in partial agreement with Savic et al.,

[21]. Women exposed to 4,16-androstadien-3-one showed non-significant right amygdala activation. In female mice exposed to urine from males, females, and cats, only male urinary volatiles induced Fos activity in the amygdala-projecting cells of the main olfactory bulb [22]. This direct pathway between the main olfactory bulb and the amygdala suggests that attractant pheromones of the opposite sex may be able to directly control reproductive behaviors [22]. The findings of this study, combined with those of

Kang, corroborate the idea that ANDRO acts as a male attractant pheromone [23].

Additionally, the amygdala plays a role in olfactory associative learning and hedonic processing of odors [24, 25]. The participants in this study were not asked about their

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In the 2M2B – CON contrast, activation of Brodmann’s areas 6, 7, and 8 are all associated with motor control. Brodmann’s area 7 has not been implicated in any olfactory processing study. Brodmann’s areas 6 and 8 were activated in response to both pleasant and unpleasant odors; however, only area 8 showed activation in response to the pleasant odor. These areas are suspected to mediate motor responses to odors. [26]. The right superior frontal gyrus (Brodmann’s area 9) plays a role in working memory, which may be its most likely implication in the current study [27].

Activation of the posterior cingulate gyrus in olfactory processing studies has proven to be inconsistent. Some olfactory tasks yield positive correlations between activation of the posterior cingulate gyrus, while others yield negative correlations [28].

Body odor is generally processed by the posterior cingulate cortex; thus it is illogical that

2M2B should activate this region and ANDRO did not [29, 30].

The findings of the present study partially support the hypothesis that ANDRO would elicit activation in the olfactory pathway; these include the insula and amygdala.

Activation of the olfactory bulb was likely missed because the proximity of the olfactory bulb to the skull resulted in its unintentional removal during skull stripping. The reason that hypothalamic activity was not observed is unclear; the most likely explanation being that the concentration of ANDRO used in this study may not have matched physiological concentrations. The direct odor delivery system in this study limited the concentration range for each odorant: low concentrations were unperceivable and high concentrations 130

Texas Tech University, Glenna M. Pirner, May 2018 were found to be aversive in pilot studies. Future research should take androstenone anosmia, sexual experience, and hedonic value of each odorant into consideration.

Conclusions

Androstenone elicited activation in the left insula and left amygdala, both regions which are associated with olfactory processing. 2-methylbut-2-enal elicited activation mainly in motor processing regions, such as Brodmann’s areas 6, 7, and 8. Evidence that either molecule activated the hypothalamus, as would be expected by a priming pheromone, was not observed. In future work consideration should be given to the physiological concentration of the odorant, which perhaps warrants a different odorant delivery system. Additionally, anosmia, sexual experience, and hedonic value of the odorants should be factored into the conclusions.

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CHAPTER VII

CONCLUSION

With millions of pet dogs in the United States alone, it is surprising how little effort has been placed into identifying and describing pheromones in domestic dogs. This knowledge can provide an understanding of the physiological basis for behavior, which will be of vast importance for owners and dogs alike. Not only can knowledge about chemical communication improve animal welfare, it can be of benefit to trainers and handlers of working dogs.

Rabbit maternal pheromone, 2-methylbut-2-enal, modifies the heart rate and behavior responses of chronically stressed dogs exposed to an acute stressor. Dogs may experience chronic stress as a result of illness or injury, as well as environmental factors.

For these animals, acute stress has a more detrimental effect than in non-stressed conspecifics. Alleviating the impact of acute stressors such as thunderstorms and car travel may vastly improve the well-being of these animals. Future research should investigate other applications of this technology, such as veterinary visits and environmental changes. Additionally, the neurological effects of the interomone should be determined to fully understand the mechanism by which 2-methylbut-2-enal influences the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal- medullary axis.

Octanal, 2-methylquinoline, methyl propyl sulfide, and 2-heptanone, which had significantly higher peak areas in intact adult male dogs, each have pheromonal properties in other species. This strongly suggests that one or more of these molecules

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Texas Tech University, Glenna M. Pirner, May 2018 may have biological activity in domestic dogs. Behavioral and physiological assays are currently underway to investigate the potential function of these molecules. It is not surprising that intact adult females had few unique molecules in the urine. Future research should address changes in the female across the phases of the estrous cycle, as attractant pheromones are likely only present during the times of proestrus and estrus.

Beyond urinary pheromones other biological fluids should be assayed, such as vaginal secretions, anal gland secretions, and saliva.

In regards to putative human pheromones, the fMRI study reported here suggests that androstenone does activate regions associated with olfactory processing. Because it is unknown if 2-methylbut-2-enal has any biological activity in humans, the finding that it elicits activity in the motor processing regions cannot be interpreted at this time. It is likely that many factors affect the way in which these molecules are processed neurologically, such as previous exposure to and the potential of a conditioned response to the molecules. The ability now exists to conduct fMRI experiments in domestic dogs; as such, the potential for conducting a similar study using 2M2B and other putative canine pheromones is extremely appealing for the future.

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