Self-Enurination in the Domesticated Male Goat (Capra Hircus)

SELF-ENURINATION IN THE DOMESTICATED MALE GOAT (CAPRA HIRCUS)

WILLIAM F. FRITZ JR.

A dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

Graduate Program in Endocrinology and Animal Biosciences

Written under the direction of

Dr. Larry S. Katz

And approved by

______

New Brunswick, New Jersey

May 2017

ABSTRACT OF THE DISSERTATION

SELF-ENURINATION IN THE DOMESTICATED MALE GOAT (CAPRA HIRCUS)

By: WILLIAM F. FRITZ JR.

Dissertation Director:

Dr. Larry S. Katz

The sexual behavior of the female goat has been researched extensively to solve reproductive efficiency issues in the agricultural setting. Traditionally, the sexual behavior of the male goat has not been as well understood. The male goat is often dismissed for having an insatiable sexual appetite and thus not a problem worthy of inquiry. More recently, studies on low-performing males in closely related domesticated species have opened the door for the study of sexual behavior in male ruminants. Among the less understood behaviors of the male ruminant, the male goat is known to display a behavior known as self-enurination. Self-enurination is displayed in male domesticated goats characterized by the downward turning of the head and neck to intercept the stream of urine from the erect penis resulting in the application of urine to the face, beard and posterior surface of the forelegs. While many hypotheses on the etiology of such a behavior have been proposed, none have been demonstrated through experimentation.

While the behavior appears to be involved in olfactory communication, it is not clear whether the behavior is directed at females or other males of the same species. Knowing that the behavior is testosterone-dependent, displayed much more frequently during the breeding season and evoked by sexual stimuli, suggests a reproductive function. On the

contrary, the behavior is elicited in the absence of females, so it is also believed to have social implications. By developing a set of social and physical environmental conditions capable of reliably producing bouts of self-enurination, hypotheses directed at understanding the behavior’s purpose have been tested. Findings are relevant to the production industry in the development of natural and less invasive breeding practices and also provide a better understanding of goat sexual behavior necessary to improve husbandry techniques. Also, this work contributes to the field of chemical communication through the study of a domesticated species still subject to natural selection, even after thousands of years of domestication in relatively intensely managed herds.

iii

DEDICATION

I dedicate this to my advisor, mentor, committee member and friend, Linda Smith, who passed away in 2015. Linda was the person who inspired me to pursue a career in science

and to attend Rutgers, where she had completed her Ph.D.

To my parents Susan and William Fritz, for supporting me and affording me the

opportunities that lead me to where I am today. They raised me to dream big, and work

hard and it has made all the difference.

To my wife Michelle, for her love, support, encouragement and for keeping me

grounded.

To my sister Jennifer, for her support and friendship

To all of my extended family and friends, for their generous love and support.

ACKNOWLEDGEMENTS

I would like to express my most sincere gratitude to my graduate research advisor,

Larry S. Katz for affording me the opportunity to pursue my degree working in his lab.

Over the years, Larry has taught me many important lessons that apply not only in the lab, but in life. He has been an invaluable teacher, mentor and friend and I will carry his wisdom with me forever.

I would also like to thank my committee members, Drs. Michael Sukhdeo,

Michael Westendorf and George Wagner for their guidance and contributions.

I would especially like to thank Susan Becker for the countless hours she has spent teaching me, helping me at the barn, organizing the lab, or just chatting about research and life. She has been jokingly referred to as my “lab mother,” but after five years together, I would call her a lifelong friend.

A special thanks to Rebecca Potosky, Laura Comerford, Felicia Kleiman and

Clint Burgher for all of their work on the farm that makes our research run smoothly.

Finally, I would like to thank all of the undergraduate research students that have assisted with my work, especially Hanna Canfield, Tonya Ternova, Meg Radford,

Cynthia Xue, Lena Sena, Caitlin Kober and Emily Convery.

TABLE OF CONTENTS

Title

ABSTRACT OF THE DISSERTATION ii

DEDICATION iv

ACKNOWLEDGMENTS v

TABLE OF CONTENTS vi

LIST OF TABLES ix

LIST OF FIGURES x

TABLE OF ABBREVIATIONS xiii

INTRODUCTION 1

CHAPTER I

Review of Literature 3

A. Purpose and scope of the literature review 4

B. Sexual behavior of the male goat 5

C. The male effect 8

D. Scent of a buck 10

E. Sebaceous gland secretions 10

F. Urine in scent-marking and sexual behavior 13

G. Caprine olfactory perception 14

H. The flehmen response 14

I. The accessory olfactory system 15

J. Taxonomic prevalence and permutations of self-enurination 17

TABLE OF CONTENTS (continued)

K. Importance of self-enurination to the goat 19

L. Behavioral scenarios that evoke self-enurination 20

M. Endocrine influence of self-enurination 20

N. Predictions on purpose and origin of self-enurination 21

O. Relevance 25

P. Research objectives and hypotheses 26

Q. References 27

Chapter II

Differential effects of androgens, estrogens and socio-sexual context on sexual behaviors in the castrated male goat (Capra hircus)

A. Abstract 37

B. Introduction 39

C. Materials and Methods 43

D. Results 48

E. Discussion 50

F. Tables and Figures 56

G. References 66

Chapter III

Domesticated male goats use self-enurination to present attractive olfactory cues to estrous females

A. Abstract 70

B. Introduction 71

vii

C. Materials and Methods 74

D. Results 77

E. Discussion 78

F. Tables and Figures 81

G. References 93

Chapter IV

Effects of Simulated self-enurination on reproductive behavior and endocrinology in male goats

A. Abstract 97

B. Introduction 99

C. Materials and Methods 109

D. Results 114

E. Discussion 117

F. Figures 124

G. References 136

Chapter V

A. Dissertation Conclusions 141

B. References 146

viii

LIST OF TABLES

Table # Title Page #

Chapter II

Table 1 Self-enurinations more likely to occur in bucks distanced from 58

does

Table 2 Courtship occurs at higher frequencies in bucks with fenceline 59

contact with does

Chapter III

Table 1 Mean frequency of investigative behaviors in experiment 2 86

LIST OF FIGURES

Figure # Title Page #

Chapter II

Figure 1. Buck exhibiting self-enurination 56

Figure 2. Behavior test apparatus for experiment 1 57

Figure 3. Androgens activate self-enurination 60

Figure 4. Estrogens activate courtship 61

Figure 5. Opportunity to court and breed estrous does negatively affects 62

self- enurination frequency in androgen-treated wethers

Figure 6. Both hormone treatment and socio-sexual conditions affect 63

courtship

Figure 7. Hormone treatments do not affect mounting frequency 64

Figure 8. Hormone concentrations do not affect frequency of ejaculation 65

Chapter III

Figure 1. Behavior test apparatus for experiment 1 81

Figure 2. Behavior test apparatus for experiment 2 82

Figure 3. Urine recycling fountain for experiment 2 83

Figure 4. Estrous females spent more time near ovx does carrying buck SE 84

urine than those carrying wether urine

Figure 5. Estrous females prefer buck SE urine over wether urine 85

Figure 6. Estrous females spent more time near buck SE urine than wether 87

urine fountain

Figure 7. Estrous females prefer buck SE urine over wether urine fountain 88

LIST OF FIGURES (continued)

Figure # Title

Figure 8. Time spent near DHT-treated wether urine vs. EB-treated wether 89

urine

Figure 9. Estrous females prefer DHT-treated wether urine over EB-treated 90

wether urine

Figure 10. Time spent near buck SE urine vs buck plain urine 91

Figure 11. Estrous females prefer Buck plain urine over buck SE urine 92

Chapter IV

Figure 1. Testosterone response to simulated SE for all bucks in experiment 124

Figure 2. Testosterone response to simulated SE for mature bucks in 125

experiment 1

Figure 3. Proportion of bucks displaying flehmen and mean time spent in 126

flehmen in experiment 2

Figure 4. LH response to simulated SE for bucks in experiment 2 127

Figure 5. LH response to simulated SE for mature bucks in experiment 2 128

Figure 6. Proportion of bucks displaying flehmen and mean time spent in 129

flehmen for experiment 3 during non-breeding season

Figure 7. LH response to simulated SE for experiment 3 during non- 130

breeding season

Figure 8. Proportion of bucks displaying flehmen and mean time spent in 131

flehmen for experiment 3 during transition into breeding season

LIST OF FIGURES (continued)

Figure # Title

Figure 9. LH response to simulated SE for experiment 3 during transition 132

into breeding season

Figure 10. Proportion of bucks displaying flehmen and mean time spent in 133

flehmen for experiment 4

Figure 11. LH response to simulated SE for experiment 4 134

Figure 12. T response to simulated SE for experiment 4 135

xii

TABLE OF ABBREVIATIONS

AOB Accessory Olfactory Bulb

Buck Domesticated male goat (Capra hircus)

DHT Dihydrotestosterone

DHT-P Dihydrotestosterone propionate

Doe Intact female domesticated goat (Capra hircus)

E Estradiol

EB Estradiol benzoate

GnRH Gonadotropin releasing hormone

HPG Hypothalamic-pituitary-gonadal

IZ Incentive Zone

LH Luteinizing hormone

MOB Main olfactory bulb

NZ Neutral zone

OVX Ovariectomized

SE Self-enurination

T Testosterone

TP Testosterone propionate

VNO Vomeronasal organ

Wether Prepubertally castrated male domesticated goat (Capra hircus)

xiii 1

INTRODUCTION

During the breeding season, the male goat (buck) urinates onto his head and front legs in a behavior known as self-enurination (SE). Though several predictions have been proposed to explain its purpose, these predictions lack empirical evidence. It is widely accepted that the behavior serves a purpose related to reproduction as it is displayed primarily during the breeding season and more frequently when males are housed in proximity to cycling females. It is also likely that the behavior serves a social purpose, as it is frequently displayed even in the absence of females. In bucks, testosterone (T) concentrations increase and reach their maximum concentration early in the breeding season and are greatly diminished in the non-breeding season (Delgadillo et al., 2004;

Muduuli et al., 1979). Unpublished work in our lab shows that the frequency of SE correlates with elevated T concentrations in the breeding season and that the behavior occurs with very low frequency outside of the breeding season when T concentrations are low. Observation of prepubertally castrated males (wethers) also suggests that the behavior is T-dependent, as wethers in our herd rarely display the behavior. Though it seems likely that the behavior is T-dependent, it is unclear how T activates the behavior.

Many ideas exist attempting to explain a purpose for SE. One idea suggests that the behavior may serve as a way of presenting attractive olfactory cues to females (Coblentz,

1976). Another opinion suggests that it is used as a way for bucks to advertise their physiological state by presenting metabolic byproducts to other members of the herd

(McCullough, 1969). By advertising his physiological state, the buck would be reducing the number of challenges with predictable outcomes, conserving energy for tasks directly associated with mating (Coblentz, 1976). Along with this position is the idea that the

2 behavior may serve as a superiority display; a sort of “chest-thumping” that may be used to display social rank separate from simple visual discrimination (Shank, 1972). To take this idea a step further, SE may also serve as a way of dispersing an aversive scent to deter other males from competition (Shank, 1972). One last opinion suggests that the behavior is a way for the buck to display primer pheromones to himself, perhaps activating his hypothalamic-pituitary-gonadal (HPG) axis for mating and reducing the duration of the post-ejaculatory refractory period (Price et al., 1986). By this hypothesis, the behavior may also serve as a way of increasing T concentrations at the onset of the breeding season and maintaining those concentrations despite the declining physiological state that is observed when bucks spend less time feeding and activity levels are increased for reproduction. While any of these predictions may independently account for the evolution of such a behavior, the evidence suggests that this behavior has evolved to serve multiple socio-sexual functions. While it may be impossible to determine which adaptation evolved first, it is possible to accept or reject these ideas by experimentation.

Understanding how and why ungulates use urine to communicate may provide new knowledge to improve husbandry and develop more natural captive breeding practices. In this dissertation, three lines of experimental inquiry were conducted in an attempt to better understand the evolutionary, ecological and physiological underpinnings of SE and to provide novel information on variation and diversity in chemical communication systems in the animal kingdom. Also, findings lead to better understanding of chemical communication by studying a domesticated species still subject to natural selection, even after thousands of years of domestication in relatively intensely managed herds.

CHAPTER I

REVIEW OF LITERATURE

A. Purpose and scope of the literature review

The purpose of this work is to investigate a behavior, which in its current form, may or may not have been altered over time by domestication via housing management and artificial selection. This research intends to further elucidate the evolutionary origin of self-enurination (SE) in the goat and determine what adaptive purpose, if any, the behavior serves today. In addition, this work will demonstrate the uniqueness of this behavior and why it is a behavior worthy of investigation. This review will present work showing that SE is indeed a novel field of investigation that can provide not only a new understanding of the behavior’s usefulness, but also a better understanding of sexual behavior in the domesticated ruminant.

This literature review served to build a foundation upon which the objectives of this dissertation work was built. The first objective was to determine the environmental and endocrine activation of SE in the goat. To provide background on this objective, the known environmental and hormonal influences on sexual behaviors in related species is explored. The second objective of this work is to determine if SE is used to display attractive chemical cues to lure females. In light of this objective, literature on olfactory communication across species and what is known about attractive chemical cues and their activation in the goat and closely related species were reviewed. In the third objective, the idea that SE is used by the buck to present chemical cues to himself, stimulating his own

HPG axis to increase T was tested. The literature reviewed for this objective includes a review of the “male effect” and how pheromones from male sheep and goats affect the

HPG axis of annovulatory females. Also, the pathways involved in the reception and and interpretation of chemical cues by the accessory olfactory system are explored.

B. Sexual behavior of the male goat

The male domesticated goat (buck; Capra hircus) is popularly noted for his high libido and sexual vigor. Statistics on the reproductive rate of goats are not often reported, as it is only in the Angora breed that reproduction presents challenges (Shelton and Groff,

1974; Van Heerden, 1963; Van Rensburg, 1971). In other breeds, reproduction rates are the highest of any domesticated ruminant species (Shelton, 1978). The average buck can breed as many as 50+ does in one season (Shelton, 1978). The buck usually reaches puberty at a very young age. Most non-Angora breeds will exhibit estrus or rut in their first breeding season (Shelton, 1978; Yao and Eaton, 1954). Live sperm in the epididymis of the dairy goat has been observed as early as 110 days post-partum (Yao and Eaton,

1954). However, it is not advisable to breed goats (especially females) until they have reached sexual maturity, usually by 9-10 months of age or 60-75% of mature bodyweight

(Shelton, 1978). The goat is a seasonal breeder, and in the northeastern United States, this means that mating usually occurs during the fall months, as photoperiod changes from long hours of light to short hours of light and longer hours of dark (Amoah et al., 1996;

Bissonnette, 1941). Coinciding with the breeding season, the buck’s circulating testosterone (T) concentrations increase and behavioral and phenotypic changes facilitate courtship and copulation (Delgadillo et al., 2004; Longpre et al., 2011; Walkden-Brown et al., 1994; Walkden-Brown et al., 1997).

Before mating, the male goat engages in several distinct behaviors that, as a whole, make up the courtship display. In the goat buck, these behaviors include vocalizations, snorts, foreleg kicks, tongue flicking, chin resting, anogenital sniffs, nuzzling. Self-enurination, while not typically displayed during courtship, may serve a

6 purpose in courtship. When tending a female, the male’s courtship display is used by the female as an honest indicator of mate quality (Longpre et al., 2011).

The activation of sexual behaviors by T in the male goat and many other species has been demonstrated extensively (Alsum and Goy, 1974; Beyer et al., 1975; Damassa et al., 1977; DeBold and Clemens, 1978; Harding et al., 1983; Hart and Jones, 1975;

Katz, 2007; Lincoln et al., 1972; Schumacher and Balthazart, 1983). However, the aromatization or reduction of T to estrogens or other androgens, respectively, accounts for most of the hormone’s observed effects on the male’s sexual behaviors in bovine species. The relative contribution of androgens and estrogens to the activation of specific sexual behaviors may vary greatly across species. In sheep and cattle, which are in the same biological family (Bovidae) as goats, activation of sexual behaviors is estrogen- dependent (D'occhio and Brooks, 1980; Dykeman et al., 1982; Parrott, 1978). In some species, activation of sexual behaviors is androgen-dependent, or a combination of androgen- and estrogen-dependent. For example, in the guinea pig only androgens activate sexual behavior (Alsum and Goy, 1974). In the rabbit (Beyer et al., 1975) and zebra finch (Harding et al., 1983), both androgens and estrogens are needed to activate sexual behaviors. In other species, such as the hamster, both androgens and estrogens independently activate certain sexual behaviors (DeBold and Clemens, 1978). In the male goat, the study of the effect of sex hormones on male sexual behavior has been limited to testosterone. The endocrine activation of SE has not been studied.

The masculinization of the male brain necessary for the display of male typical sexual behaviors occurs prenatally. Phoenix et al (1959) first discovered that T secreted prenatally by the fetal testes caused the organization or differentiation of neural tissues

7 mediating the display of male mating behaviors postnatally (Phoenix et al., 1959). The aromatization hypothesis was later described, stating that T secreted by the fetal testes, was aromatized to estradiol, which binds to estrogen receptors in the brain, causing masculine structural organization (Naftolin et al., 1974). It was later confirmed that alpha-fetoprotein prevented the masculinization of the female brain by binding to prenatal estrogens (Bakker et al., 2006). However, prenatal masculinization of the brain does not simply activate the display of male-specific sexual behaviors throughout life; T must be continuously secreted.

In a seasonal breeder, such as the male goat, circulating T concentrations increase during the breeding season, causing behavioral changes that facilitate courtship and copulation (Delgadillo et al., 2004; Longpre et al., 2011; Walkden-Brown et al., 1994;

Walkden-Brown et al., 1997). However, there is significant variation in the degree of and duration to cessation of sexual behaviors following castration. In goats, though obvious decreases in sexual behaviors are noted within a few weeks following castration, the ejaculatory response may remain for over a year following castration. Courtship and mounting, though diminished in frequency soon after castration, may still be presented occasionally throughout the lifespan, even if the procedure is performed prior to puberty

(Hart and Jones, 1975). This information suggests that changes to the brain during development may affect the display of sexual behaviors later in life, independent of the absence or presence of sex hormones at the time the behavior is displayed.

8 physical environment is his surroundings; humidity, temperature, housing, topography, etc. His social environment is comprised of sexual, competitive, aggressive and communicative interactions with parents, siblings, other conspecifics, or animals of different species, including humans (Katz, 1987). The buck’s endocrine and nervous systems make up his internal environment and also play a critical role in the activation of his sexual behaviors (Katz, 1987). It is, of course, both the buck’s internal and external environments that influence behaviors via highly complex interactions.

C. The male effect

The male effect is a phenomenon that calls on information from multiple sensory systems. In the sheep and goats, following reception of visual, olfactory, tactile or auditory cues from the male, females may display stimulation and synchronization of sexual activity during the anestrous period. This response is known as the “male effect” or “buck effect” in goats and the “ram effect” in sheep. Many studies have shown that the aforementioned male sexual cues will cause a surge in luteinizing hormone (LH) concentrations in the female, which in some cases may result in ovulation. (Chemineau,

1987; Chemineau et al., 1986; Knight et al., 1978; Lindsay, 1966; Martin et al., 1986;

Pearce and Oldham, 1984; Schinckel, 1954b; Shelton, 1960; Shelton, 1980; Underwood et al., 1944; Walkden-Brown et al., 1993).

The most effective way to induce the male effect in the anestrous female is direct contact with a sexually active male, especially when contact is prolonged to four or more days (Shelton, 1980; Walkden-Brown et al., 1993). It has also been demonstrated that a period of separation before introduction of the male or introduction of a novel male increases the likelihood of females to induce ovulation (Cushwa et al., 1992; Schinckel,

1954a; Underwood et al., 1944). Buck’s with higher libido are more likely to induce the male effect (Fulkerson et al., 1981; Signoret et al., 1982). Taking this one step further, many studies have attempted to describe the relative contributions of the multiple male- derived sensory cues. It appears that olfactory cues play an important role in mediating the male effect. In sheep and goats, the male’s scent is capable of increasing the frequency of LH pulses and inducing ovulation in the female (Knight and Lynch, 1980;

Over et al., 1990). However, this response is not as consistent as demonstrated with full male contact (Claus et al., 1990; Walkden-Brown et al., 1993).

An experiment by Shelton (1980), addressed the relative contributions of exteroceptive factors (visual, olfactory and tactile) to the initiation of estrus and ovulation in Angora goats. He found that while does were stimulated by bucks separated by a fence, this degree of contact (lacking tactile) was not equal to the effect of having the male in the pen (Shelton, 1980). It was also noted that when the distance of separation between buck and doe was too great (150 yds in this case), there was no effect (Shelton,

1980). Olfactory contact produced only a slight effect on the number of Angora females cycling (Shelton, 1980). He concluded that the stimulating influence of the ram for the male effect results from a combination of the aforementioned exteroceptive factors

(Shelton, 1980). It was also noted that induced anosmia reduced, but did not entirely suppress the ovulation of anovular goat does when bucks were introduced (Chemineau et al., 1986). Another study by Delgadillo et al. 2006, added that although goat buck behavior and odor primarily induce ovulation in anovular does, vocalizations seem to facilitate the display of the does’ estrus.

D. Scent of a buck

In the buck, chemical signaling remains a primary mode of communicating a state of reproductive “readiness.” Goat buck fleece alone is capable of inducing an ovulatory response in females (Claus et al., 1990; Hamada et al., 1996; Walkden-Brown et al.,

1993). However, reproductive chemical communication from the male’s perspective is not as well understood. It has been suggested that the male goat (buck) possesses a unique olfactory “profile” that is used to attract females and may play a role in maintaining a herd’s socio-sexual hierarchy (Coblentz, 1976). This strong, characteristic scent of the buck during the breeding season comes from two bodily fluids: sebum and urine (Coblentz, 1976; Jenkinson et al., 1967; Pocock, 1910).

E. Sebaceous gland secretions

The strong characteristic scent of the buck is androgen-dependent, thus it is most prominent during the breeding season when T is elevated (Claus et al., 1990; Iwata et al.,

2000a; Iwata et al., 2000b). In prepubertally castrated male goats (wethers), the scent associated with intact males during the breeding season is absent at all times. Treatment of castrated males or ovariectomized (ovx) females with T or dihydrotestosterone (DHT) promotes pheromone activity in the skin of certain regions, particularly the head and rump (Iwata et al., 2000b). This pheromone activity is capable of increasing the LH pulse frequency and inducing estrus in females (Iwata et al., 2000b; Kakuma et al., 2007).

In the goat, production and release of pheromones are associated with scent glands, which are defined as localized regions of the hide that are densely populated with sebaceous glands in the upper dermal layer of the skin (Hay and Hodgins, 1978; Iwata et al., 2000a; Jenkinson et al., 1967; Pocock, 1910). In the goat, scent glands are located in

11 tissue caudal and medial to the base of the horn (Jenkinson et al., 1967; Pocock, 1910;

Sar and Calhoun, 1966), neck (Jenkinson et al., 1967) forefeet (Pocock, 1910; Ryder,

1966), beneath the tail (Ryder, 1966), at the junction of the hoof and skin (Sar and

Calhoun, 1966) and in the perianal region (Sar and Calhoun, 1966). Histological and biochemical evidence points to the glands located on the head and neck as being the primary source of the buck’s rutting odor (Jenkinson et al., 1967).

Consistent with sebaceous gland localization are the significantly higher expression levels of the genes stearoyl-CoA desaturase 1 (SCD1) and long chain fatty acids member 5 (ELOVL5) in pheromone-producing samples from active scent glands

(Momozawa et al., 2007), suggesting that these genes may play a role in the production of the male-effect pheromone(s). Not surprisingly, this regional specificity of pheromone production is correlated with expression of 5-α reductase enzyme converting T to DHT

(Wakabayashi et al., 2000). While the endocrine pathway for creating the characteristic scent of a buck is clearly androgen-dependent (particularly DHT), the chemical structures of many of the acting pheromones have yet to be identified. It is known that both the fatty acid and non-fatty acid components of buck fleece contain pheromone activity (Birch et al., 1989; Claus et al., 1990). Surprisingly, 4-ethyloctanoic acid, which is largely responsible for the strong odor of bucks during the breeding season does not possess pheromone activity (Birch et al., 1989; Chemineau et al., 2006). However, recent studies show that after being left out at room temperature for several months, 4-ethyloctanoic acid is able to activate gonadotropin releasing hormone (GnRH) pulse generator activity in females (Iwata et al., 2003). This, along with the knowledge that more than 25 fatty acids are found in the lipid fraction of buck fleece that are not present in male castrate or

12 female fleece (Hillbrick et al., 1995; Jenkinson et al., 1967; Sugiyama et al., 1986) demonstrates that there are likely many compounds responsible for the buck effect.

By the classical definition, a pheromone may be any chemical factor that, when secreted or excreted, elicits a social response from member(s) of the same species

(Karlson and Lüscher, 1959). The use of pheromones for reproductive success many species has been widely recognized and studied (reviewed in (Wyatt, 2003). The capability of pheromones to alter the physiology of the recipient suggests their importance in reproduction and social interaction. Primer pheromones are pheromones that produce a modification of physiological processes in the recipient (McClintock,

2000). In the male goat, several androgen-dependent primer pheromones have been identified with the use of a non-traditional bioassay for pheromone detection. By stereotaxically implanting recording electrodes in the median eminence/arcuate region,

Mori et al. (1991) were able to record real-time multiple-unit activity (MUA) volleys that reflected GnRH pulse generator activity (Iwata et al., 2000a; Iwata et al., 2000b; Mori et al., 1991; Okamura and Mori, 2005). Using this model, they found that intact males and

T- and DHT-treated castrates as well as ovx does produced pheromones capable of inducing GnRH pulse generator activity in estradiol-primed ovx females (Iwata et al.,

2000a; Iwata et al., 2000b; Okamura and Mori, 2005).

While the presence of these primer pheromones in sebum has been well documented, their presence in urine has been neither confirmed nor studied. In the ram, it has been demonstrated that only the ram’s wool, not the urine, is capable of inducing an

LH response, but this work has not been repeated in the goat (Claus et al., 1990; Cohen-

Tannoudji et al., 1994; Knight and Lynch, 1980; Over et al., 1990). Since urine-marking

13 behavior is not uniform among different species of superorder ungulata, it should not be assumed that primer pheromone production and activity are not present in the goat’s urine.

F. Urine in scent-marking and sexual behavior

The second component of the characteristic scent of the male goat during the breeding season is urine. Males of many caprine species exhibit a behavior during the breeding season in which parts of the body are impregnated with the scent of their own urine. Self-enurination (O'Brien, 1982), scent-urination (Coblentz, 1976), or urine marking (Shank, 1972) as it is often called, involves direct urination into areas in which hair is the longest (Coblentz, 1976). In the domesticated goat, the behavior is identified by the downward turning of the head and shoulders towards the hindquarters and the emission of urine from the erect penis onto the face, beard and front legs (Price et al.,

1986). It has even been suggested that the goat’s distinct beard may have evolved to serve as a reservoir for the presentation of chemical signals in the urine (Coblentz, 1976). The urine may be delivered in a manner ranging from a spray to a precise stream and can be released in small increments or in a consistent, steady flow (Price et al., 1986). By achieving a convex curvature of the spine, the goat is able to place his mouth and nose in direct contact with the stream, often lapping the urine with his tongue (Price et al., 1986).

The event usually concludes with the individual grooming the penis with his tongue after the stream has ended (Price et al., 1986). Though many ideas have been presented to explain the necessity of such a behavior, none have been successfully demonstrated by experimentation. These hypotheses will be presented later in this review.

G. Caprine olfactory perception

Perhaps the most striking evidence of the importance of chemical communication in the goat is the complexity of the olfactory system. The domesticated goat has two distinct olfactory pathways through which chemical signals in the environment are received. The main olfactory system begins at the nares and relays information to the brain via the main olfactory bulb (MOB). The sensory organ of this system is the olfactory epithelium (OE). This system is used to detect volatile (gaseous) chemical cues and is most similar to the olfactory system found in humans. The accessory olfactory system begins at the nasopalatine ducts located on the roof of the mouth and communicates with the accessory olfactory bulb (AOB). The sensory organ of the accessory olfactory system in the goat is known as the vomeronasal organ (VNO or

Jacobson’s organ) and is vestigial in humans. It is now known that these two distinct olfactory systems work in tandem to detect chemical cues in the environment and on other individuals of the same species (Keller and Lévy, 2012). In ungulates, the accessory olfactory system appears to play a larger role in detecting signals necessary for reproduction, thus it will be discussed in greater detail.

H. The flehmen response

Detection of non-volatile chemical cues via the accessory olfactory system begins with the flehmen response. The flehmen response is a sexually dimorphic behavior

(displayed more frequently by males than females during sexual encounters) characterized by an upward turning of the head, neck extension and curling of the upper lip, which is typically maintained for 5-60 seconds (Ladewig et al., 1980; Melese- d'Hospital and Hart, 1985). It occurs most often after investigation of recently voided

15 urine or vaginal secretions and facilitates transport of materials from the mouth to the vomeronasal organ (VNO) via the nasopalatine ducts.

I. The accessory olfactory system

Detection of non-volatile chemical cues is carried out by the accessory olfactory system. Unlike the main olfactory system, chemical signals enter via the oral cavity.

Entry of materials begins at the nasopalatine ducts, which present as two medial openings in the rostral hard palate caudal to an incisive papilla. The two cigar-shaped, blind-ended, mucus-filled VNO ducts run parallel and branch caudally from the nasopalatine ducts

(Ladewig et al., 1980). The VNO is embedded in the hard palate along the junction of the palate and nasal septum (Besoluk et al., 2001; Ladewig and Hart, 1980; Ladewig et al.,

1980; Melese-d'Hospital and Hart, 1985). The goat VNO is approximately 7cm long and

3-4mm wide and is protected by a thin cartilaginous layer (Besoluk et al., 2001). The sensory epithelium of the VNO is located along the medial wall of the medial and posterior segments of the VNO lumen (Ladewig and Hart, 1980). Surrounding the lumen of the VNO is erectile tissue, which has been associated with a pumping mechanism used to draw materials into the lumen (Salazar and Quinteiro, 1998). It is believed that by constricting the nares during flehmen, the goat creates a vacuum in the VNO upon inhalation. This vacuum effect, combined negative pressure caused by relaxation of the erectile tissue surrounding the lumen, causes materials from the oral cavity to be drawn into the VNO lumen (Estes, 1972; Ladewig and Hart, 1980; Ladewig et al., 1980). Two experiments using fluorescent dye have determined that the flehmen response is necessary for the detection of non-volatile materials via the VNO. Only goats that performed the flehmen response when presented with non-volatile fluorescent materials

16 showed fluorescence past the anterior portion of the VNO lumen (Ladewig and Hart,

1980; Melese-d'Hospital and Hart, 1985).

Once inside the VNO, pheromones act as ligands to activate vomeronasal receptor neurons. Vomeronasal receptor neurons are bipolar primary sensory neurons that possess a dendrite that extends to the surface of the sensory epithelium (Døving and Trotier,

1998; Halpern and Martı́nez-Marcos, 2003; Witt and Wozniak, 2006). From the basal pole of the cell body extends a long axon that travels to the AOB (Døving and Trotier,

1998; Halpern and Martı́nez-Marcos, 2003; Witt and Wozniak, 2006). The ligand binds to G protein-coupled putative pheromone receptors located on the apical microvilli of the terminal dendrite’s terminal knob, ultimately causing a change in membrane potential, sending a signal to the AOB (Døving and Trotier, 1998; Halpern and Martı́nez-Marcos,

2003; Wakabayashi et al., 2002; Witt and Wozniak, 2006). The bed nucleus of the stria terminalis (BNST) innervates the AOB and is also innervated by some areas of the amygdala and the supraoptic nucleus (Døving and Trotier, 1998; Halpern and Martı́nez-

Marcos, 2003; Witt and Wozniak, 2006).

It is proposed that through these connections, primer pheromones influence physiological function, as well as social and sexual behavior. The influence of pheromones on sexual behavior is a vast field of inquiry and has been reviewed extensively by (Tirindelli et al., 2009). It is generally true across species that both the

VNO and main olfactory system are activated by chemicals with known pheromonal effects (Tirindelli et al., 2009; Wyatt, 2003). The degree to which certain pheromones influence behavior via the main or accessory olfactory systems appears to vary across species. The first experiments to examine the effects of pheromones on sexual physiology

17 and behavior in ungulates were performed on sheep. Studies on the effects of pheromones on the “ram effect” in sheep was reviewed by (Signoret, 1991). In sheep and goats, pheromones in the male’s integument can increase the frequency of LH pulses and induce ovulation in the female (Claus et al., 1990; Knight and Lynch, 1980; Over et al., 1990).

This induction of estrus results in female-specific appetitive and copulatory behaviors associated with the estrus state. Rams use odors present in the urine of ewes to detect estrous state, which influence the likelihood of the male to court and mount (Blissitt et al., 1990). Ablation of the main olfactory system in Creole goat does was found to decrease the likelihood of ovulation and estrous behavior of anovulatory does usually induced by the presence of males (Chemineau et al., 1986). A study by Hamada et al.

(1996) concluded that buck pheromones induced the stimulation of the hypothalamic

GnRH pulse generator in goat does, through the aforementioned pathways (Hamada et al., 1996). While the influence of pheromones from the integument and urine in sheep and goats on sexual behavior has been studied to some extent, further study is needed to understand how the olfactory systems of the male goat are used to interpret pheromones to influence the endocrine system and sexual behavior, especially as it pertains to SE.

J. Taxonomic prevalence and permutations of self-enurination

While SE is not restricted to members of the Caprinae subfamily (Signoret, 1991), it does not appear to present itself outside of the order Artiodactyla (even-toed ungulates).

Interspecies variation is quite great when it comes to methods of self-marking with urine.

Many caprine species (and a several additional species of even-toed ungulates) perform

SE as described in the domesticated goat. These species include ibex (C. ibex) (Hediger,

1950), bezoar goats (C. aegagrus) (Schaller and Laurie, 1974), markhor (C. falconeri)

(Geist, 1971; Schaller and Mirza, 1971), red deer (C. elaphus) (Darling, 1937), bontebok

(Damaliscus pygargus) (David, 1973), barbary sheep (Ammotragus lervia) (Haas, 1958), blue sheep (Pseudois nayaur) (Schaller, 1972), wildebeest (Connochaetes taurinus)

(Estes, 1969), feral goats (Coblentz, 1976; Shank, 1972), and bighorn sheep (Ovis canadensis) (Geist, 1971). Of the genus Rupicapra, chamois (Rupicapra rupicapra) shake side to side causing urine to be spread the urine about the flanks (Kramer, 1969;

Shank, 1972). In another interesting permutation, the camel (Camelus dromedaries) urinates directly onto the tuft of long hair at the tip of the tail and swings it side to side, again spreading the urine about his flanks (Coblentz, 1976). The white-tailed deer

(Odocoileus virginianus) (Hirth, 1973), mule deer (O. hemionus) (Muller-Schwarze,

1971) and the reindeer (Rangifer tarandus) (Espmark, 1964) urinate directly onto the hind legs during the rut. The mountain goat (Oreamnos americanus), a species whose phylogeny is not directly connected to that of the domesticated goat, has been observed to urinate directly onto itself, but only while digging a wallow (Coblentz, 1976). Wallowing is quite common in members of bovidae and cervidae and involves rolling or relaxing in a shallow hole containing mud, water or dust. These behaviors likely serve the functions of avoiding biting insects, keeping cool, or spreading scent about the body. While wallowing does not always include urination, the bison (Bison bison) (Lott, 1971) and the moose (Alces alces) (Geist, 1963) will often roll on the ground where their urine was recently voided. Similarly, the hog deer (Axis porcinus) will urinate on its bedding before lying down in it (Schaller, 1967). It should be noted that the domesticated goat does not engage in wallowing.

Besides application to themselves, urine is also used by several species of hoofed mammals for territorial marking. The blackbuck antelope (Antilope cervicapra) (Schaller,

1967), springbok (Antidorcas marsupialis) (David, 1973), vicuna (Lama vicugna)

(Koford, 1957), and Thomson’s (Gazella thomsoni) (Estes, 1969) and Grant’s (Gazella granti) (Estes, 1969) gazelles have all been observed to urinate and defecate together regularly at certain locations in their territory.

K. Importance of self-enurination to the goat

Without an obvious purpose, it may be tempting to explain the buck’s display of

SE by deeming it an “evolutionary remnant” or a behavior that has lost its purpose through domestication. However, there exists evidence of its continued importance to the goat. In signaling theory, the theory of an honest signal suggests that if the signal unintentionally provides the signaler with some degree of harm or danger, continuation demonstrates the signal’s necessity or importance. The classic example of an honest signal is the colorful tail feathers of the male peacock. The ornate plumage is believed to attract females despite making himself more susceptible to predation. Self-enurination appears to be an honest signal because it is continually elicited despite cost to the buck.

Most scent-marking behaviors have little cost besides leaving or emitting a scent that may be detected by predators, but in the case of self-enurination, the buck will continue to display the behavior even though it physically harms him. Over time, the urine’s caustic nature causes hair loss, irritation, and in some cases, open wounds which may become infected. These effects appear primarily on the top of the nose as well as the posterior surface of the forelegs. None of these consequences appear to affect the frequency of the behavior and they certainly do not prevent its continuation (personal

20 observation). It has been speculated that housing location (i.e. proximity to cycling females) may alter the prevalence or degree of the urine burns, as this has been shown to affect the frequency of the behavior itself. In the wild, it seems plausible that most does would be successfully bred and become pregnant early in the breeding season, greatly reducing the duration of the buck’s exposure to cycling females. This may account for the lack of documentation of urine burns in non-domesticated self-enurinating ungulate species.

L. Behavioral scenarios that evoke self-enurination

Some clues to the need for SE may lie in the behavioral context during which the behavior is most often displayed. Price et al. (1986) came to the conclusion that there is a direct, positive correlation between SE and the amount of sexual activity in a heterosexual bout of mating in the goat. This study also concluded that SE was primarily exhibited before or after primary courtship and copulation (Price et al., 1986). In addition, they noticed that a buck allowed visual and auditory contact with an estrous female without direct contact for courtship and copulation often displayed SE (Price et al., 1986). These findings suggest that a degree of sexual arousal is necessary to increase the likelihood of the behavior.

M. Endocrine influence of self-enurination

Perhaps one of the more complicated aspects of sexual behavior is how sex steroids are involved. Our lab has previously demonstrated that treating prepubertally castrated male goats (wethers) with T activates the behavior of self-enurination.

However, it is unlikely that the observed effects can be attributed to testosterone alone, as it has several fates in the body. To further complicate the issue, it appears that there is

21 sexual dimorphism and interspecies variation in how sex steroids influence sexual behavior. In the steer (prepubertally castrated bull), estradiol activates male sexual behaviors to a greater extent than testosterone (Dykeman et al., 1982). In the same species, DHT appears to exert little influence on male sexual behaviors (Dykeman et al.,

1982). Interestingly, these effects are sexually dimorphic as DHT does not activate sexual behavior in ovariectomized heifers, but in steers DHT slightly activated sexual and aggressive behaviors (Dykeman et al., 1982). Consistent with the findings in cattle, castrated male sheep, wethers, did not display sexual behavior when treated with DHT

(D'occhio and Brooks, 1980; Parrott, 1978). Interspecies variation exists in male guinea pig (Alsum and Goy, 1974), Rhesus monkey (Phoenix, 1974) and hamster (Whalen and

Debold, 1974) castrates, in which DHT stimulates increased sexual activity. Our lab hypothesized that in wethers, treatment with DHT was most likely responsible for the activation of secondary sexual characteristics, such as scent production, secondary beard growth, and buck-like body composition, while estrogens activated sexual behaviors such as courting, mounting and SE. While some of the hypotheses about the effects of DHT were confirmed, in a preliminary study estrogen treatment did not appear to activate sexual behavior. The role sex steroids play in influencing the display of SE is a gap that this work intends to fill.

N. Predictions on purpose and origin of self-enurination

While the etiology of the behavior and its variations remain unclear, SE appears to provide olfactory cues supportive to reproduction, mate selection and possibly social rank (Coblentz, 1976; Price et al., 1986; Shank, 1972). The pulsing erection, the entry of the penis into the oral cavity and the increased frequency of the behavior during the

22 breeding season suggests that the behavior serves a purpose for reproduction and that sexual stimuli are involved in its onset. While the behavior is widely assumed to have olfactory significance, it is not clear whether the behavior is directed towards females, other males, or both. McCullough (McCullough, 1969) presented the idea that the urine carries information pertinent to the individual’s physiological condition. He cites the presence of metabolic by-products in the urine, which may advertise a buck’s physiological state to other members of the herd (McCullough, 1969). From an evolutionary standpoint, a dominant buck that advertises his physiological condition would be allowing himself to conserve energy for tasks directly related to reproduction by decreasing the likelihood of challenges with predictable outcomes (Coblentz, 1976).

This use would also benefit subordinates by providing a clue as to when to challenge higher-ranking males that may otherwise be hiding a declining physiological state. It is possible that similar to the way in which the odor of urine changes with diet, it may change when the male begins to metabolize tissues other than fat for energy

(McCullough, 1969). In the goat, dominant males rarely eat enough to maintain their weight since tending females requires constant attention. This leads to rapid weight loss, which may lead to a change in urine odor that would suggest increased odds of winning a challenge to a subordinate male. Similar to this prediction is the notion that the behavior serves as an “olfactory rank symbol” that is used to indicate dominance in a different way than simple visual recognition (Shank, 1972). Supporting this prediction is the fact that in the wildebeest (Estes, 1969) and bontebok (David, 1973) the behavior is displayed as part of the challenge ritual. However, this is not true in the goat.

The idea that SE is simply a way of dispersing territorial chemical signals throughout the environment has not been discounted. When a buck mounts a female, his beard and posterior surface of the front legs come in contact with the female’s backside.

Since these are areas commonly marked with urine by the buck, it is likely that his urine is transferred to the doe’s hair and skin during mounting. Perhaps it is possible that the presence of dominant buck urine on the integument of a female would communicate to other males that she has already been bred and that a breeding attempt may result in conflict with a dominant buck. Male goats are often seen rubbing their heads on objects in the environment, again suggesting the potential for spreading signals in urine. While female attractant and male aversive properties of urine have been observed in rodents

(Jones and Nowell, 1973, 1974; Jones and Nowell, 1989; Sawyer, 1978), similar experiments have not been conducted in the goat.

While these predictions may explain the evolution of such a behavior, SE also appears to play some role in attracting the opposite sex. A bout of SE is often witnessed during courtship displays or immediately following ejaculation, suggesting the necessity of a reproductive incentive. In addition to attraction, self-enurination may also aid females in identifying dominant males. By finding and mating with only the dominant males, the female would decrease the chances of interference from other males (Coblentz,

1976). Separate from attraction is the idea that, like buck hair, pheromones contained in the buck’s urine may hasten and synchronize the onset of estrus in females. In a species in which dominant male turnover is quite high, this would benefit the male by allowing him to breed a greater number of females while he is the dominant buck. In turn, females that respond to the male’s stimulus would ensure breeding by the most dominant males

(Coblentz, 1976). In summary, a buck may use self-enurination to quickly synchronize all of the females (at least the healthy ones) in a herd, ensuring that they would all be bred by the most dominant buck and thus increasing reproductive success for both sexes

(Coblentz, 1976).

A final prediction suggests that the behavior is involved in a form of sexual stimulation or arousal directed at the individual eliciting the behavior (Price et al., 1986).

It has been postulated that the male goat may have evolved a mechanism to detect primer pheromones present in his own urine that may be used to affect his own endocrine physiology. This idea of pheromones acting on the self has only been demonstrated in one other instance in which male rats respond to their own alarm pheromone by releasing stress hormones (Inagaki et al., 2012). It has been shown that buck fleece (containing urine) is capable of stimulating increased LH pulse frequency in females, so it is plausible that similar physiological pathways may exist in the male. From an evolutionary standpoint, being able to increase testosterone concentrations would be a distinct advantage for reproductive success. To take this hypothesis one step further, the male may be using this mechanism as a positive feedback loop to increase testosterone in preparation for the breeding season. Since T is known to trigger the behavior, the behavior’s ability to increase T would complete a positive feedback loop. Perhaps the strongest point of evidence for this hypothesis is the male’s tendency to display flehmen following a bout of SE. By displaying flehmen, it is assumed that the buck is receiving his own scent via the accessory olfactory system. Current literature does not suggest a purpose for the buck’s flehmen response to his own SE. Included in this hypothesis is the idea that perhaps such a behavior could be used to hasten the duration of the refractory

25 period or accelerate the onset of ejaculation prior to mounting by affecting post- ejaculatory hormones or their receptors. Again, this type of endocrine modification would result in increased reproductive success for the male.

Since experimentation has not yet confirmed one hypothesis for the purpose of SE in the male goat, it is seems likely that the behavior has evolved to serve multiple purposes over time. For experimental evaluation, preference will be given to hypotheses based on a socio-sexual function, as these appear to have the most evidence.

O. Relevance

In addition to being one of the first animals to be domesticated, the goat is one of the most versatile livestock animals available. Their small manageable size makes them easy to care for and their wide range of natural habitats makes them suitable for livestock farmers in all corners of the world. Like the cow, the goat is used for both dairy and meat production as well as having a hide and fiber that lends itself well to textile manufacturing. As an additional bonus, the goat possesses a manure that is easy to work with and quite fertile. The goat was once considered the livestock choice of mostly third world agricultural systems, but it has recently become more popular in more developed countries that promote awareness of sustainable and economical agricultural practices. Its ability to utilize a wide variety of vegetation (more than sheep and cattle) make it suitable for almost any environment. This same quality has also made them a natural alternative to herbicide for clearing large areas of unwanted brush or vegetation. With a growing demand for goats and their products, it is important to have an understanding of their husbandry, reproductive physiology and behavior. Experiments in this proposal may contribute to the advancement of more natural breeding techniques exercised in farms

26 around the world. Physiological discoveries may lead to a greater understanding of human olfactory physiology and how it affects and is affected by reproductive behaviors.

P. Research Objectives and Hypotheses

1) Define the effects and relative contributions of androgens, estrogens and socio-

sexual context on sexual behaviors, especially SE in the castrated male goat

Hypothesis: Self-enurination and appetitive sexual behaviors are activated by estrogens and the frequency of these behaviors increases when the male is unable to access the female(s).

2) Determine if male goat urine voided during the breeding season is attractive to female goats and if so, which hormones are responsible for the attractive properties

Hypothesis:Male goat urine voided during the breeding season is attractive to females and this attractiveness is activated by androgens.

3) Determine if the male goat uses chemical cues present in his urine to stimulate his HPG axis to increase testosterone concentrations

Hypothesis: The action of chemical cues on the accessory olfactory system by SE causes an increase in LH and testosterone concentrations.

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CHAPTER II

DIFFERENTIAL EFFECTS OF ANDROGENS, ESTROGENS AND

SOCIO-SEXUAL CONTEXT ON SEXUAL BEHAVIORS IN THE

CASTRATED MALE GOAT

ABSTRACT

The behavioral and endocrine activation of sexual behaviors exhibited by male goats, especially self-enurination (SE), is poorly understood. In the first experiment, to assess the influence of socio-sexual context on SE in bucks, the effects of distance from does, the presence of estrous versus non-estrous does and the presence of another buck on

SE and courtship frequencies of intact male goats (bucks; n=12) were tested using a unique behavior test apparatus. For experiments 2 and 3, to test the relative contributions of sex steroid hormones and socio-sexual context on SE, castrated male goats (wethers; n=20) were randomly divided into five groups and injected for seven weeks with one of the following: 25 mg testosterone propionate (T), 25 mg dihydrotestosterone propionate

(DHT), 100 µg estradiol benzoate (E), 100 µg E and 25 mg DHT (E+DHT), or oil

(CON). The effects of these treatments on frequency of SE and courtship were assessed using the behavior test apparatus (social scenarios) adapted from the findings in experiment 1. In one scenario, a wether could observe (from 4.6 m) a buck and estrous female (doe) together in a wire mesh holding pen. In a different scenario, the wether could observe (from the same distance) a buck that could only court the estrous doe through a wire mesh barrier. Finally, to observe the effects of steroid treatment on mounting and ejaculation frequencies, in addition to SE and courtship, each wether was placed loose in a pen with an estrous doe for 10 min. After a five-week, treatment- washout period, wethers were randomly assigned to different treatment groups and retested. In experiment 1, bucks that were distanced from females displayed more SEs than those with fenceline contact, while those with fenceline contact displayed more bouts of courtship (P<0.05). In experiments 2 and 3, courtship frequencies displayed in

38 all three scenarios were greater than CON only for groups exposed to estrogen directly or via aromatization (T, E+DHT, E; P<0.05). Frequencies of SE exhibited during behavior tests in which the wether was watching were greater than CON only for androgen-treated groups (T, E+DHT, DHT; P<0.05). In contrast, when the wether was free to interact with the female, only the DHT group displayed SE at a higher frequency than CON (P<0.05).

Treatment had no effect on mount frequencies in this test scenario, however ejaculation frequencies were highest for T and E+DHT (P<0.05). These studies suggest that the courtship behaviors of the male goat are estrogen-dependent. However, SE appears to be activated by androgens. It was also demonstrated that social context contributes as much to behavior expression as steroid treatment, as in social scenario 2 some sexual behaviors were displayed in similar frequencies across groups, despite differing sex steroid treatments.

INTRODUCTION

In the domesticated male goat (buck), testosterone (T) rises at the onset of the breeding season, and falls several months later (Delgadillo et al., 2004; Muduuli et al.,

1979; Walkden-Brown et al., 1994). Coinciding with this increase, the buck displays sexual behaviors at a higher frequency than in the non-breeding season (Hart and Jones,

1975). These behaviors include courtship behaviors such as tongue flicks, foreleg kicks, snorts, vocalizations, or any combination of these, as well as mounting and self- enurination (SE).

Self-enurination is characterized by turning the head and shoulders downwards towards the hindquarters and emitting urine from the erect penis onto the face, beard and front legs (figure 1). The urine is delivered in a manner ranging from a spray to a narrow stream, and can be released in short pulses or in a continuous, steady flow. Self- enurination frequently includes the buck lapping his urine and may conclude with the goat grooming the penis with his tongue and the flehmen response.

The adaptive value of SE to the buck remains unclear though several hypotheses exist. In several studied species, male-typical sexual behaviors including scent marking are regulated by T, suggesting such behaviors serve reproductive functions (Bronson and

Whitten, 1968; Ferkin et al., 1994; Hau, 2007; Yahr et al., 1979). Further, the presence of an erection and the increase in the behavior’s frequency in the presence of females also suggest a reproductive function. Experiments in our lab have sought to demonstrate the most popular hypothesis that SE is used to attract estrous females. While estrous females seem to prefer buck urine compared to prepubertally castrated male (wether) urine, this may not be the only function of the behavior.

Observational evidence suggests that attracting females is not the only function

SE serves for the buck. Other bucks show interest in the urine voided by a buck during

SE and these occurrences often result in the flehmen response in both the buck displaying

SE and the buck investigating the urine (Coblentz, 1976; O'Brien, 1982; Shackleton and

Shank, 1984; Shank, 1972). The flehmen response is characterized by the curling of the upper lip with the nares partially closed with neck and head reaching forward and upward

(Schneider, 1930). Flehmen causes non-volatile chemical substances to enter the vomeronasal organ (Ladewig and Hart, 1980). While the flehmen response has been ascribed to the confirmation of the diestrus or proestrus state of females, the adaptive significance of the flehmen response following SE remains a mystery (Ladewig et al.,

1980). It has been suggested that flehmen in response to urine voided by SE or plain urination of other bucks serves to assess dominance or physical vigor, though this hypothesis lacks empirical evidence (Coblentz, 1976; McCullough, 1969). The lapping of his urine and grooming of his penis often displayed by the buck during or after SE may tempt one into relating the behavior to masturbation. Though, this explanation is anthropomorphic and the absence of ejaculation during these occurrences differentiate the behavior from masturbation as it is described in other species (Price et al., 1986).

Findings presented by Price et al. (1986) suggest that the behavior is more likely to occur when the buck can view, but not breed an estrous female. This may occur when the buck is separated from a female by a fence, or when the male is in the post- ejaculatory refractory period. It was also found that distance from the stimulus and the presence of another buck (especially when that buck is tending the female) increase the likelihood of SE, perhaps suggesting that “frustration” of the sex drive may increase the

41 likelihood of the behavior (Price et al., 1986). These findings were used to design behavioral test scenarios to increase the likelihood of SE, which is ordinarily displayed with relatively low frequency.

The objective of the first study (Experiment 1: Effects of social scenario on SE and courtship in bucks) was to determine the environmental conditions (specifically

42 socio-sexual) that increase the likelihood of SE. Findings from this study would then be used to create experiments in which we could measure the relative contributions of androgen (non-aromatizable), estrogen and external cues on the frequency of sexual behaviors in hormone-treated prebubertally castrated males (wethers). In the second experiment (Experiment 2: Hormones and social scenario affect sexual behaviors in wethers), the effect of the aforementioned hormones on the frequency of SE was addressed using social scenarios derived from experiment 1. Finally, in the third experiment, we sought to tease apart the contributions of these hormones and degree of sexual stimulation to the activation and frequency of mounting and ejaculation in addition to SE and courtship, when the wethers were allowed full access to estrous does.

MATERIALS AND METHODS

Animals

All animals were Alpine goats between the ages of 2-9 years, and received a diet consisting of grass hay and grain, and had ad libitum access to water and mineral salt blocks. Diet and husbandry was in compliance with the Consortium Guide for the Care and Use of Agricultural Animals in Agricultural Research and Education (FASS, 2010).

Research was conducted as approved by the Rutgers University Animal Care and

Facilities Committee. Male and female goats were housed on the NJ Agricultural

Experiment Station Research Farm in New Brunswick, NJ (40°29′10″N/74°27′8″W) in barns with free access to outdoor exercise areas. Experiment 1 was conducted during the

2012 breeding season and experiment 2 was conducted during the 2014 breeding season, which for Alpine goat begins in mid-August and terminates near the end of January in the northern hemisphere. Focal goats in experiment 1 were gonadally intact males (bucks; n=12). Stimulus estrous females were estrus-synchronized intact females (does; n=14) and stimulus non-estrous females were ovariectomized females (OVX does; n=6). In experiment 2, focal goats were prepubertally castrated males (wethers; n=20). Teaser bucks (n=3) were gonadally intact males and stimulus females were estrus-synchronized intact does (n=24) or estrus-induced OVX does (n=8).

Hormone Treatment

Subject wethers in experiment 2 were randomly divided into five treatment groups, controlling for age. Each group was injected subcutaneously three times weekly, for eight weeks, with one of the following; sesame oil (vehicle control; CON), 25 mg testosterone propionate (T), 100 µg estradiol benzoate (E), 25 mg dihydrotestosterone

44 propionate (DHT), and a mixture of 100 µg estradiol benzoate and 25 mg dihydrotestosterone (E+DHT). After a five-week washout period, wethers were randomly reassigned to new treatment groups and injected for another eight weeks.

Estrus Synchronization

Estrus-synchronized stimulus does in experiment 1 were drawn from a herd of 14 and estrus-induced or synchronized stimuli does in experiment 2 were drawn from a herd of 8 and 24, respectively. The herds were divided into two groups, which were estrus- induced or synchronized on alternating weeks. For experiments conducted as females entered the breeding season, estrus was induced in OVX females by providing exogenous progestins and estradiol from the protocol developed by Billings and Katz (1997). Estrus synchronization in the intact herd was accomplished using a sequential treatment of prostaglandin (PGF2α). During the breeding season each female received an injection of

10 mg PGF2α (dinoprost tromethamine, i.m.) 60 h prior to preference test as modified from Ott et al. (1980). Standing estrus was detected prior to each behavior test. A non- experimental buck was brought into the females' home pen and allowed to mount females but not allowed to intromit. If the female stood to be mounted, she was considered to be in estrus. If the female rejected the male, she was considered non-estrous.

Behavior Testing

Experiment 1: Effects of social scenario on SE and courtship in bucks

In experiment 1, a 5-minute behavior test was used to assess the contributions of distance from stimulus does, the presence of another buck and the estrous state of stimulus does on the frequency of SE and courtship in bucks (figure 2). Each buck was tested in all eight possible combinations of the following conditions; in the pen 6.1m

45 from stimulus does (far), in the pen with fenceline contact with stimulus does (near), with another buck in the opposite buck pen (paired), without a buck in the opposite buck pen

(alone), with estrous stimulus does (estrous does) or with non-estrous stimulus does (non- estrous does). Thus, the eight conditions were; 1) Far, paired, estrous does, 2) Far, paired, non-estrous does, 3) Far, alone, estrous does, 4) Far, alone, non-estrous does, 5) Near, paired estrous does, 6) Near, paired, non-estrous does, 7) Near, alone, estrous does, 8)

Near, alone, non-estrous does. Total bouts of courtship and total number of SEs were recorded for each buck in each of his eight, 5-minute tests.

Experiment 2: Hormones and social scenario affect sexual behaviors in wethers

At the end of each 8-week course of steroid treatment, wethers were tested in three 10-minute social scenarios. In social scenarios 1a and 1b, the test wether had no access to a female and was watching the female from a distance of 4.6m. In 1a, the wether watched a buck courting and mounting an estrous female, and in 1b, he watched a buck courting an estrous female through a fence barrier, thus no mounting occurred. In social scenario 2, the test wether was allowed to interact freely with an estrous female for the duration of the test. In all social scenarios, SE and courtship frequencies were recorded. In Scenario 2, in addition to SE and courtship, mount and ejaculation frequencies were also recorded.

Subject wether watched a buck courting and mounting an Social Scenario 1A estrous female Subject wether watched a buck courting an estrous female Social Scenario 1B through a fence barrier

Social Scenario 2 Wether was allowed to interact freely with an estrous female

Statistical Analysis

Experiment 1: Effects of social scenario on SE and courtship in bucks

In experiment 1, the effect of three conditions (distance from stimulus does, presence of another buck, and estrous state of stimulus does) were assessed for SE and courtship frequencies. Courtship and SE frequencies from each buck, tested four times for each comparison, were combined (ignoring the other two conditions), as overall frequencies were relatively low. For example, to assess the effect of distance from stimulus does on SE, the SE frequency for bucks in the far pen from tests where he was

1) alone with estrous does, 2) alone with non-estrous does, 3) paired with estrous does and 4) paired with non-estrous does were summed and divided by the total number of

SEs in all eight conditions (SEs in near and far pens combined). This generated a proportion of SEs occurring in the far pen to SEs occurring in both the near and far pens.

Combined frequencies of SE and proportion of bucks displaying SE for each of the three conditions were analyzed by chi-squared test to determine if frequencies differed from chance (50%). Differences were deemed significant at P<0.05.

Experiment 2: Hormones and social scenario affect sexual behaviors in wethers

Mean SE frequencies in social scenarios 1 and 2 and mean courtship frequencies, in social scenario 2 underwent SQRT(x+1) transformation before analysis. These and all other behavior frequencies, except courtship frequency in social scenario 1, were analyzed by one-way analysis of variance (ANOVA) and mean behavior frequencies between treatments were compared using Fisher’s LSD Test. Treatment effect was deemed significant at P<0.05. Since courtship frequencies in social scenario 1 were highly variable among individuals, these data were not normally distributed. These data

47 were thus analyzed by Welch’s test of means allowing for unequal variances and medians were compared by Kruskal-Wallis Multiple-Comparison Z-value test. Differences between treatments were compared by Dunn’s test and deemed significant at Z>1.96.

RESULTS

Experiment 1: Effects of social scenario on SE and courtship in bucks

Table 1 displays total SE frequencies for the 12 bucks in each of the eight conditions. In this experiment, more total SEs occurred in the far pen (17/24; 71%), than were displayed in the near pen (7/24; 29%; P<0.05). Of the instances in which a buck was in the far pen, SE was displayed in 14/48 (29%) of tests and when they were in the near pen, it was displayed in 6/48 (13%) of tests. SEs occurred in 14/24 (58%) of tests when the buck was in the apparatus with another buck in the opposite buck pen and 10/24

(42%) occurred when he was alone. Of the tests where a buck was in the apparatus with another buck present, there were SEs in 11/48 (23%) of tests and 9/48 (19%) of tests when the buck was alone. When estrous does were used as the stimulus, 14/24 (58%) of the total number of SEs were recorded and SE was displayed in 10/48 (21%) of tests.

When non-estrous does served as the stimulus, 10/24 (42%) of total SEs were displayed and in 10/48 (21%) of tests SE occurred.

Table 2 displays total courtship frequencies for the 12 bucks in each of the eight conditions. For courtship, as expected, more courtships occurred in the near pen

(1570/1618; 97%) than in the far pen (48/1618; 3%; P<0.05). In tests where the buck was in the near pen, bucks displayed courtship in more tests (44/48; 92%), than they did in tests in the far pen (7/48; 15%; P<0.05). When the stimulus does were in estrous, bucks displayed courtship in higher frequencies (1227/1618; 76%) than in the presence of non- estrous does (391/1618; 24%; P<0.05). However, the number of tests in which bucks displayed courtship when does were in estrus was not greater (28/48; 58%) than the number of tests where bucks displayed courtship in the presence of non-estrous does

(25/48; 58%). The presence of another buck in the apparatus did not have an effect on the total number of courts (815/1618; 50%) when compared to total courts when bucks were alone (803/1618; 50%). It also did not have an effect on the number of bucks that displayed courtship in tests where they were with another buck (25/48; 52%) when compared to being in the apparatus alone (28/48; 58%).

Experiment 2: Hormones and social scenario affect sexual behaviors in wethers

Social Scenarios 1A and 1B

Results from social scenario 1A and 1B were combined for analysis, as their results did not differ. In both scenarios, wethers treated with T, E, and E+DHT courted more frequently than CON- or DHT-treated wethers (fig. 3; P<0.05). Wethers treated with T, DHT, and E+DHT exhibited SE more frequently than CON- or E-treated wethers

(fig. 4; P<0.05). In summary, groups whose treatments contained E or T, which is aromatized to estradiol, courted at higher frequency than CON- or DHT-treated wethers.

Groups whose treatments contained androgen exhibited SE in greater frequency than those treated with CON or E only.

Social Scenario 2

In social scenario 2, wethers treated with T, E, and E+DHT displayed courtship in higher frequency than CON wethers or those treated with DHT (fig. 5; P<0.05). Wethers treated with DHT were the only group to display SE in greater frequency than CON (fig.

6; P<0.05). No differences between treatment groups for mount frequencies were observed (fig. 7). Only wethers treated with T and E+DHT exhibited ejaculations in greater frequency than CON (fig. 8; P<0.05).

DISCUSSION

The goat provides a unique model for the study of sexual behavior because it has been subjected to thousands of years of domestication. For those behaviors in which human intervention overcomes natural selection pressure, domestication can “relax” said pressure on some behaviors, possibly resulting in a loss of adaptive significance (Price,

1984). This makes assessing the contexts in which behaviors occur and understanding the selective processes contributing to the expression of behavior in the goat both difficult and interesting.

The sexual behaviors of the male goat are dependent upon highly complex interactions between his internal and external environment. The buck’s external environment is comprised of his physical surroundings and social interactions. His physical environment is his surroundings; humidity, temperature, housing, topography, etc. His social environment is comprised of sexual, competitive, aggressive and communicative interactions with parents, siblings, other conspecifics, or animals of different species, including humans (Katz, 1987). The buck’s endocrine and nervous systems make up his internal environment and also play a critical role in the activation of his sexual behaviors (Katz, 1987). It is, of course, both the buck’s internal and external environments that influence behaviors via highly complex interactions. This experiment expands upon how these factors (particularly endocrine profile and social environment) influence the activation and frequency of sexual behaviors in the male goat.

The masculinization of the male brain necessary for the display of male typical sexual behaviors occurs prenatally. Phoenix et al. (1959) first discovered that T secreted prenatally by the fetal testes caused the organization or differentiation of neural tissues

51 mediating the display of male mating behaviors postnatally. The aromatization hypothesis was later described, stating that T secreted by the fetal testes, was aromatized to estradiol, which binds to estrogen receptors in the brain, causing masculine structural organization (Naftolin et al., 1974). It was later confirmed that alpha-fetoprotein prevented the masculinization of the female brain by binding to prenatal estrogens

(Bakker et al., 2006). However, prenatal masculinization of the brain does not simply activate the display of male-specific sexual behaviors throughout life; testosterone must be continuously secreted.

(Hart and Jones, 1975). These findings were consistent with our finding that sexual behaviors were displayed by CON wethers in this experiment.

Similar to other bovids, courtship behaviors in the male goat were activated by estrogens, regardless of social scenario. While the non-aromatizable androgen DHT has been shown to increase sebaceous gland secretions in the goat buck (Iwata et al., 2000b), results from this study show that it also activates SE. This finding perhaps suggests that

52 the behavior evolved for the purpose of scent marking because in several related species, scnet marking behaviors are androgen-dependent. Prior to this study, the relative roles of estrogens and non-aromatizable androgens in the activation of SE had not been studied. It has been shown in sheep and cattle, that appetitive sexual bevaiors are activated by estrogens aromatized from T. Activation of SE by a non-aromatizable androgen is a unique finding and suggests that SE is not a typical appetitive sexual behavior even though it is associated with sexual arousal (Price et al., 1986).

Clarke et al. (1976) conducted a study comparing the effects of T delivered to ewes at different times during pregnancy on sexual behaviors of the offspring.

Interestingly, though offspring of ewes treated with T prior to 90 days of gestation displayed a male-like urination posture (standing normally, voiding urine in pulses), they found that ewe lambs exposed to T from day 90 to 140 of gestation displayed the normal posture of female urination (arched the back and slightly crouched hind legs) (Clarke et al., 1976). An interesting assessment of these findings presented by Katz (1987), stated that these differences may be attributed to the relative contributions of the differentiation of peripheral structures and the differentiation of the central nervous system. Early in development, differentiation of peripheral structures would account for the pulsatile release of urine by the development of urethral musculature caused by androgen stimulation, while differentiation of the central nervous system at this time would be more likely to account for the urinary posture, which is likely a result of behavioral masculinization (Katz, 1987). Thus, it is plausible that androgens influence both the peripheral structures and the central nervous system early in development to enable the

53 behavior to present itself. This would account for the observation of an occasional SE in untreated wethers, as displayed by a CON-treated wether in social scenario 1 (fig. 3).

Further evidence presented in other species show that androgens, particularly

DHT, which binds with a high affinity to androgen receptors, act on the central nervous system to influence behaviors. For example, in rats Hart (1968) and Breedlove and

Arnold (1980) found accumulations of androgens and their metabolites in regions of the spinal cord associated with erection and ejaculation reflexes. Current understandings are that penile erections are caused by creating a change in the activity of efferent autonomic pathways to erectile tissues from the spine (Giuliano and Rampin, 2000). These spinal neurons are activated by peripheral and supraspinal cascades originating from genital sensory information, receptors in the spinal cord, brainstem and hypothalamic nuclei

(Giuliano and Rampin, 2000). In light of this information, our finding that SE is androgen-dependent could suggest that SE is a behavior displayed in response to an erection occurring with the inability to engage in copulation, but this idea needs further study.

The frequency of the male goat’s sexual behaviors is heavily influenced by his environment. The overall frequency of courtship in groups that courted (T, E, E+DHT) in social scenarios 1A and 1B was greater than the same groups in social scenario 2 (figs. 4,

6). To explain this difference, courting bucks in social scenario 1A and 1B were observed courting more heavily at the onset of the test period and appeared to lose interest in the stimulus over time. This was likely due to learned helplessness caused by distance and containment. In social scenario 2, courting bucks’ courtship frequency was limited only by their own libido.

Wethers in scenario 1A and 1B that self-enurinated (T, DHT, E+DHT), did so more frequently than the same groups in scenario 2 (figs. 3, 5). This difference is explained by the context in which SE occurs. In preliminary studies directed at finding scenarios that increased the likelihood of SE, it was observed that SE does not usually occur when the buck is tending a female. Instead, SE tends to occur when he is sexually aroused, but not able to court directly and copulate with an estrous female. Examples of this include a buck courting an estrous female through a barrier, or during the post- ejaculatory refractory period. It was observed that in social scenario 2, wethers that courted and self-enurinated in scenarios 1A and 1B (T, E+DHT), chose to spend time engaged in courtship and copulation and only self-enurinated occasionally following an ejaculation (fig. 3). In scenario 2, DHT-treated wethers rarely courted the estrous females, more often placing them in a situation where SE was likely to occur (figs. 5, 6).

Relative contributions of social context and endocrine milieu to the activation of sexual behaviors were teased apart in this experiment. While the likelihood of the male to display courtship behaviors or SE is dependent on the presence of androgens and/or estrogens acting either prenatally (organization) or during adulthood (activation), the social context plays a critical role. For the goat the susceptibility to contextual influence appears to be specific behavior-dependent. For example, wethers would not SE when they were able to court and mount an estrous doe freely, even though the same hormone treatment activated the behavior in other sexually relevant contexts, i.e limited direct contact with females (fig. 5). In contrast, mounting occurred regardless of steroid treatment, including none, if the context allowed, i.e., estrous female present (fig. 7).

These males were exposed to androgens prenatally and postnatally before castration was

55 performed, so the brain was masculinized to some extent. These findings suggest that both context and endocrine profiles affect the likelihood of a buck to display SE and other sexual behaviors. It is likely that contextual clues modify which behaviors are more likely to be expressed.

Figure 1. Buck exhibiting self-enurination

Figure 2. Behavior test apparatus for experiment 1:.Stimulus does were placed at one end of the apparatus, in separate pens. Depending on the test conditions, a buck was either “alone” or “paired” with a buck in the opposite male pen. A buck in the “far” pen was 6.1m from stimulus does and in the “near” pen had fenceline contact with does.

Observations were made through a fence barrier in the aisle adjacent to the lower barrier in the diagram.

Buck Condition Sum of SEs/5 min Sum of Bucks Displaying SE

Far, Alone, Estrous does 4 3

Far, Alone, Non-estrous does 3 3

Far, Paired, Estrous does 6 4

Far, Paired, Non-estrous does 4 4

Near, Alone, Estrous Does 2 2

Near, Alone, Non-estrous does 1 1

Near, Paired, Estrous does 2 1

Near, Paired, Non-estrous does 2 2

Table 1. Self-enurinations more likely to occur in bucks distanced from does: Sum of self-enurinations and number of bucks that displayed self-enurination during a 5-min test

(n=12). Far bucks were 6.1m away from and near bucks had fenceline contact with stimulus does. Bucks were alone when they were the only buck in the apparatus and paired when another buck was in the opposite pen (near or far). Stimulus does were estrous or non-estrous.

Buck Condition Sum of CTs/5 min Sum of Bucks Displaying CT

Far, Alone, Estrous does 33 3

Far, Alone, Non-estrous does 6 2

Far, Paired, Estrous does 3 2

Far, Paired, Non-estrous does 6 2

Near, Alone, Estrous Does 545 12 Near, Alone, Non-estrous 219 11 does Near, Paired, Estrous does 646 11 Near, Paired, Non-estrous 160 10 does

Table 2. Courtship occurs at higher frequencies in bucks with fenceline contact with does: Sum of courtships (CT) and number of bucks that displayed courtship during a 5- min test (n=12). Far bucks were 6.1m away from and near bucks had fenceline contact with stimulus does. Bucks were alone when they were the only buck in the apparatus and paired when another buck was in the opposite pen (near or far). Stimulus does were estrous or non-estrous.

Figure 3. Androgens activate self-enurination: Self-enurination frequency (Mean ±

SEM) displayed by the wethers treated with CON (vehicle control), T (testosterone propionate), E+DHT (estradiol benzoate+ dihydrotestosterone propionate), E (estradiol benzoate), and DHT (dihydrotestosterone propionate) during a 10-min test. In social scenario 1, wethers watched stimulus from 4.6m. For each treatment, n=8. Bars with different superscripts differ (P<0.05).

Figure 4. Estrogens activate courtship: Courtship frequency (mean ± SEM) displayed by the wethers treated with CON (vehicle control), T (testosterone propionate), E+DHT

(estradiol benzoate+ dihydrotestosterone propionate), E (estradiol benzoate), and DHT

(dihydrotestosterone propionate) during a 10-min test. In social scenario 1, wethers watched stimulus from 4.6 m. For each treatment n=8. Bars with different superscripts differ (P<0.05).

Figure 5. Opportunity to court and breed estrous does negatively affects self- enurination frequency in androgen-treated wethers: Self-enurination frequency

(Mean ± SEM) displayed by the wethers treated with CON (vehicle control), T

(testosterone propionate), E+DHT (estradiol benzoate+ dihydrotestosterone propionate),

E (estradiol benzoate), and DHT (dihydrotestosterone propionate) during a 10-min test.

In social scenario 2, wethers interacted freely with estrous does. For each treatment n=8.

Bars with different superscripts differ (P<0.05).

Figure 6: Both hormone treatment and socio-sexual conditions affect courtship:

Courtship frequency (Mean ± SEM) displayed by the wethers treated with CON (vehicle control), T (testosterone propionate), E+DHT (estradiol benzoate+ dihydrotestosterone propionate), E (estradiol benzoate), and DHT (dihydrotestosterone propionate) during a

10-min test. In social scenario 2, wethers interacted freely with estrous does. For each treatment n=8. Bars with different superscripts differ (P<0.05).

Figure 7. Hormone treatments do not affect mounting frequency: Mount frequency

(Mean ± SEM) displayed by the wethers treated with CON (vehicle control), T

(testosterone propionate), E+DHT (estradiol benzoate+ dihydrotestosterone propionate),

E (estradiol benzoate), and DHT (dihydrotestosterone propionate) during a 10-min test.

In social scenario 2, wethers interacted freely with estrous does. For each treatment n=8.

Figure 8. Hormone concentrations do not affect frequency of ejaculation: Ejaculation frequency (Mean ± SEM) displayed by the wethers treated with CON (vehicle control), T

(testosterone propionate), E+DHT (estradiol benzoate+ dihydrotestosterone propionate),

E (estradiol benzoate), and DHT (dihydrotestosterone propionate) during a 10-min test.

In social scenario 2, wethers interacted freely with estrous does. For each treatment n=8.

Bars with different superscripts differ (P<0.05).

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Schneider, K.M., 1930. Das Flehmen. Akad. VerlagGes. Schumacher, M., Balthazart, J., 1983. The effects of testosterone and its metabolites on sexual behavior and morphology in male and female Japanese quail. Physiology & behavior 30, 335-339. Shackleton, D.M., Shank, C.C., 1984. A Review of the Social Behavior of Feral and Wild Sheep and Goats. Journal of Animal Science 58, 500-509. Shank, C.C., 1972. Some Aspects of Social Behaviour in a Population of Feral Goats (Capra hircus L.). Zeitschrift für Tierpsychologie 30, 488-528. Walkden-Brown, S., Restall, B., Norton, B., Scaramuzzi, R., Martin, G., 1994. Effect of nutrition on seasonal patterns of LH, FSH and testosterone concentration, testicular mass, sebaceous gland volume and odour in Australian cashmere goats. Journal of Reproduction and Fertility 102, 351-360. Walkden-Brown, S., Restall, B., Scaramuzzi, R., Martin, G., Blackberry, M., 1997. Seasonality in male Australian cashmere goats: long term effects of castration and testosterone or oestradiol treatment on changes in LH, FSH and prolactin concentrations, and body growth. Small Ruminant Research 26, 239-252. Yahr, P., Newman, A., Stephen, D.R., 1979. Sexual behavior and scent marking in male gerbils: Comparison of changes after castration and testosterone replacement. Hormones and behavior 13, 175-184.

CHAPTER III

DOMESTICATED MALE GOATS (CAPRA HIRCUS) USE SELF-

ENURINATION TO PRESENT ATTRACTIVE OLFACTORY CUES

TO ESTROUS FEMALES

ABSTRACT

Two experiments were conducted in order to determine if estrous female goats

(does) are attracted to male goat (buck) urine and to determine if androgens activate the attractive chemical signals in his urine. During the breeding season, the male goat urinates onto his head and front legs in a behavior known as self-enurination (SE). While it has been suggested that the behavior may be used by the buck to display chemical signals in the urine to attract estrous does for mating, this hypothesis lacks empirical evidence. In the first experiment, urine was presented on ovariectomized does. Intact estrous does (n=13) were placed in a 5-minute preference test our hypothesis that they preferred buck SE urine over prepubertally castrated male (wether) urine. In the second experiment, urine choices (buck plain urine vs. wether urine, dihydrotestosterone-treated wether urine vs. estradiol-treated wether urine and buck SE urine vs. buck plain urine) were presented in recycling fountains. In the first experiment, estrous does displayed preference for buck SE urine over wether urine (P<0.05). In the second experiment, does displayed preference for buck plain urine over wether urine (P<0.05). In subsequent two- choice tests does appeared to lose interest in the urine fountains, and thus did not show meaningful preference in the other comparisons.

INTRODUCTION

During the breeding season, the domesticated male goat (buck) displays a behavior known as scent-urination, urine-marking, or self-enurination (SE) (Coblentz,

1976; O'Brien, 1982; Shank, 1972). Self-enurination is characterized by turning the head and shoulders downwards towards the hindquarters and emitting urine from the erect penis onto the face, beard and front legs. Self-enurination may include the buck lapping his urine and/or grooming his penis. The flehmen response is often exhibited following

SE, and is often used by both sexes in different sexual contexts (Hart and Jones, 1975;

Ladewig et al., 1980). The flehmen response is displayed more often by males but is also displayed by females and its frequency is at its highest during the breeding season

(O'Brien, 1982; Shank, 1972). It is used to detect non-volatile chemical signals via the accessory olfactory system and characterized by the upward turning of the head and curling of the upper lip (Ladewig and Hart, 1980; Schneider, 1930). Outside of the breeding season and occasionally during the breeding season, the buck will urinate in an ordinary manner. A “plain” urination is when a buck urinates in the absence of an erection and does not investigate the urine as it is being voided. Whether or not the composition and scent of plain urine differs from that of urine voided during an SE remains unknown.

In the rat (Brown, 1977), mouse (Johnston and Bronson, 1982), hamster (Petrulis and Johnston, 1995), elephant (Rasmussen et al., 1997), and rhesus macaque (Keverne and Michael, 1970) it was shown that chemical cues can function as sexual attractants and are capable of influencing sexual arousal and appetitive sexual behaviors. In several studied species, male-typical scent marking behaviors are regulated by testosterone (T)

72 and occur at a greater frequency during the breeding season, suggesting such behaviors serve reproductive functions (Bronson and Whitten, 1968; Ferkin et al., 1994; Hau, 2007;

Yahr et al., 1979). In ungulates, it was initially believed that scent marking was used exclusively for territoriality (Owen-Smith, 1977), though observation of scent marking behavior in non-territorial species suggests that it may be used in other contexts (Johnson,

1973; Ralls, 1971).

Though the function of SE in the goat is unknown, one hypothesis suggests that

SE is used to apply urine to the body in an effort to display olfactory cues to attract estrous females for mating (Coblentz, 1976). Further, the signals may serve to hasten the onset of and/or synchronize estrus in females at a time when the buck is in peak condition

(Coblentz, 1976). In sheep (Knight and Lynch, 1980), the frequency of ovulation has been shown to increase in ewes exposed to a ram’s fleece containing his chemical cues.

While observations in moose (Miquelle, 1991) and goats (Coblentz, 1976) have suggested that urine marking serves to attract females and possibly synchronize their estrus, these hypotheses have not been tested.

It is unclear whether or not buck urine voided during the breeding season differs chemically from buck urine voided outside of the breeding season or urine voided from prepubertally castrated males (wethers). It is known that the presence of testosterone activates sebaceous gland secretions, giving the buck a characteristic scent that has been shown to be capable of hastening the onset of the breeding season in the doe (Claus et al.,

1990). It has also been shown that goat bucks and T- and DHT-treated castrates and ovariectomized (ovx) does produce pheromones capable of inducing GnRH pulse generator activity in estradiol-primed ovx females (Iwata et al., 2000a; Iwata et al.,

2000b; Okamura and Mori, 2005). While the presence of these primer pheromones in sebum has been well documented, their presence in goat urine has been neither confirmed nor studied. In the ovine ram, it has been demonstrated that only the ram’s wool, not the urine, is capable of inducing an LH response, but this work has not been repeated in the goat (Claus et al., 1990; Cohen-Tannoudji et al., 1994; Knight and Lynch, 1980; Over et al., 1990). In light of this shortcoming, we would like to test the hypothesis that buck urine has a different chemical makeup, which is more attractive to the estrous female than urine voided by a prepubertally castrated male. Further, we believe that urine voided by the buck during the breeding season when T concentrations are elevated differs chemically from that voided during the non-breeding season when T concentrations are lower.

Unpublished work in our lab has identified social scenarios that increase the likelihood of SE, which were used to collect SE urine. Using findings presented by (Price et al., 1986), we have confirmed that the behavior is more likely to occur when the buck can view, but not breed an estrous female. The objective of this study was to determine if buck urine is attractive to estrous females, and if SE may be used to emit the attractive signals in the urine. Further, we sought to determine if the attractive properties of buck urine are activated by androgens or estrogens and if buck urine voided during an SE differs chemically from that voided during a plain urination.

MATERIALS AND METHODS

Animals

All animals were Alpine goats between the ages of 2-9 years, which received a diet consisting of grass hay and grain, and had ad libitum access to water and mineral salt blocks. Diet and husbandry was in compliance with the Consortium Guide for the Care and Use of Agricultural Animals in Agricultural Research and Education (FASS, 2010).

Research was conducted as approved by the Rutgers University Animal Care and

Facilities Committee. Male and female goats were housed on the NJ Agricultural

Experiment Station Research Farm in New Brunswick, NJ (40°29′10″N/74°27′8″W) in barns with free access to outdoor exercise areas. All goats used in the experiments were acclimated to their respective test apparatuses in the weeks prior to the experiments.

Estrus Synchronization and Detection

Estrus induced or synchronized goats used as sexual stimuli in experiments 1 and

2 were females drawn from a herd of 18 and 12, respectively. The herds were divided into two groups, which were estrus synchronized on alternating weeks. Estrus synchronization was accomplished using a sequential treatment of prostaglandin-F2α

(PGF2α), as modified from Ott et al. (Ott et al., 1980). During the breeding season, when these experiments were conducted, each female received an injection of 10 mg PGF2α

(dinoprost tromethamine, i.m.) 60 h prior to the preference test. Standing estrus was detected prior to each behavior test. A non-experimental buck was brought into the females' home pen and allowed to mount females but not allowed to intromit. If the female stood to be mounted, she was considered to be in estrus. If the female rejected the male, she was considered non-estrous and was not tested on that day.

Urine Collection

Buck SE urine and plain buck urine were collected during the breeding season in the weeks prior to behavior testing and frozen for use in the experiments. Plain urine was collected by attaching a collection apparatus to the goat’s underside. Self-enurination urine was collected by leading bucks to a pen containing does and allowing them to court.

After courting, the buck was led away from the does and SE would likely follow. The SE was intercepted by a student with a urine collecting apparatus. Urine from hormone- treated wethers used in experiment 2 (conducted in Nov. and Dec. 2016) was collected in the 2014 breeding season. Treated wether urine pools consisted of plain urine collected from eight wethers injected subcutaneously three times per week for eight weeks with either 25mg dihydrotestosterone propionate or 100µg estradiol benzoate. Urine was pooled, frozen and stored for use in experiment 2.

Statistical Analysis

For all two-choice tests, data were analyzed by time spent in the incentive zones

(IZ) and preference score. Animals that did not enter either IZ were excluded from analysis. A mean of time spent or preference score between the first run and switchback were calculated for each doe to account for any side bias. Mean time spent was compared by paired t-test and preference scores were compared by one-sample t-test to determine if the preference scores differed from chance (50%). For all analyses, results were significant at p<0.05. Preference scores were calculated as follows:

�� #1 �� = × 100 �� #1 + �� #2

Experimental Design

Experiment 1

In this experiment, estrous does were tested in a two-choice test to determine if preference was displayed for SE urine collected from an intact buck during the breeding season. Self-enurination urine’s attractiveness was compared to that of wether urine.

Focal goats were estrus-synchronized intact females (does; n=16). Urine was applied to ovariectomized females (ovx does; n=4) to simulate how urine would “naturally” be displayed by a buck following SE. Urine was applied to the ovx does by soaking a rag in the urine and rubbing it on the head, beard, shoulders and flanks before each doe was tested. Estrous does were tested randomly, one at a time in a 5-minute two-choice preference test in the apparatus depicted below (fig. 1). Time spent in IZs and investigative behaviors were recorded. After all 16 does were tested, the choice locations were switched to opposite sides and the experiment was repeated.

Experiment 2

Focal goats were estrus-synchronized intact females (does; n=10). Ovx does (n=4) were placed in separate pens located outside of the test area to keep the test does from being alone in the test apparatus, as goats are social animals and may become distressed when placed alone in an unfamiliar area. Does were tested randomly, one at a time in a 5- minute two choice preference test in the apparatus depicted below (fig. 2). Fountains were set up at opposite ends, each with one of the two urine choices flowing at a similar and constant rate (fig 3). Time spent in IZs when the doe was tending to a fountain and investigative behaviors were recorded. After all 10 does were tested, the apparatus was cleaned and the choice locations were switched to opposite sides and the experiment was repeated.

RESULTS

Experiment 1

Mean time spent in IZ (fig. 4) and mean preference scores (fig. 5) are displayed for the 13 does tested in the first experimental protocol. Both time spent in IZ and preference scores were higher for ovx does carrying SE buck urine than wether urine

(p<0.05). Overall frequency of investigative behaviors was low and thus, not depicted.

Experiment 2

Mean frequencies of investigative behaviors for all three comparisons in experiment 2 are depicted in table 1. Mean time spent in IZ and mean preference score are depicted for buck SE urine vs. wether urine (fig. 6, 7), DHT-treated wether urine vs

EB-treated wether urine (fig. 8, 9) and buck SE urine vs. buck plain urine (fig. 10, 11).

Both time spent in IZ and preference score were greater for buck SE urine than wether urine (p<0.05). For both DHT-treated wether urine vs. EB-treated wether urine and buck

SE urine vs. buck plain urine, less total time was spent investigating the choices.

Preference scores for DHT-treated wether urine were greater than those for EB-treated wether urine (fig. 9; p<0.05), but mean time spent in IZs did not differ (fig. 8). Similarly, preference scores for buck plain urine were greater than those for buck SE urine (fig. 11; p<0.05), but mean time spent in IZs also did not differ (fig. 10).

DISCUSSION

In both experimental protocols, estrous does displayed preference for buck urine over wether urine. In experiment 1, estrous does displayed fewer investigative behaviors than in experiment 2. This marked difference may be explained by the difference in urine presentation. In experiment 1, the urine was displayed on the fleece of ovx does, which stood behind a fence barrier. In this protocol, the does were not able to directly interact and investigate the urine choices at all times, though the scent was still present and detectable in the IZ. In contrast, experiment 2 allowed the does to freely interact with the urine choices as if it they were being voided by a live goat. Behaviors characteristic of olfactory investigation of urine such as sniffing, touching, lapping with their tongues, and displaying flehmen were displayed by test does while investigating the urine fountains.

These types of interactions were observed at much lower frequencies in the first experimental protocol. The frequency of investigative behaviors in experiment 2 differed between some urine choices. Most notably, mean touches and flehmen lip curls during a

5-minute test were greater for does investigating plain buck urine than for those investigating wether urine (P<0.05)

In the next two comparisons (DHT-treated wether urine vs. EB-treated wether urine and buck SE urine vs. buck plain urine), time spent in IZs was decreased. Using the same does for all three comparisons, the does began to lose interest in the fountain apparatus and thus spent less time investigating the choices. While differences in preference scores and frequencies of investigative behaviors may be deemed statistically significant, less time investigating the choices makes it unclear whether or not the observed discriminations were meaningful.

The comparison of DHT-treated wether urine and EB-treated wether urine was performed to determine if androgens or estrogens were responsible for the attractive properties of buck urine. Treatment of castrated males or ovariectomized (ovx) females with testosterone (T) or dihydrotestosterone (DHT) promotes pheromone activity in the skin of certain regions, particularly the head and rump (Iwata et al., 2000b). More than 25 fatty acids are found in the lipid fraction of buck fleece that are not present in male castrate or female fleece (Hillbrick et al., 1995; Jenkinson et al., 1967; Sugiyama et al.,

1986). Pheromones in buck fleece are capable of increasing the luteinizing hormone (LH) pulse frequency and inducing estrus in females (Iwata et al., 2000b; Kakuma et al., 2007).

Regional specificity of pheromone production is correlated with expression of 5-α reductase converting T to DHT (Wakabayashi et al., 2000). The presence or absence of 5-

α reductase in the urinary tract or of pheromones in goat urine have not been studied or identified. Several volatile compounds that may serve as chemical cues have been identified in the urine of other species, such as male red deer (Bakke and Figenschou,

1990), female white-tailed deer (Jemiolo et al., 1995), female buffalo (Rajanarayanan and

Archunan, 2011), female Asian elephants (Rasmussen et al., 1997) and domesticated cows (Dehnhard et al., 1991). While pheromone activity in the sebaceous glands in male goats has been shown to be DHT-dependent, endocrine influence and identification of urinary pheromones in goats and other ungulates has not been studied.

It is unknown whether or not urine voided from a buck during SE differs chemically from urine voided during a plain urination. Since this was the final comparison presented to the does, they appeared to spend the least time investigating

80 these choices. At this time, we have not determined if the composition of buck SE urine differs from that of plain buck urine and if that difference is detectable by does.

Olfactory cues, in the absence of all other sensory stimuli, do not account for all of the properties of the buck that are attractive to estrous does. Without the buck’s entire presentation of visual, auditory and tactile cues, does do not have enough context to make discrete sexual choices (Longpre and Katz, 2011). However, the scent of a buck’s urine was detectable by test does and in some cases, it was preferable or more engaging even in the absence of the buck. In both presentation formats (experiments 1 and 2), estrous does displayed preference for buck SE urine when compared to wether urine. Thus, it appears that urine is an important part of the buck’s suite of sexual cues and is capable of attracting an estrous female. It is thus plausible that SE is used by the buck to display olfactory cues in his urine to attract females. However, this may not be the only purpose of SE, as it does not explain the display of flehmen that is frequently shown by males after investigating their own urine during SE.

7.6m

1.5m 1.5m 3.1m 1.5m OVX OVX 3.7m 0.6m

Incentive Zone Neutral Zone Incentive Zone OVX OVX

Figure 1. Behavior test apparatus for experiment 1: Two ovx does were placed at each end of the apparatus, in separate pens, carrying the urine choices. Both ovx does on one side carried the same urine type. Observations were made through a fence barrier in the aisle adjacent to the lower barrier in the diagram.

4.6m

IZ Neutral Zone IZ 1.5m

1.5m 3.1m 3.7m

Observation Area 1.5m OVX OVX

Figure 2. Behavior test apparatus for experiment 2: The square with circle inside at either end of the test area represents the fountain depicted below. Observations were made in the observation area. OVX does were placed in respective pens to decrease isolation stress in test does.

Figure 3. Urine recycling fountain for experiment 2: A funnel is inside the wooden box and below it is a container that collects urine falling through the funnel and holds the pump. Vinyl tubing carried urine up and out of a routing pipe, pumping a constant stream cycling back into the funnel.

Buck SE Urine vs. Wether Urine n=13

140 *

120

100

40 Mean Time in IZ (sec)

0 Buck SE Wether

Figure 4. Estrous females spent more time near ovx does carrying buck SE urine than those carrying wether urine: Mean (± SEM) time spent in buck SE urine IZ vs. wether urine IZ for estrous does (n=13) during 5-min preference test for experiment 1.

Urine choices were presented on the integument of OVX does. Asterisk denoted significant difference (p<0.05).

Buck SE Urine vs. Wether Urine n=13

100

80 *

Mean Preference Score (%) 20

0 Buck SE Wether

Figure 5. Estrous females prefer buck SE urine over wether urine: Mean (± SEP) preference scores of estrous does (n=13) for buck SE urine vs. wether urine presented on the integument of OVX does during 5-min preference test for experiment 1. Asterisk denotes significant difference (p<0.05).

Experiment 2: Mean frequency of investigative behaviors Urine Choice IZ Entries Examinations Touches Flehmens Urinations

Buck SE 4.1 ± 0.4 *2.3 ± 0.4 1.0 ± 0.3 *1.4 ± 0.5 0.3 ± 0.1 Wether plain 3.5 ± 0.5 1.4 ± 0.3 0.8 ± 0.3 0.4 ± 0.2 0.0 ± 0.0

DHT-treated wether 3.0 ± 0.5 *1.6 ± 0.3 0.6 ± 0.2 0.6 ± 0.2 0.1 ± 0.1 EB-treated wether 2.7 ± 0.5 0.7 ± 0.2 0.6 ± 0.5 0.4 ± 0.4 0.2 ± 0.1

Buck SE 2.6 ± 0.4 1.0 ± 0.4 0.9 ± 0.7 0.3 ± 0.3 0.2 ± 0.1 Buck plain 2.7 ± 0.5 2.2 ± 0.7 0.5 ± 0.2 *1.5 ± 0.5 0.4 ± 0.2

Table 3. Mean frequency of investigative behaviors in experiment 2: Frequency of behaviors are expressed as the mean (± SEM) number of behaviors occurring over a 5- minute behavior test period for estrous does (n=10). Only data from does that visited at least one of the choices were included. Asterisks denote significant differences (p<0.05).

Buck SE Urine vs. Wether Urine n=10

40 *

Mean Time in IZ (sec) 10

0 Buck SE Wether

Figure 6. Estrous females spent more time near buck SE urine than wether urine fountain: Mean (± SEM) time spent by estrous does in buck SE urine fountain IZ vs. wether urine fountain IZ for estrous does (n=10) during 5-min preference test in experiment 2. Asterisk denotes significant difference (p<0.05).

Buck SE Urine vs. Wether Urine n=10

100 * 80

Mean Preference Score (%) 20

0 Buck SE Wether

Figure 7. Estrous females prefer buck SE urine over wether urine fountain: Mean (±

SEP) preference scores of estrous does (n=10) for buck SE urine vs. wether urine presented in fountains during 5-min preference test in experiment 2. Asterisk denotes significant difference (p<0.05)

DHT-treated Wether Urine vs. EB-treated Wether Urine n=8

Mean Time in IZ (sec) 10

0 DHT Wether EB Wether

Figure 8. Time spent near DHT-treated wether urine vs. EB-treated wether urine:

Mean (± SEM) time spent for estrous does (n=8) in DHT-treated wether urine fountain IZ vs. EB-treated wether urine fountain IZ during 5-min preference test in experiment 2.

DHT-treated Wether Urine vs. EB-treated Wether Urine n=8

100

* 80

Mean Preference Score (%) 20

0 DHT Wether EB Wether

Figure 9. Estrous females prefer DHT-treated wether urine over EB-treated wether urine: Mean (± SEP) preference scores of estrous does (n=8) for DHT-treated wether urine vs. EB-treated wether urine fountains during 5-min preference test in experiment 2.

Asterisk denotes significant difference (p<0.05)

Buck SE Urine vs. Buck Plain Urine n=8

Mean Time in IZ (sec) 10

0 Buck SE Buck Plain

Figure 10. Time spent near buck SE urine vs buck plain urine: Mean (± SEM) time spent for estrous does (n=8) in buck SE urine fountain IZ vs. buck plain urine fountain IZ during 5-min preference test in experiment 2.

Buck SE Urine vs. Buck Plain Urine n=8

100 * 80

Mean Preference Score (%) 20

0 Buck SE Buck Plain

Figure 11. Estrous females prefer Buck plain urine over buck SE urine: Mean (±

SEP) preference scores of estrous does (n=8) for buck SE urine fountain vs. buck plain urine fountain during 5-min preference test in experiment 2. Asterisk denotes significant difference (p<0.05)

REFERENCES

Bakke, J.M., Figenschou, E., 1990. Volatile compounds from the red deer (Cervus elaphus). substances secreted via the urin. Comparative Biochemistry and Physiology Part A: Physiology 97, 427-431. Bronson, F., Whitten, W., 1968. Oestrus-accelerating pheromone of mice: assay, androgen-dependency and presence in bladder urine. Journal of reproduction and fertility 15, 131-134. Brown, R.E., 1977. Odor preference and urine-marking scales in male and female rats: Effects of gonadectomy and sexual experience on responses to conspecific odors. Journal of Comparative and Physiological Psychology 91, 1190. Claus, R., Over, R., Dehnhard, M., 1990. Effect of male odour on LH secretion and the induction of ovulation in seasonally anoestrous goats. Animal Reproduction Science 22, 27-38. Coblentz, B.E., 1976. Functions of scent-urination in ungulates with special reference to feral goats (Capra hircus L.). American Naturalist 110, 549-556. Cohen-Tannoudji, J., Einhorn, J., Signoret, J.P., 1994. Ram sexual pheromone: First approach of chemical identification. Physiology and Behavior 56, 955-961. Dehnhard, M., Claus, R., Pfeiffer, S., Schopper, D., 1991. Variation in estrus-related odors in the cow and its dependency on the ovary. Theriogenology 35, 645-652. Ferkin, M.H., Sorokin, E.S., Renfroe, M.W., Johnston, R.E., 1994. Attractiveness of male odors to females varies directly with plasma testosterone concentration in meadow voles. Physiology & behavior 55, 347-353. Hart, B.L., Jones, T.C., 1975. Effects of castration on sexual behavior of tropical male goats. Hormones and Behavior 6, 247-258. Hau, M., 2007. Regulation of male traits by testosterone: implications for the evolution of vertebrate life histories. BioEssays 29, 133-144. Hillbrick, G., Tucker, D., Smith, G., 1995. The lipid composition of cashmere goat fleece. Crop and Pasture Science 46, 1259-1271. Iwata, E., Wakabayashi Y Fau - Kakuma, Y., Kakuma Y Fau - Kikusui, T., Kikusui T Fau - Takeuchi, Y., Takeuchi Y Fau - Mori, Y., Mori, Y., 2000a. Testosterone- dependent primer pheromone production in the sebaceous gland of male goat. Biology of Reproduction 62, 806-810. Iwata, E., Wakabayashi Y Fau - Matsuse, S., Matsuse S Fau - Kikusui, T., Kikusui T Fau - Takeuchi, Y., Takeuchi Y Fau - Mori, Y., Mori, Y., 2000b. Induction of primer pheromone production by dihydrotestosterone in the male goat. Jemiolo, B., Miller, K., Wiesler, D., Jelinek, I., Novotny, M., Marchinton, R., 1995. Putative chemical signals from white-tailed deer (Odocoileus virginianus). Urinary and vaginal mucus volatiles excreted by females during breeding season. Journal of chemical ecology 21, 869-879. Jenkinson, D.M., Blackburn, P., Proudfoot, R., 1967. Seasonal changes in the skin glands of the goat. The British veterinary journal 123, 541-549. Johnson, R.P., 1973. Scent marking in mammals. Animal Behaviour 21, 521-535. Johnston, R., Bronson, F., 1982. Endocrine control of female mouse odors that elicit luteinizing hormone surges and attraction in males. Biology of Reproduction 27, 1174-1180.

Kakuma, Y., Ichimaru T Fau - Yonezawa, T., Yonezawa T Fau - Momozawa, Y., Momozawa Y Fau - Hashizume, C., Hashizume C Fau - Iwata, E., Iwata E Fau - Kikusui, T., Kikusui T Fau - Takeuchi, Y., Takeuchi Y Fau - Ohkura, S., Ohkura S Fau - Okamura, H., Okamura H Fau - Mori, Y., Mori, Y., 2007. Androgen induces production of male effect pheromone in female goats. Journal of Reproduction and Development 53. Keverne, E., Michael, R., 1970. Annual changes in the menstruation of rhesus monkeys. Journal of Endocrinology 48, 669-670. Knight, T.W., Lynch, P.R., 1980. Source of ram pheromones that stimulate ovulation in the ewe. Animal Reproduction Science 3, 133-136. Ladewig, J., Hart, B.L., 1980. Flehmen and Vomeronasal organ function in male goats. Physiology and Behavior 24, 1067-1071. Ladewig, J., Price, E.O., Hart, B.L., 1980. Flehmen in male goats: role in sexual behavior. Behavioral and Neural Biology 30, 312-322. Longpre, K.M., Katz, L.S., 2011. Estrous female goats use testosterone-dependent cues to assess mates. Hormones and behavior 59, 98-104. Miquelle, D.G., 1991. Are moose mice? The function of scent urination in moose. The American Naturalist 138, 460-477. O'Brien, P.H., 1982. Flehmen: its occurrence and possible functions in feral goats. Animal Behavior 30, 1015-1019. Okamura, H., Mori, Y., 2005. Characterization of the Primer Pheromone Molecules Responsible for the ‘Male Effect’ in Ruminant Species. Chemical Senses 30, i140-i141. Ott, R., Nelson, D., Hixon, J., 1980. Peripheral serum progesterone and luteinizing hormone concentrations of goats during synchronization of estrus and ovulation with prostaglandin F2 alpha. American journal of veterinary research 41, 1432- 1434. Over, R., Cohen-Tannoudji, J., Dehnhard, M., Claus, R., Signoret, J.P., 1990. Effect of pheromones from male goats on LH-secretion in anoestrous ewes. Physiology and Behavior 48, 665-668. Owen-Smith, N., 1977. On territoriality in ungulates and an evolutionary model. The Quarterly Review of Biology 52, 1-38. Petrulis, A., Johnston, R.E., 1995. A reevaluation of dimethyl disulfide as a sex attractant in golden hamsters. Physiology & behavior 57, 779-784. Price, E.O., Smith, V.M., Katz, L.S., 1986. Stimulus conditions influencing self- enurination, genital grooming and flehmen in male goats. Applied Animal Behaviour Science 16, 371-381. Rajanarayanan, S., Archunan, G., 2011. Identification of urinary sex pheromones in female buffaloes and their influence on bull reproductive behaviour. Research in veterinary science 91, 301-305. Ralls, K., 1971. Mammalian scent marking. Science 171, 443-449. Rasmussen, L., Lee, T.D., Zhang, A., Roelofs, W.L., Daves, G.D., 1997. Purification, identification, concentration and bioactivity of (Z)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus. Chemical Senses 22, 417-437. Schneider, K.M., 1930. Das Flehmen. Akad. VerlagGes.

Shank, C.C., 1972. Some Aspects of Social Behaviour in a Population of Feral Goats (Capra hircus L.). Zeitschrift für Tierpsychologie 30, 488-528. Sugiyama, T., Matsuura, H., Sasada, H., Masaki, J., Yamashita, K., 1986. Characterization of Fatty Acids in the Sebum of Goats According to Sex and Age (Biological Chemistry). Agricultural and biological chemistry 50, 3049-3052. Wakabayashi, Y., Iwata, E., Kikusui, T., Takeuch, Y., Mori, Y., 2000. Regional differences of pheromone production in the sebaceous glands of castrated goats treated with testosterone. The Journal of Veterinary Medical Science 62, 1067- 1072. Yahr, P., Newman, A., Stephen, D.R., 1979. Sexual behavior and scent marking in male gerbils: Comparison of changes after castration and testosterone replacement. Hormones and behavior 13, 175-184.

CHAPTER IV

EFFECTS OF SIMULATED SELF-ENURINATIONS ON

CIRCULATING CONCENTRATIONS OF LUTEINIZING

HORMONE AND TESTOSTERONE IN THE MALE GOAT

ABSTRACT

Self-enurination (SE), self-marking with urine, in male goats (bucks) is androgen- dependent and occurs primarily during the breeding season. The behavior is characterized by the downward turning of the head and shoulders towards the hindquarters, immediately followed by the emission of urine from the erect penis onto the face, beard, and front legs. The urine is delivered in a manner ranging from a spray to a narrow stream. SE frequently includes the buck lapping his urine, and may conclude with him grooming his penis with his tongue. The flehmen response is almost always displayed immediately following SE. The flehmen response is used by the goat to facilitate the entry of non-volatile chemicals into the accessory olfactory system. The adaptive significance of both SE and the flehmen that usually follows is unknown. Unpublished work in our lab demonstrated that SE may be used to present attractive olfactory cues to females. SE may also serve as a self-excitatory sexual behavior, whereby urine or chemicals therein stimulate the hypothalamo-pituitary-gonadal (HPG) axis via accessory olfactory system excitation. A series of experiments were conducted to test this hypothesis using simulated self-enurinations. A simulated SE involves spraying urine through a modified 22 gauge needle attached to a syringe to deliver a titratable spritz directed at the buck’s nose. In the first three experiments, T or LH were measured in blood samples taken following spritz(es) with SE urine or 0.9% saline. In the first experiment, conducted during the transition out of the breeding season, bucks were exposed to a single spritz and a testosterone increase resulted (P<0.05). Next, bucks were tested in a similar experimental design conducted during the breeding season, in which an

LH response to SE urine was not significant. This may have been due to LH’s

98 suppression by elevated T during the breeding season. Next, a series of four spritzes were used during the nonbreeding and transition into the breeding seasons. Serum LH concentrations tended to be higher in bucks receiving SE urine during the nonbreeding season and were higher following SE urine spritzes during the transition into the breeding season (P<0.05). Finally, the protocol was modified to include additional baseline samples, samples for LH as well as T, and an air control instead of saline in an experiment conducted during the transition into the breeding season. While the LH response was not significant, a significant T response was measured (P<0.05). In all experiments, except experiment 2, bucks that received SE urine were more likely to display flehmen and in experiments where flehmen response duration was recorded, the average flehmen lip curl duration was greater following treatment with SE urine

(P<0.05). While modifications in methodology are necessary for a clearer conclusion, it appears that SE may stimulate the neuroendocrine system of the buck to increase LH and

T for reproductive success.

INTRODUCTION

Males of species in the subfamily Caprinae exhibit a behavior during the breeding season in which parts of the body are impregnated with the scent of their own urine. Self- enurination (O'Brien, 1982), scent-urination (Coblentz, 1976), or urine marking (Shank,

1972) as it is often called, involves direct urination into areas in which hair is the longest

(Coblentz, 1976). In the male domesticated goat (buck), the behavior is identified by the downward turning of the head and shoulders towards the hindquarters and the emission of urine from the erect penis onto the face, beard and front legs (Price et al., 1986). It has even been suggested that the buck’s characteristic beard may have evolved to serve as a reservoir for the presentation of chemical signals in the urine (Coblentz, 1976). The urine may be delivered in a manner ranging from a spray to a precise stream and can be released in small increments or in a consistent, steady flow (Price et al., 1986). By achieving a convex curvature of the spine, the goat is able to place his mouth and nose in direct contact with the stream, often lapping the urine with his tongue (Price et al., 1986).

The event sometimes concludes with the individual grooming the penis with his tongue after the stream has ended (Price et al., 1986). Self-enurination (SE) is usually followed by the flehmen response, which allows for the detection of non-volatile chemical signals via the vomeronasal organ (VNO) and is characterized by the upward turning of the head and curling of the upper lip (Ladewig and Hart, 1980; Schneider, 1930).

In signaling theory, an honest signal is described as a signal that is continually elicited despite some degree of harm or danger. Most scent-marking behaviors have little cost besides leaving or emitting a scent that may be detected by predators, but in the case of SE, the buck will continue to display the behavior even though it physically harms

100 him. Over time, the urine’s caustic nature causes hair loss, irritation, and in some cases, open wounds which may become infected. While the importance of SE is demonstrated by its honesty, its adaptive significance is not clear.

Contextual evidence may provide some clues as to the purpose SE serves to the buck. Self-enurination is displayed in its highest frequencies during the breeding season, when T is elevated. Also, unpublished work in our lab has shown SE to be activated by androgens, specifically the non-aromatizable androgen dihydrotestosterone. Price et al.

(1986) came to the conclusion that there is a direct, positive correlation between SE and the amount of sexual activity in a heterosexual bout of mating in the goat. This study also concluded that SE was primarily exhibited before or after primary courtship and copulation (Price et al., 1986). In addition, they noticed that a buck allowed visual and auditory contact with an estrous female without direct contact for courtship and copulation often displayed SE (Price et al., 1986). These findings suggest that a degree of sexual arousal is necessary to increase the likelihood of the behavior. However, SE is also observed in bucks when females are not present and other males in the herd may show interest in urine being voided during SE by lapping it with their tongues and displaying flehmen.

Though many hypotheses have been presented to explain the adaptive significance of such a behavior, none have been successfully demonstrated by experimentation. While the etiology of the behavior remains unclear, the flehmen response suggests SE may provide olfactory cues supportive to reproduction, mate selection and possibly social rank (Coblentz, 1976; Price et al., 1986; Shank, 1972). The pulsing erection, the entry of the penis into the oral cavity and the increased frequency of

101 the behavior during the breeding season and during sexual arousal suggest that the behavior serves a reproductive purpose. While the behavior is widely assumed to have olfactory significance, it is not clear whether the behavior is directed towards females, other males, or both. McCullough (1969) presented the idea that the urine carries information pertinent to the individual’s physiological condition. He cites the presence of metabolic by-products in the urine, which may advertise a buck’s physiological state to other members of the herd (McCullough, 1969). From an evolutionary standpoint, a dominant buck that advertises his physiological condition would be allowing himself to conserve energy for tasks directly related to reproduction by decreasing the likelihood of challenges with predictable outcomes (Coblentz, 1976). It is possible that similar to the way in which the odor of urine changes with diet, it may change when the male begins to metabolize tissues other than fat for energy (McCullough, 1969). In the goat, dominant males rarely eat enough to maintain their weight since tending females requires constant attention. This leads to rapid weight loss, which may lead to a change in urine odor that would suggest increased odds of winning a challenge to a subordinate male.

SE, it is likely that his urine is transferred to the doe’s hair and skin during mounting.

Perhaps it is possible that the presence of dominant buck urine on the integument of a female would communicate to other males that she has already been bred and that a breeding attempt may result in conflict with a dominant buck. Male goats are often seen

102 rubbing their heads on objects in the environment, again suggesting the potential for spreading signals in urine. While female attractant and male aversive properties of urine have been observed in rodents (Jones and Nowell, 1973, 1974; Jones and Nowell, 1989;

Sawyer, 1978), the field has been mostly unexplored in goats and other domesticated animals.

While these predictions may explain the evolution of such a behavior, SE also appears to play some role in attracting the opposite sex. A bout of SE is often witnessed before or after courtship displays or immediately following ejaculation, suggesting the necessity of a reproductive incentive. Unpublished work in our lab has shown that estrous female goats show preference for buck urine from an SE or plain urination when compared to wether urine. In addition to attraction, SE may also aid females in identifying dominant males. By finding and mating with only the dominant males, the female would decrease the chances of interference from other males (Coblentz, 1976).

Separate from attraction is the idea that, like buck hair, pheromones contained in the buck’s urine may hasten and synchronize the onset of estrus in females. In a species in which dominant male turnover is quite high, this would benefit the male by allowing him to breed a greater number of females while he is the dominant buck. In turn, females that respond to the male’s stimulus would ensure breeding by the most dominant males

(Coblentz, 1976). In summary, a buck may use SE to quickly synchronize all of the females (at least the healthy ones) in a herd, ensuring that they would all be bred by the most dominant buck and thus increasing reproductive success for both sexes (Coblentz,

1976). While the behavior may have evolved to attract or synchronize female conspecifics, its pervasiveness following thousands of years of domestication where

103 mate-seeking is facilitated or eliminated suggests that the behavior may have evolved to serve multiple purposes. Additionally, this explanation does not explain the significance of the flehmen response that often follows.

A final hypothesis, which is the one that is tested in this study, suggests that the behavior is involved in a form of sexual stimulation or arousal directed at the individual eliciting the behavior (Price et al., 1986). It has been postulated that the male goat may have evolved a mechanism to detect primer pheromones present in his own urine that may be used to affect his own endocrine physiology (Price et al., 1986). The idea of pheromones acting on the self has only been demonstrated in one other instance in which male rats respond to their own alarm pheromone by releasing stress hormones (Inagaki et al., 2012). It has been shown that buck fleece (containing urine) is capable of stimulating increased luteinizing hormone (LH) pulse frequency in females, so it is plausible that similar physiological pathways may exist in the male (Chemineau, 1987; Chemineau et al., 1986; Knight et al., 1978; Lindsay, 1966; Martin et al., 1986; Pearce and Oldham,

1984; Schinckel, 1954b; Shelton, 1960; Shelton, 1980; Underwood et al., 1944;

Walkden-Brown et al., 1993). From an evolutionary standpoint, being able to increase testosterone (T) concentrations would be a distinct advantage for reproductive success.

To take this theory one step further, the male may be using this mechanism as a positive feedback loop to increase T in preparation for the breeding season. Since T is known to activate the behavior, the behavior’s ability to increase T would complete a positive feedback loop. Perhaps the strongest point of evidence for this theory is the male’s tendency to display flehmen following a bout of SE. By displaying flehmen, it is assumed that the buck is receiving his own scent via the accessory olfactory system.

104

Detection of non-volatile chemical cues via the flehmen and VNO is carried out by the accessory olfactory system. Unlike the main olfactory system, chemical signals enter via the oral cavity, beginning at the nasopalatine ducts, which present as two medial openings in the rostral hard palate caudal to an incisive papilla. The two cigar-shaped, blind-ended, mucus-filled VNO ducts run parallel and branch caudally from the nasopalatine ducts (Ladewig et al., 1980). The VNO is embedded in the hard palate along the junction of the palate and nasal septum (Besoluk et al., 2001; Ladewig and Hart,

1980; Ladewig et al., 1980; Melese-d'Hospital and Hart, 1985). The goat VNO is approximately 7cm long and 3-4mm wide and is protected by a thin cartilaginous layer

(Besoluk et al., 2001). The sensory epithelium of the VNO presents along the medial wall of the medial and posterior segments of the VNO lumen (Ladewig and Hart, 1980).

Surrounding the lumen of the VNO is erectile tissue, which has been associated with a pumping mechanism used to draw materials into the lumen (Salazar and Quinteiro,

1998). It is believed that by constricting the nares during flehmen, the goat creates a vacuum in the VNO upon inhalation (Salazar and Quinteiro, 1998). This vacuum effect, combined with negative pressure caused by relaxation of the erectile tissue surrounding the lumen, causes materials from the oral cavity to be drawn into the VNO lumen (Estes,

1972; Ladewig and Hart, 1980; Ladewig et al., 1980). Two experiments using fluorescent dye have determined that the flehmen response is necessary for the detection of non- volatile materials via the VNO. Only goats that performed the flehmen response when presented with non-volatile fluorescent materials showed fluorescence past the anterior portion of the VNO lumen (Ladewig and Hart, 1980; Melese-d'Hospital and Hart, 1985).

105

In the female goat, studies of the “male effect” have described pathways in which non-volatile chemical cues invoke endocrine responses in the recipient. We postulate that these signaling pathways are present in the male goat as well and are used by the buck to influence his own endocrine system, using pheromones present in his own urine. In the

“male effect,” these pheromones, once inside the VNO, act as ligands to activate vomeronasal receptor neurons. Vomeronasal receptor neurons are bipolar primary sensory neurons that possess a dendrite that extends to the surface of the sensory epithelium (Døving and Trotier, 1998; Halpern and Martı́nez-Marcos, 2003; Witt and

Wozniak, 2006). From the basal pole of the cell body extends a long axon that travels to the AOB (Døving and Trotier, 1998; Halpern and Martı́nez-Marcos, 2003; Witt and

Wozniak, 2006). The ligand binds to G protein-coupled putative pheromone receptors located on the apical microvilli of the terminal dendrite’s terminal knob, ultimately causing a change in membrane potential, sending a signal to the AOB (Døving and

Trotier, 1998; Halpern and Martı́nez-Marcos, 2003; Wakabayashi et al., 2002; Witt and

Wozniak, 2006). The bed nucleus of the stria terminalis (BNST) innervates the AOB and is also innervated by some areas of the amygdala and the supraoptic nucleus (Døving and

Trotier, 1998; Halpern and Martı́nez-Marcos, 2003; Witt and Wozniak, 2006). It is proposed that through these connections, primer pheromones influence physiological function, as well as social and sexual behavior, as demonstrated in studies of the “male effect” in sheep and goats. In this case, the buck may be using pheromones present in his own urine to stimulate the GnRH pulse generator through these pathways as the response has been successfully demonstrated by does in response to buck hairs or isolated buck pheromones (Hamada et al., 1996; Mori et al., 1991).

106

Included in this self-stimulatory prediction is the idea that perhaps such a behavior could be used to shorten the duration of the post-ejaculatory refractory period or accelerate the onset of ejaculation prior to mounting by affecting post-ejaculatory hormones or their receptors. Price et al. (1986) concluded that SE was primarily exhibited before or after primary courtship and copulation and reliably during the post-ejaculatory refractory period. Again, this type of endocrine modification would result in increased reproductive success for the male. The objective of this experiment is to test the hypothesis that the buck uses chemical cues in his urine that he presents to himself by SE to stimulate his hypothalamic-pituitary-gonadal (HPG) axis to increase T concentrations for reproductive success via flehmen and the accessory olfactory system.

107

MATERIALS AND METHODS

Animals

Research was conducted as approved by the Rutgers University Animal Care and

Facilities Committee. Bucks were housed on the NJ Agricultural Experiment Station

Research Farm in New Brunswick, NJ (40°29′10″N/74°27′8″W) in barns with free access to outdoor exercise areas. The breeding season for Alpine goat begins in mid-August and terminates near the end of January in the northern hemisphere. Focal goats in all experiments were gonadally intact males.

Urine Collection

Self-enurination urine was collected using a specially designed urine catching apparatus with a bag attached to the end of a wooden dowel. The apparatus was used to intercept the stream of urine during a bout of SE. Self-enurination was stimulated for collection by leading bucks up to a pen containing does, allowing the bucks to court (and in experiment 1, mount and intromit), then isolating them from the does, which would often lead to an SE. All urine used in these experiments was collected between October and December of the breeding season prior to the respective experiment. In experiment 1, bucks were spritzed with their own SE urine, and in all other experiments, pooled SE urine was used.

Simulated Self-enurination

108

Simulated self-enurination (spritz) was performed by delivering a measured quantity of SE urine or a control through a syringe with a modified 22 gauge needle. The modified needle is modeled after the buck’s erect penis; bending the tip of the needle upwards to resemble the urethral process. Similar to how urine from the buck during SE exits the urethra and hits the urethral process, dispersing the urine, depression of the syringe’s plunger causes emission of the urine or control from the needle’s cylinder, which collides with the upward tip to create a mist or spray. Prior to delivery, the syringe and its contents were warmed by placing under the armpit or on a hand-warmer to bring them closer to the temperature of recently voided goat urine. The spritz was delivered at a distance of approximately 30cm and aimed at the nose at the facial midpoint, just above the nasal openings. The reliable display of prolonged flehmen by bucks following simulated self-enurinations with urine or saline, confirms that detection via the VNO had taken place.

Luteinizing Hormone Radioimmunoassay (RIA)

Blood was allowed to clot at room temperature for one hour. Serum was separated by centrifugation at 1600 g for 15 min and stored at -20ºC until analysis of LH concentrations was performed. Luteinizing hormone radioimmunoassays were performed in the laboratory of our collaborator Brenda Alexander, Ph.D. at the University of

Wyoming. Concentrations of serum LH in all samples were determined by RIA (Forest et al., 1981) using a tisera described by Adams et al. (1975). Radioiodinated LH (LER

1268-1) was used as the labeled antigen. Unknowns were expressed as nanograms of

NIH-LH-S18 reference preparation. Inter-assay coefficient of variation was 2.4% in

109 experiment 2, 12.4% in experiment 3, and 5.6% in experiment 4. The minimum detection limit was 0.2 ng/mL in experiments 2, 3 and 4.

Tetsosterone Radioimmunoassay (RIA)

Blood was allowed to clot at room temperature for one hour. Serum was separated by centrifugation at 1600 g for 15 min and stored at -20ºC until analysis of concentrations of T was performed. Serum T concentrations were determined by commercial RIA kits validated in our laboratory for goat serum. In experiment 1, the RIA kit used was

Siemens Healthcare Diagnostics Inc. Coat-A-Count® Total Testosterone (Los Angeles,

CA) and in experiment 4, the MP Biomedicals ImmuChem® Double Antibody kit for

Testosterone (Santa Ana, CA) was used. The minimum detection limit of the experiment

1 assay was 0.01 ng/ml and the inter-assay coefficient of variation was 3.1%. In experiment 4, the minimum detection limit was 0.02 ng/ml, the inter-assay coefficient of variation was 5.6%.

Experiment 1: T during the transition out of the breeding season

In this experiment, the change in T concentrations following a simulated SE was analyzed using bucks (n=12; age 2-9 yr) during January-February 2013 (transition out of the breeding season). Self-enurination urine used was collected from an SE after the buck intromitted and ejaculated. Bucks were treated in a switchback design in which each buck received one spritz and a series of blood sampling for each of the two treatments; his own

SE urine or 0.9% saline. In Week 1, six bucks were tested on one day, with three bucks receiving SE urine and three receiving saline. The other six bucks were tested similarly on a different day that week. In Week 2, the switchback for each group of six was performed. Testing was performed at the same time each day in week 1 and week 2 to

110 control for time of day fluctuations of testosterone concentrations. The order in which the bucks were tested on a given day was randomized, but that randomized order was repeated for the switchback to control for time of day. Blood was collected by jugular venipuncture from each buck at the following time points relative to SE urine or saline application: -20, 0, 20, 40, 60, and 80 minutes. At the zero-time point, blood was collected prior to application of SEU or SAL.

Experiment 2: LH during the breeding season

In this experiment, the presence of a luteinizing hormone (LH) response following a simulated SE was analyzed using bucks (n=14; age 2-9 yr) during October-November

2013 (during the breeding season). Self-enurination urine used was collected from an SE following courtship towards does, but different from experiment 1, only if the buck had not intromitted and ejaculated. Bucks were treated in a switchback design, with the same experimental design as in experiment 1, and the same control (0.9% saline) was used.

Different from experiment 1, a 2mL spritz was used instead of 1mL to provide longer exposure to treatments. In this experiment, treatments were administered in isolated, designated areas to prevent cross-contamination. Bucks were only tested if they had not self-enurinated in the 20 minutes prior to the start of their test and SE was blocked or deterred if an attempt was made by the buck during a test. Blood was collected by jugular venipuncture from each buck at the following time points relative to SE urine or saline application: -10, -5, 0, 5, 10, 15, and 20 minutes. At the zero time point, blood was again collected prior to application of SEU or SAL.

Experiment 3: LH during the non-breeding and transition into the breeding season

111

Similar to experiment 2, the presence of an LH response following a simulated SE was analyzed in a two-part experiment using bucks (n=14; age 2-7 yr) from June 23-July

9, 2015 (the non-breeding season) and from July 28-August 13, 2015 (the transition into the breeding season. Some methodological changes were made after findings from experiment 2 were analyzed in an effort to further simulate the natural occurrence of SE and better detect an LH response, if present. Self-enurination urine used was collected from an SE following courtship towards does, as in experiment 2, but SE was pooled for treatment in this experiment. Bucks were treated in a switchback design, with the same experimental design as in experiment 1 and 2, and the same control (0.9% saline) was used. Differing from experiment 1, four 1mL spritzes were administered 15 minutes apart to further prolong and repeat exposure to treatments to further mimic SE as it would occur in a natural setting. Again, treatments were administered in isolated, designated areas to prevent cross-contamination and bucks were only tested if they had not self- enurinated in the 20 minutes prior to the start of their test and SE was blocked or deterred if an attempt was made by the buck during a test. Blood was collected beginning at 0700 hr (0900 hr in experiments 1 and 2) as findings in rams showed that an LH response was best detected early in the morning, close to sunrise (Gonzalez et al., 1988b; Signoret,

1991). Blood was collected by jugular venipuncture from each buck at the following time points relative to the fourth and final SE urine or saline spritz: -45, 5, 10, 15, 20, 25 minutes.

Experiment 4: LH and T during the transition into the breeding season

In the final experiment, the presence of the hypothesized LH and subsequent T response following a simulated SE was tested using bucks (n=14; age 2-7 yr) from

112

August 1-24, 2015 (the transition into the breeding season). Self-enurination urine used was again collected from an SE following courtship towards does and pooled. Bucks were treated in a switchback design, with a similar experimental design as in experiment

1 and 2, but the switchbacks occurred over a span of three weeks to avoid days with unusually high temperatures during a heat wave that occurred in our region. In this experiment, air was used as the control to prevent the re-liquefying urine residue on the bucks’ faces that may have occurred from treatment with saline. Again, four 1mL spritzes were administered 15 minutes apart, in isolated, designated areas. In this experiment, an additional control measure was added: the treatment administered in each area was switched each day, so that each buck received his SE urine and air treatments in the same isolated area, and half of the bucks received both treatments in one area and the other half in the other. Again, bucks were only tested if they had not self-enurinated in the 20 minutes prior to the start of their test and SE was blocked or deterred if an attempt was made by the buck during a test. Blood was again collected beginning at 0700 hr by jugular venipuncture from each buck at the following time points relative to the fourth and final SE urine or air spritz: -65, -55, -45, 5, 10, 20, 30, 45 and 60 minutes. To collect the additional baseline and T response samples, a 2.5% lidocaine/2.5% prilocaine cream was applied to the skin over the jugular veins and left on the skin for one hour prior to the first baseline sample. Cream was applied at an approximate rate of 7.5 g per side of neck

(based on covering a 9-inch length and 1.5 inch width shaved area). Before the baseline sample, the cream was wiped off and the neck was cleaned with a 70% ethanol rinse and gauze.

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Statistical Analysis

Flehmen duration was analyzed by independent sample t-test. Flehmen probability was analyzed using the chi-square test for independence. Luteinizing hormone and T concentrations were analyzed by repeated measures analysis of variance

(ANOVA), with means compared using Fisher’s LSD test. Area under curve (AUC) was compared by paired t-test.

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RESULTS

Experiment 1: T during the transition out of the breeding season

In experiment 1, a preliminary experiment was performed during the transition out of the breeding season to test the hypothesis that SE is used by bucks to increase T.

Simulated SE (spritz) with SE urine was more likely to elicit the flehmen response than a spritz with saline (fig. 1; 11/12 vs. 4/12 bucks, respectively; P<0.05). For all bucks, regardless of treatment, serum concentrations of T were higher at 40, 60 and 80 minute time points than at -20, 0, and 20 minutes (fig. 2; P<0.05). In sexually and socially mature bucks (older than 3 years of age; n=8), T was higher in response to SE urine than to saline at 80 min (fig. 3; P<0.05).

Experiment 2: LH during the breeding season

Next, a second experiment was conducted to test the same hypothesis, this time moving up the HPG axis to test the effects of a simulated SE on LH concentrations. In this experiment, treatment with SE urine resulted in a longer mean time spent in flehmen than did saline (fig. 4; 43.8 and 20.2 sec, respectively; P<0.05). This time, a spritz with

SE urine was not more likely to elicit a flehmen response than one with saline (fig. 4;

13/13 vs. 10/13, respectively). In all bucks (fig. 5; 2-9 years of age; n=13) and sexually and socially mature bucks (fig. 6; older than 5 years of age; n=6), serum concentrations of LH in samples taken after the spritz, were not greater than samples taken before the spritz for either treatment. Also, serum LH concentrations did not differ by treatment at any individual time point.

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Experiment 3: LH during the non-breeding and transition into the breeding season

In this experiment, the effects of multiple simulated self-enurinations on LH concentrations were analyzed during both the non-breeding season and the transition into the breeding season. Spritzes with SE urine resulted in a higher probability of flehmen occurrence in both the non-breeding season and the transition into the breeding season

(fig. 6, 42/56 vs 18/56 and fig. 8, 51/56 vs. 24/56, respectively). Self-enurination urine spritzes also resulted in greater mean flehmen duration than treatment with saline, in both the nonbreeding season and the transition into the breeding season (fig. 6, 26.8 vs 7.8 sec and fig. 8, 14.3 vs. 8.3 sec, respectively; P<0.05). In the nonbreeding season, mean serum

LH concentrations and AUC in samples following treatment tended to be higher for bucks treated with SE urine than saline (fig. 7; P=0.06 and P=0.11, respectively). During the transition into the breeding season, mean serum LH concentrations and AUC in samples following treatment were significantly higher for bucks treated with SE urine than saline (fig. 9; P<0.05).

Experiment 4: LH and T during the transition into the breeding season

In the final experiment, the effects of multiple spritzes of SE urine or air on LH and T during the transition into the breeding season were assessed. In this experiment, SE urine was more likely to elicit flehmen than air (fig. 10; 47/48 vs. 0/48, respectively).

Mean flehmen duration following SE urine spritzes was also higher than following spritzes with air (fig. 10; 21.3 vs. 0 sec). Treatment with SE urine resulted in a greater T increase from baseline than treatment with air at 5 min (388% vs. 133%, respectively;

P<.05) and at 20 min (349% vs. 159%, respectively; P<.05). Luteinizing hormone concentration AUC was lower following treatment with SE urine than after treatment

116 with air (57.6 and 74.1 ng/ml x min for SEU and AIR, respectively; P <.1) when T was elevated.

117

DISCUSSION

In this series of four experiments, we tested the hypothesis that the buck uses SE to stimulate his own HPG axis thus increasing his circulating concentrations of T. To test this hypothesis, we used the previously described simulated SE and measured serum T and LH concentrations in samples taken based on time points when we expected to see the respective endocrine responses. Many forms of sexual activity have been shown to be associated with increases in serum LH and T concentrations. Repeated mounting and intromission by male mice and rats was shown to increase serum LH concentrations and decrease pituitary LH concentrations (Taleisnik et al., 1966). Several minutes of pre- copulatory behavior and intromission in the male rat has been shown to increase serum T concentrations (Purvis and Haynes, 1972). Increased serum T and gonadotropin concentrations were observed in the male rabbit when placed in the presence of a female

(Saginor and Horton, 1968).

In the bull, a slight elevation in serum LH and T were observed following either ejaculation on the first mount or ejaculation after three to four mounts not resulting in intromission (Convey et al., 1971; Smith et al., 1973). Greater increases in serum LH and

T concentrations have been observed in the bull following visual observation of a cow or mounting without intromission (Katongole et al., 1971). In male sheep (rams), mounting with intromission, mounting without intromission and visual contact with estrous ewes resulted in increased concentrations of serum LH and T (Gonzalez et al., 1991b;

Gonzalez et al., 1988a; Moore et al., 1978; Sanford et al., 1974; Schanbacher et al.,

1987). Similar findings suggest that this phenomenon, known as the “female effect” is also present in the domesticated goat (Gonzalez et al., 1991b; Howland et al., 1985;

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Walkden-Brown et al., 1994). These finding show that the HPG axis in male bovine species is subject to influence by sexual stimuli. While findings in the ram suggest that the urine or fleece of estrous ewes do not cause a release of LH or a subsequent increase in serum T concentrations, the stimulation of the HPG axis of the buck by similar means or in response to signals in his own urine have not been studied (Gonzalez et al., 1991a).

Studies in bucks and rams have provided useful information on the timing of detecting an LH release and subsequent increase in serum T concentrations following the presentation of a sexual stimulus. These findings were used to design our sampling protocol to test our hypothesis that SE causes stimulation of the HPG axis via the accessory olfactory system in the buck displaying the behavior. In rams (Sanford et al.,

1974; Schanbacher and Ford, 1976) and bulls (Katongole et al., 1971), 40-60 minutes after an LH peak, a peak in serum T concentration was observed.

In the male pygmy goat, Muduuli et al. (1979) found that serum T concentrations were increased at 30 minutes following an LH peak and reached peak concentrations within 60 minutes. The observed LH peak gradually decreased to baseline levels within

90 minutes (Muduuli et al., 1979). This pattern was more obvious during June (the non- breeding season) than in October (the breeding season), when mean concentrations of both hormones were at their greatest (Muduuli et al., 1979). In Australian cashmere bucks, an experiment was conducted to measure the effects of exposure to estrous does on the timing and degree of LH and T responses. Following the introduction of estrous does, an increase in LH concentration was observed within 10 minutes and remained elevated over the 8 hour sampling period during which contact with the does was

119 sustained (Walkden-Brown et al., 1994). A well-defined T pulse was observed 45 ± 1.5 minutes following an LH pulse (Walkden-Brown et al., 1994).

Higher probability of flehmen and greater flehmen duration following SE urine exposure may stimulate greater involvement of the accessory olfactory system which can then recruit other CNS systems responsible for both reproductive behavior expression and HPG activity. For this reason, both total flehmen duration and probability of flehmen were used as indicators of accessory olfactory system recruitment. The significance of the greater duration of flehmen following treatment with SE urine than saline or air is unknown, though it may indicate interest or an attempt to interpret signals detected therein.

In experiment 1 (T during the transition out of the breeding season), it appeared that a T response was observed at 40, 60 and 80 minutes for both treatments for all bucks.

The T response to our control (saline) may have been due to the switchback design implemented in this experiment. Since we randomized the order in which bucks would receive each of the two treatments, it seems possible that bucks that received treatment with SE urine in the first week may have anticipated the urine exposure despite the treatment with saline in the second week; perhaps resulting in a “false positive” treatment effect. This idea is supported by our observation of bucks receiving saline the second week approaching the syringe for treatment with a high level of interest. This did not appear to occur in bucks receiving SE urine in week 2, after having received saline in week one. Another possibility is that since treatment occurred in the same room in an enclosed area and both treatments were administered on any given test day, some bucks receiving saline may have been exposed to a lingering scent of SE urine. It is also

120 plausible that a spritz with saline may have re-liquified any urine residue that may have been present on the buck’s face. However, despite these potential complications a significant difference between mean serum T concentrations following SE urine and saline treatment was observed at the 80-minute time point, suggesting that a treatment effect may have been present. Unfortunately, T concentrations were highly variable.

In experiment 2 (LH during the breeding season), significant methodological changes were made to address shortcomings in experiment 1. First, since SE is displayed in its highest frequency during the breeding season, this is when this experiment was conducted, postulating that bucks may be more responsive to the hypothesized signals during this time. Next, spritzes were administered in isolated locations to prevent cross- contamination. Finally, since T concentrations were highly variable, LH was measured, moving upstream in the HPG axis to decrease variation. This experiment did not confirm our hypothesis. Whereas the optimal time to study SE is during the breeding season when it occurs at the greatest frequency, detecting small changes in LH concentrations proved difficult because LH is suppressed at this time by high concentrations of circulating T.

Taking into account the high frequency of SE in a natural setting, a prolonged or repeated exposure (in this case four spritzes, every 15 minutes) was used in experiment 3

(LH during the non-breeding and transition into the breeding season) to test our hypothesis. This experiment was repeated both later in the non-breeding season and during the transition into the breeding season as it was thought that this may have been the time SE was most useful to a buck, facilitating the increase in T concentrations observed during the transition into the breeding season. Since it was described in rams that the best time to detect a change in LH concentration was soon after sunrise, bleeding

121 in this experiment began at 0700 hr (0900 hr in experiments 1 and 2) (Gonzalez et al.,

1988b; Signoret, 1991). Since LH concentrations following spritzes with SE urine tended to be higher during the breeding season and were higher during the transition into the breeding season than those following treatment with saline, it seems that the transition into the breeding season is the period when bucks are most responsive to the hypothesized signals. This period and the time of day adjustment may have also been better suited for detection of an LH response. Unfortunately, due to the limitations of jugular venipuncture and the stress it may cause to the bucks, modifications made it necessary to only take a single baseline sample from each buck. For this reason, it seems unclear how LH following treatment compared to baseline concentrations.

To address these limitations, in experiment 4, a 2.5% lidocaine/2.5% prilocaine cream was applied to the skin over the jugular veins prior to beginning the experiment to reduce or eliminate the pain and discomfort caused by jugular venipuncture, so that additional baseline samples, as well as additional samples following treatment could be added to assess both LH and T. Also, air instead of saline was used as a control to prevent the re-liquefaction of residual urine that may have been present on the bucks’ faces. Since the previous protocol appeared to be successful for the detection of an LH response to simulated SE, no additional methodological changes were made. It appeared that treatment with SE urine resulted in a T response at the 5- and 20-minute time points.

While this may seem early for a T response, these time points were relative to the fourth and final spritz and the observed response may have been induced by an earlier spritz.

Not sampling blood after each spritz likely complicated interpretation of the LH data.

However, in bucks treated with SE urine the involvement of the HPG axis was reflected

122 by a decrease in LH area under the curve when T was elevated, perhaps due to suppression of LH by the T response. It is unclear why an LH response to SE urine was observed in bucks in experiment 3, but not at the same time points in experiment 4. Many variables between these experiments, including different bucks, less frequent sampling for LH, a different control and high ambient temperatures during a heat wave in experiment 4 may have accounted for these discrepancies.

While these experiments certainly provide insight into a potential explanation of the adaptive significance of SE in the domesticated buck, experimental limitations made it difficult to accept or reject the hypothesis. Unfortunately, the use of jugular venipuncture and the significant individual variation in hormone concentrations makes the detection of small LH or T responses quite difficult. Ideally, blood sampling should be performed using a remote catheter to take samples following a naturally occurring SE.

Unfortunately, jugular catheterization of goats in previous experiments conducted in our lab has proved challenging. The goat is curious, agile and energetic by nature, and catheterization often fails by kinking of the tube or removal or dislodging of the catheter by the goat. While the simulated SE is a unique and novel bioassay for assessing the effects of non-volatile chemical cues on the physiology of not only goats, but other species that display flehmen, it is only a small part of SE as it occurs in a natural setting.

Without environmental influence, the erection, posture, penis grooming that sometimes follows and other elements of the behavior as it naturally occurs, the effects of SE on the

HPG axis may have been diminished or lost.

Another limitation is the relatively small sample size used to assess such an effect.

Observations in our lab have shown that there is a high degree of individual variation in

123 the frequency of SE display. Experiments conducted on high and low performing rams and rams with and without sexual experience have shown the plasticity in the ability to display an endocrine response to a sexual stimulus. Low performing or sexually inactive rams display lesser or absent LH and T responses to sexual stimuli than high performing or sexually active males (Borg et al., 1992; Perkins et al., 1992). While low performing males tend not to be as common in goats as they are in sheep, male goats certainly display variations in libido and frequency of SE. Perhaps experimentation with a greater number of bucks known to exhibit SE in greater frequency would yield clearer results, as they may be more responsive to these signals. Due to these limitations, further study is needed to determine if the domesticated goat buck uses SE to stimulate his own HPG axis for reproductive success.

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Figure 1. Testosterone response to simulated SE for all bucks in experiment 1: Mean

(± SEM) serum testosterone concentrations at -20, 0, 20, 40, 60 and 80 minutes relative to SE urine or saline spritz are displayed for all bucks (2-9 years of age; n=14) in experiment 1 (T during the transition out of the breeding season). Axis break represents when spritz was administered.

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Figure 2. Testosterone response to simulated SE for mature bucks in experiment 1:

Mean (± SEM) serum testosterone concentrations at -20, 0, 20, 40, 60 and 80 minutes relative to SE urine or saline spritz are displayed for mature bucks (5-9 years of age; n=8) in experiment 1 (T during the transition out of the breeding season). Axis break represents when spritz was administered. Asterisk denotes difference between treatments for mean serum testosterone at that time point (P<0.05).

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Figure 3. Proportion of bucks displaying flehmen and mean time spent in flehmen in experiment 2: Mean (± SEM) time spent in flehmen following SE urine or saline spritzes are displayed for all bucks (2-9 years of age; n=13) in experiment 2 (LH during the breeding season). Proportions of bucks displaying flehmen following a spritz are displayed within bars. Asterisk denotes difference between treatments for mean time spent in flehmen (P<0.05).

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Figure 4. LH response to simulated SE for bucks in experiment 2: Mean (± SEM) serum LH concentrations at -10, -5, 0, 5, 10, 15 and 20 minutes relative to SE urine or saline spritz are displayed for all bucks (2-9 years of age; n=13) in experiment 2 (LH during the breeding season). Axis break represents when spritz was administered.

128

Figure 5. LH response to simulated SE for mature bucks in experiment 2: Mean (±

SEM) serum LH concentrations at -10, -5, 0, 5, 10, 15 and 20 minutes relative to SE urine or saline spritz are displayed for mature bucks (5-9 years of age; n=6) in experiment 2

(LH during the breeding season). Axis break represents when spritz was administered.

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Figure 6. Proportion of bucks displaying flehmen and mean time spent in flehmen for experiment 3 during non-breeding season: Mean (± SEM) time spent in flehmen following SE urine or saline spritzes are displayed for bucks (2-7 years of age; n=14) in experiment 3 (LH during the non-breeding and transition into the breeding season).

Proportions of bucks displaying flehmen following a spritz are displayed within bars.

Asterisk denotes between treatments for mean time spent in flehmen (P<0.05). Dagger denotes difference between treatments for proportion of flehmen responses to total number of spritzes (P<0.05).

130

Figure 7. LH response to simulated SE for experiment 3 during non-breeding season: Mean (± SEM) serum LH concentrations at -45, 5, 10, 15 and 20 minutes relative to SE urine or saline spritzes are displayed for bucks (2-7 years of age; n=14) in experiment 3 (LH during the non-breeding and transition into the breeding season). Axis break represents when four spritzes was administered.

131

Figure 8. Proportion of bucks displaying flehmen and mean time spent in flehmen for experiment 3 during transition into breeding season: Mean (± SEM) time spent in flehmen following SE urine or saline spritzes are displayed for bucks (2-7 years of age; n=14) in experiment 3 (LH during the non-breeding and transition into the breeding season). Proportions of bucks displaying flehmen following a spritz are displayed within bars. Asterisk denotes between treatments for mean time spent in flehmen (P<0.05).

Dagger denotes difference between treatments for proportion of flehmen responses to total number of spritzes (P<0.05).

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Figure 9. LH response to simulated SE for experiment 3 during transition into breeding season: Mean (± SEM) serum LH concentrations at -45, 5, 10, 15, 20 and 25 minutes relative to SE urine or saline spritzes are displayed for bucks (2-7 years of age; n=14) in experiment 3 (LH during the non-breeding and transition into the breeding season). Axis break represents when four spritzes were administered. Asterisks denote difference between treatments for mean serum testosterone at individual time points

(P<0.05).

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Figure 10. Proportion of bucks displaying flehmen and mean time spent in flehmen for experiment 4: Mean (± SEM) time spent in flehmen following SE urine or air spritzes are displayed for bucks (2-8 years of age; n=12) in experiment 4 (LH and T during the transition into the breeding season). Proportions of bucks displaying flehmen following a spritz are displayed within bars. Asterisk denotes between treatments for mean time spent in flehmen (P<0.05). Dagger denotes difference between treatments for proportion of flehmen responses to total number of spritzes (P<0.05).

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Figure 11. LH response to simulated SE for experiment 4: Mean (± SEM) serum LH concentrations at -65, -55, -45, 5, 10, 20, 30, 45 and 60 minutes relative to SE urine or air spritzes are displayed for bucks (2-8 years of age; n=12) in experiment 4 (LH and T during the transition into the breeding season). Axis break represents when four spritzes were administered.

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Figure 12. T response to simulated SE for experiment 4: Mean (± SEM) serum testosterone concentrations at -65, -55, -45, 5, 20, 45 and 60 minutes relative to SE urine or air spritzes are displayed for bucks (2-8 years of age; n=12) in experiment 4 (LH and T during the transition into the breeding season). Axis break represents when four spritzes were administered. Asterisk denotes difference between treatments for mean serum testosterone at individual time points (P<0.05).

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Wakabayashi, Y., Mori, Y., Ichikawa, M., Yazaki, K., Hagino-Yamagishi, K., 2002. A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats. Chemical senses 27, 207-213. Walkden-Brown, S., Restall, B., Norton, B., Scaramuzzi, R., Martin, G., 1994. Effect of nutrition on seasonal patterns of LH, FSH and testosterone concentration, testicular mass, sebaceous gland volume and odour in Australian cashmere goats. Journal of Reproduction and Fertility 102, 351-360. Walkden-Brown, S.W., Henniawati, Restall, B.J., 1993. The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male. Animal Reproduction Science 32, 55-67. Witt, M., Wozniak, W., 2006. Structure and function of the vomeronasal organ.

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CHAPTER V

DISSERTATION CONCLUSIONS

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DISSERTATION CONCLUSIONS

Self-enurination (O'Brien, 1982), scent-urination (Coblentz, 1976), or urine marking (Shank, 1972) behavior has been described in several bovine and cervine species, but the understanding of the activation and adaptive significance of the behavior remains very limited. Only one study was conducted gathering empirical data on the behavior in the goat by Price et al. (1986) and findings were limited to the environmental context in which the behavior occurred. The goal of the research presented in this dissertation was to increase the understanding of SE in the goat, and perhaps similar behaviors that have been described in other species.

The findings presented herein contribute to a greater understanding of how and why ungulates use urine to communicate and provides new knowledge, which may be used to improve husbandry and develop more natural captive breeding practices. Also, these findings provide a better understanding of animal behavior in a domesticated species still subject to natural selection, even after thousands of years of domestication in relatively intensely managed herds. The goat provides a unique model for the study of sexual behavior because it has been subjected to thousands of years of domestication. For those behaviors in which human intervention overcomes the pressure of natural selection, domestication can “relax” said pressure on some behaviors, possibly resulting in a loss or change of adaptive significance (Price, 1984). This makes assessing the contexts in which behaviors occur and understanding the selective processes contributing to the expression of behavior in the goat both difficult and interesting. This work investigates a behavior, which in its current form, may or may not have been altered over time by domestication via housing management and artificial selection.

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This research intends to further understand the evolutionary origin of SE in the goat and for what adaptive purpose, if any, the behavior exists today. To study the behavior, the internal and external influences that work in tandem to promote the display of the behavior had to be understood. From there, it was possible to test popular predictions on the adaptive significance of this behavior in the domesticated goat. In this dissertation, three lines of experimental inquiry were conducted in an attempt to better understand the evolutionary, ecological and physiological underpinnings of SE and to provide novel information on variation and diversity in chemical communication systems in the animal kingdom.

It has been described extensively that T activates sexual behaviors in many species (Alsum and Goy, 1974; Beyer et al., 1975; Damassa et al., 1977; DeBold and

Clemens, 1978; Harding et al., 1983; Hart and Jones, 1975; Katz, 2007; Lincoln et al.,

1972; Schumacher and Balthazart, 1983). However, the aromatization or reduction of T to estrogens or other androgens, respectively, accounts for most of the hormone’s observed effects on the male’s sexual behaviors in bovine species. The relative contribution of androgens and estrogens to the activation of specific sexual behaviors may vary greatly across species. Additionally, environmental influences, especially socio-sexual context may greatly influence the display and frequency of these behaviors.

The objectives of the studies in Chapter II were to determine the environmental and endocrine factors that activate and influence SE and other sexual behaviors. The results demonstrated that unlike sexual behaviors in other species, SE is activated by androgens, not estrogens. Like other species, it was noted that courtship behaviors were activated by estrogens aromatized from testosterone. Finally, it was found that context rather than

143 endocrine profile, may have a greater influence on the activation and frequency of sexual behaviors in the male goat.

Using findings from Chapter II, chapters III and IV sought to test predictions on the adaptive significance of SE as displayed by today’s domesticated goat. A hypothesis first presented by Coblentz (1976) stated that the buck uses SE to apply olfactory cues to his integument for the purpose of attracting females. This popular hypothesis was tested in Chapter III. Findings in this chapter showed that the urine voided from a buck during

SE is more attractive to an estrous female than plain wether urine. Findings also suggested that androgens may be responsible for the attractive properties of the buck’s urine, but further study is needed for confirmation. While these findings suggest that the buck may indeed use SE to apply attractive olfactory cues to his body, this hypothesis does not explain the obvious sexual arousal associated with the behavior (erection, penis grooming, etc.) or the adaptive purpose of the flehmen often displayed following SE.

Chapter IV tested a hypothesis presented by Price et al. (1986), that the buck uses

SE to stimulate his HPG axis to increase his testosterone concentrations. The idea of pheromones acting on the self is a unique one and has only been demonstrated in one other instance in which male rats respond to their own alarm pheromone by releasing stress hormones (Inagaki et al., 2012). It has been proven that buck fleece (containing urine) is capable of stimulating increased luteinizing hormone (LH) pulse frequency in females, so it is plausible that similar physiological pathways may exist in the male

(Chemineau, 1987; Chemineau et al., 1986; Knight et al., 1978; Lindsay, 1966; Martin et al., 1986; Pearce and Oldham, 1984; Schinckel, 1954b; Shelton, 1960; Shelton, 1980;

Underwood et al., 1944; Walkden-Brown et al., 1993). The flehmen response delivers

144 non-volatile chemical cues to the VNO and accessory olfactory system (Estes, 1972;

Ladewig and Hart, 1980; Ladewig et al., 1980; Melese-d'Hospital and Hart, 1985), which has been shown to be capable of eliciting an endocrine response via the GnRH pulse generator (Hamada et al., 1996; Mori et al., 1991). A study by Wysocki et al. (1983) found that male mice with ablation of the VNO failed to show the typical T response when exposed to females. Findings presented in this chapter neither confirm or deny the use of SE for this purpose. However, they do show that the behavior may be used for this purpose at certain times of the year, but methodological limitations in our laboratory made it difficult to detect the type of response necessary for confirming the hypothesis.

Bucks certainly spent more time investigating SE urine via flehmen than our controls, though the significance of this finding remains unknown.

Together, these studies contribute to the understanding of SE in the domesticated goat. Though future studies are needed to fully explain the adaptive purpose of the behavior, it is clear that SE is androgen dependent and is highly influenced by socio- sexual context. The behavior likely serves multiple functions to the buck. Future studies to test the hypothesis studied in Chapter IV should involve more frequent blood sampling and simulate SE in a more natural setting. Further study is also needed to assess the role of SE in male-male interactions. In addition to its display in sexual contexts, bucks also display SE in the presence of other males only. Additionally, other males in the herd often show great interest in urine voided by another buck during SE; drinking, lapping and displaying flehmen following its investigation. While SE remains a behavior with an unknown evolutionary significance, it is likely that domestication has caused its adaptive

145 purpose to change. Study of SE or similar behaviors in wild species, may provide further insight on the need for such a behavior.

146

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