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HORMONES, STRESS AND AGGRESSION
IN THE SPOTTED HYENA
(CROCUTA CROCUTA)

presented by

Page E. Van Meter

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HORMONES, STRESS AND AGGRESSION IN THE SPOTTED HYEN A
(CROC UTA CROC UTA)

By

Page E. Van Meter

A DISSERTATION
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
DOCTOR OF PHILOSHOPHY

Zoology

2009

ABSTRACT

HORMONES, STRESS AND AGGRESSION N THE SPOTTED HYEN A
(CROC UTA CROCUTA)

By
Page E. Van Meter

In this dissertation, I used data from almost two decades of behavioral
observations of wild spotted hyenas (Crocuta crocuta), coupled with non-invasive
techniques to measure steroid hormone concentrations from these same individuals, to
investigate the behavioral endocrinology of this unique species. Female Spotted hyenas
are morphologically and behaviorally masculinized. These traits make this species
particularly interesting subjects to investigate the interaction between hormones and
behavior, and how this interaction is inﬂuenced by an individual’s social or ecological
environment.

Our lab has measured concentrations of androgens, glucocorticoids, and now,
estrogens present in fecal samples collected from free-living, individually recognized
hyenas. I ﬁrst use these data to investigate both naturally occurring and anthropogenic
inﬂuences on stress physiology in spotted hyenas. From our longitudinal study, I
demonstrated that spotted hyenas are well adapted to variation in their natural ecology,
including seasonal ﬂuctuations in prey and rainfall. However, unpredictable events, such
as sudden periods of instability in the social hierarchy, do inﬂuence Spotted hyena stress
physiology. Interestingly, adult male hyenas experienced an impact of increasing human
disturbance on their stress physiology. This result was strengthened by a cross-sectional

comparison that demonstrated that this population of hyenas, which is now exposed to

high levels of human disturbance, had elevated stress hormone concentrations when
compared to hyenas exposed to relatively low levels of human disturbance.

I next evaluated behavior likely to be mediated by steroid hormones in this
species, focusing on aggressive behavior. Using careful behavioral rate calculations that
controlled for variation in opportunity to behave aggressively, I Showed that rates of
aggression emitted by adult females toward all conspeCiﬁcs are higher than those emitted
by adult males, which is opposite mammalian norms. While rates of intrasexual
aggression did not differ signiﬁcantly between the two sexes, the quality of this
aggression did, and attacks among females were of higher-intensity than those seen
among males. My results also demonstrated that rates of aggression emitted by females as
adults were positively correlated with variation in their prenatal androgen exposure,
supporting the organizational hypothesis of steroid hormone control of behavior.

Finally, we investigated two functional hypotheses to explain a particular type of
aggression observed among adult female hyenas. Unprovoked aggression describes
attacks between individuals that are not related to maternal defense or control of
resources. My results suggest that females use unprovoked aggression to test existing
rank relationships. However, we did not ﬁnd evidence supporting the notion that

aggression is preferentially directed at females to suppress their reproductive efforts.

ACKNOWLEDGEMENTS

Dr. Kay Holekamp’s understanding of the ﬁeld of animal behavior makes her an
outstanding teacher. Her ability to weave ideas from the leading edge of this ﬁeld into the
study of hyenas makes her an excellent researcher. And her devotion to the scientiﬁc
method makes her an exceptional scientist. But it is her ability and willingness to
translate these skills to her students that makes her a respected adviser. Kay has been an
invaluable role model and has provided me with a high standard that I will apply to my
endeavors for the rest on my life. I wish to thank her for her criticism, which made me a
better student; her patience when I didn’t get it the ﬁrst time, or the second; and her
conﬁdence, which She always gave when I needed it the most.

I was very lucky to have worked with a number of remarkable scientists during
my graduate work, and if I did one thing right in this time, it was in picking my defense
committee: Dr. Tony Nunez always brought a fresh perspective to the table. Dr. Laura
Smale contributed a keen understanding of hormones, behavior, and hyenas (but I
especially thank her for her compassion and encouragement). Dr. Jeff French instructed
and oversaw all of my hormone work, gave me all the time and space I needed in his
beautiful shiny lab, provided me the opportunity to get out of Lansing (no really, Omaha
is lovely) and, most important, he was a continual source of humor and perspective.
Thanks also to the members of his lab, who generously put me up and gave of their time
whenever I visited the University of Nebraska, Omaha.

I also extend my thanks to Steve Glickman and his colleagues at the University of

California Berkeley Field Station. They graciously collected and stored a large quantity

iv

of fecal samples for my research. I thank Peggy Ostrom and Hasand Gandhi for
instruction and use of their lyophilizer. And thanks to Janine Brown of the Conservation
and Research Center of the Smithsonian Institution, who did our HPLC analyses in
Chapter 2. Special thanks to Dr. Kim Wallen for introducing me to hormone research and
encouraging me to pursue my career in science.

I would also like to thank the Brain and Behavior Group for being such a helpful
and responsive community. Thanks to the MSU Department of Zoology for providing me
with resources and funding. And thanks to the Neuroscience Program, speciﬁcally Marc
Breedlove, for vital ﬁnancial support. I received funding through a National Institutes of
Health training grant, from the Department of Zoology, and the Society for Behavioral
Neuroscience. The Mara Hyena project was‘supported in part by grants from the National
Science Foundation.

AS another testament to the esteem in which Kay is held by the academic
community, the people associated with her and this project are of a very high caliber and
I count myself lucky to have had them along with me on this journey. Thanks to Heather
Watts and Joe Kolowski, who contributed tremendously to Chapter 3. Jaime Tanner,
Kevin Theis, Soﬁa Wahaj, Russ Van Horn, Eva-Maria Muecke, Tern' McElhinny, and
Stephanie Dlonaik, who not only served as role models but also became friends. Thanks
to Jennifer Smith and Michelle Fisher for all their hard work with the aggression
database. Thanks to Suzanne La Croix, Sarah Benson-Amram, Katy Califf, Greg Stricker,
Andy Flies, Wiline Pangle, Leslie Curren, and Kate Shaw for their'camaraderie. I thank
Pat Bills for his humor and tolerance as I forced him to be my guide in all things

computational.

Although my own time in Kenya was short, my appreciation for all those who
have spent many years in the ﬁeld there is immense. My dissertation could not have been
accomplished without the hard work and foresight of others. So I extend my gratitude to
all former and current contributors to the Mara Hyena Project, with special thanks to
those who made my stay in Kenya a dream come true: Audrey DeRose-Wilson, Kim
Wooten, John Keshe, Moses Sairowa, and James Kerembe.

I was also blessed to have several friends who have stuck with me through the
years, and doubly blessed to have made news ones. Special thanks to Joe Lonstein and
Stephen Thomas for home-cooked meals, movies, trash TV, board games, occasionally a
bed, and two pairs of shoulders to lean on. I became fast friends with Jaime Tanner and
Soﬁ Wahaj during our overlapping time in East Lansing. I also thank Mike Schwartz,
Erich Ottem, Mary Martin, and Nate Miller for their friendship.

I would like to thank both sets of my parents, Sue Bramhall, Ed Van Meter, Silvio
Calabi, and Teana Van Meter, for their support, love, and tolerance as I pursued my
education in science. Although not always understanding what I was doing or why I was
doing it, they gave me an ear when I needed to rant, a bed when I needed a place to stay
(for just a few months, really), hugs, and encouragement. I’d like to particularly thank my
mom, Sue, for instilling a sense of independence in me, even when it contradicted every
maternal instinct she had (and for not dragging me home from Indonesia after 9/11), and
for giving me the best advice possible: Use good judgment! I would like to thank my dad,
Ed, for both boosting my ego with his pride in my accomplishments and for keeping me
humble by frequently correcting my spelling. Thanks to Teana for caring for me like her

own daughter, for taking such good care of my dad, and for traveling halfway around the

vi

world to visit me. Thanks to Silvio for his wit, calm demeanor, and the best advice I got
for my trip to Kenya: Even shallow puddles can hide a croc!

I would like to thank Mark Bellncula, who demonstrated that having fun is not the
antithesis of hard work, but that the two can co-exist. He has also been a tremendous role
model, showing me that you can and should make a life out of doing work that you love.
I also thank him for his patience, while I rambled around in Michigan and Kenya, and his
constant reassurance that getting a Ph.D. was a good idea. He has been my friend,
conﬁdant, cheerleader, and my rock.

Finally, after thanking my mentors, my friends, and my family, there is one
person who encompasses all three: Julia Zehr. As a mentor, she took a chance on a young
undergrad who didn’t have much more to offer than an interest in animals. She was my
ﬁrst guide to the inner workings of the scientiﬁc community. Looking back, I can now
appreciate her selﬂessness as she helped to train and prepare me for my future in science,
all while she was still working on her own. But this altruism is just a reﬂection of her as a
person. As a friend she is always willing to give of herself in any way that is needed (and
as a friend I often needed a lot). The ebb and ﬂow of our past careers predict our future
relationship, and, although our personal pursuits will take us in separate directions, I
know they will also inevitably bring us back together in the future, and I look forward to

it.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................. ix
LIST OF FIGURES . ........................................... x
GENERAL INTRODUCTION ...................................... 1
Overview of chapters .......................................... 12
References ................................................. 16
CHAPTER 1

N ON-INVASIVE MEASUREMENT OF FECAL ESTROGENS IN THE SPOTTED
HYENA(CROCUTACROCUTA)... . . . .. . . . .. . . . . .. . ...24

Introduction ............................................... 25

Methods. ................................................ 27

Results. ................................................. 34

Discussion ................................................ 45

References ................................................ 50
CHAPTER 2

FECAL GLUCOCORTICOIDS REFLECT SOCIO-ECOLOGICAL AND
ANT HROPOGENIC STRESSORS IN THE LIVES OF WILD SPOTTED

HYENAS ........................................................... 54
Introduction ............................................... 55
Methods. ................................................ 59
Results. ................................................. 72
Discussion ................................................ 78
References ................................................ 84

CHAPTER 3

INTER- AND INTRASEXUAL AGGRESSION AMONG ADULT SPOTTED

HYENAS ................................................... 89
Introduction ............................................... 89
Methods. ................................................ 95
Results. ................................................ 103
Discussion ............................................... 1 18
References...............................................128

CHAPTER 4

FUNCTIONS OF UNPROVOKED AGGRSSION AMONG FEMALE SPOTTED

HYENAS. ................................................. 139
Introduction .............................................. 139
Methods. ............................................... 144
Results. ................................................ 149
Discussion ............................................... 159
References ............................................... 167

viii

LIST OF TABLES

Table 1.1. Free-living hyena plasma estradiol (pg/ml): Individual and mean (1: standard

errors) response patterns to LHRH challenge .................................. 38
Table 1.2. Captive hyena fecal estrogen (ng/g feces): Individual and mean (i standard

errors) response patterns to LHRH challenge .................................. 39
Table 2.1. Ecological features of parks and clans ............................... 65

Table 2.2. Summary table showing relative ecological and anthropogenic differences
among parks and clans, and the respective sets of predictions generated for our nested
cross-sectional analysis. The ﬁrst prediction was based on differences in the ecological
conditions experienced by hyenas in each clan and park (H1), the second was based on
variation in lion density among clan territories (H2), the third was based upon variation in
tourism (H3), and the fourth was based on variation in pastoralist activity (H4). Only the
last of these predictions was conﬁrmed, as shown in Figure 2.2 ................... 70

Table 2.3. Models (GLMM) investigating methodological sources of variation in fGC
concentrations. Males and females were tested in separate models; all variables included
in the models are shown. Individual identity was entered as a random variable ....... 73

Table 2.4. Parameter estimates from the selected models (GLMM) explaining fGC

concentrations among each group. Individual identity was entered as a random factor in
all models ............................................................. 74

ix

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LIST OF FIGURES

Figure 1.1. HPLC results from A) pregnant and B) non-pregnant female hyenas.
Fractions were assayed for immunoreactivity with both estradiol (open circles) and
estrone conjugate (gray circles) enzyme immunoassays, and immunoreactivity is
expressed on the right-hand axes. Elution of radiolabeled standards (black circles),

estrone (E1) and estrone sulfate (ElS), are expressed as counts-per—minute (CPM) per
fraction on the left-hand axis ............................................... 36

Figure 1.2. Relative dose vs. percent of antibody bound for standards and serial dilutions
of samples from three hyenas. Slopes of all curves are parallel in this range (20—80%
binding; P = 0.18) ....................................................... 37

Figure 1.3. Percentage of nulliparous females sampled in each 4-month age interval
whose mean fecal estrogen concentrations exceeded 100 ng/g feces. Numbers over bars
represent females sampled in each age group. Multiple samples collected from individual
females within age interval were averaged .................................... 41

Figure 1.4. Within-female comparisons between A) reproductively immature and mature
states, and B) pregnant and lactating states for plasma estradiol (black) and fecal
estrogens (gray). Means and standard errors are presented, and signiﬁcance was tested
using paired t-tests. Note that the scales represented on the two y-axes differ between
sampling techniques ..................................................... 43

Figure 1.5. Estrogen concentrations in plasma (black) and fecal material (gray) across
trimesters of pregnancy, averaged across females. Signiﬁcant differences among mean
(iSEM) plasma estradiol and fecal estrogen concentrations are indicated by different
letters, and were tested using Tukey’s post-hoe test. Note that the scales represented on
the two y-axes differ between sampling techniques ............................. 44

Figure 2.1. Map of parks and clan territories .................................. 64

Figure 2.2. Mean (1 SEM) morning fGC concentrations for adults from four hyena clans.
A nested ANOVA indicated a signiﬁcant effect of clan. Different letters above bars
reﬂect signiﬁcant differences indicated by the Fisher’s LSD analysis ............... 77

Figure 3.1. Mean (i standard error) percentage of all attacks (n = 10098) directed toward
all adult conspeciﬁcs by adult females (n = 73; black bars) and males (n = 51; gray bars)
observed in various contexts. The “other” context refers to sex-speciﬁc situations like
mating and maternal defense. The sexes only differed signiﬁcantly with respect to
aggression directed toward “pesky” conspeciﬁcs (p = 0.005) .................... 105

Figure 3.2. Mean (1 standard error) lifetime aggression rates, controlled for numbers of
available targets in each observation session, directed by adult females (n = 73; black
bars) and males (n = 58; gray bars) toward adult and juvenile conspeciﬁcs. Adults of both
sexes were signiﬁcantly more aggressive to adults than to juveniles, and adult females
were signiﬁcantly more aggressive to both adults and juveniles than were adult males. 106

Figure 3.3. Mean (1 standard errors) rates of aggression directed toward adult
conspeciﬁcs by adult male (gray bars) and female (black bars) spotted hyenas. Rates of
aggression emitted by females (11 = 71) varied signiﬁcantly among target types (females
higher-ranking, females lower-ranking, and males). Rates of aggression emitted by males
(n = 57) also varied signiﬁcantly among target types (males higher-ranking, males lower-
ranking, and females). Asterisks indicate signiﬁcant sex differences in rates of aggression
directed toward target types .............................................. 108

Figure 3.4. Mean percent (:1: standard errors) of all aggressive acts that were of high-
intensity ([1 = 1232). Not all males were seen to emit high-intensity aggressions, and the
numbers under the bars indicate number of individuals used in this analysis. Letters
above the bars indicate signiﬁcant differences among groups .................... 109

Figure 3.5. Mean (1 standard errors) rates of (A) intrasexual aggression toward lower-
ranking conspeciﬁcs, (B) and inter-sexual aggression. Because feeding aggression could
only occur when food was present, rates were calculated for this context on a different
scale than for all other contexts. Asterisks indicated signiﬁcant differences. (Contexts are
abbreviated such that: F = feeding, U = unprovoked, P = pesky, and O = “other”). . . . 111

Figure 3.6. Mean percent (1 standard errors) of all intrasexual aggressive acts that were
of high-intensity (n = 925). Aggression among females was of signiﬁcantly higher
intensity in every context. (Contexts are abbreviated such that: F = feeding, U =
unprovoked, P = pesky, and O = “other”) .................................... 112

Figure 3.7. Mean (i standard error) rates of aggression directed by females toward lower-
ranking female targets, and adult males (A) during pregnancy and lactation (n = 40
females), and (B) during successive trimesters of pregnancy (n = 28 females). Females
were signiﬁcantly more aggressive toward other adult females during lactation than
during pregnancy (p = 0.004). During pregnancy, rates of aggression emitted by females
did not vary among successive trimesters of pregnancy. Females were more aggressive
toward males than toward females during all trimesters of pregnancy, although this
difference was signiﬁcant only for the ﬁrst trimester (p = 0.003) ................. 114

Figure 3.8. Relationship between maternal fA during gestation and rates of offspring (n =

14) aggression in adulthood toward (A) all other adults, (B) adult males, and (C) adult
females. All offspring included here were adult females ........................ 117

xi

Figure 3.9. Mean (i standard error) rates of intrasexual aggression emitted by adult
females. Rates are calculated as total aggressive acts among females, divided by the
number of study subjects, over the total observation time for the study period. Primate
data were adapted from Erhart and Overdorff (2008), and the numbers over each bar
indicates the number of species represented in each group ...................... 122

Figure 4.1. Percent of all unprovoked aggressive acts (n = 614) observed at each
location .............................................................. 151

Figure 4.2. Mean (i standard error) rates of unprovoked aggression received by females
(11 = 31) when they were in each reproductive state, where P1, P2, and P3 refer to
successive trimesters of pregnancy, L refers to early lactation, and C indicates the month
prior to a known conception. Different letters above the bars represent signiﬁcant
differences among the states .............................................. 152

Figure 4.3. Percent of total aggressive acts in each intensity category directed by females
(n = 14) towards higher-ranking recipients (black bars) or towards lower-ranking
recipients (gray bars). Asterisk indicates a signiﬁcant difference (p = 0.015) ........ 156

Figure 4.4. Frequency distribution showing rank distances within 26 dyads in which the
aggressor was lower-ranking than the recipient. Rank distance was calculated for each
dyad as the difference between aggressor and recipient in a) absolute rank in the clan, and
b) relative rank among females present in the immediate subgroup ............... 157

Figure 4.5. Frequency distribution showing rank distances within 444 dyads in which the
aggressor was higher-ranking than the recipient. Rank distance was calculated for each
dyad as the difference between aggressor and recipient in a) absolute rank in the clan, and
b) relative rank among females present in the immediate subgroup ............... 158

xii

GENERAL INTRODUCTION

In the natural world, animals are faced with an ever-changing environment that
presents them with both predictable (e. g. seasonal changes in climate or daylight) and
unpredictable events (8. g. predation or storms). An individual’s success frequently hinges
on its ability to perceive and adapt to these changes, often by altering its own behavior or
physiology. Neuroendocrine and endocrine systems provide the basis for a ﬂuid response
mechanism whereby animals can respond with a certain amount of ﬂexibility to
environmental stimuli. Steroid hormones in particular play a key role in mediating
behavioral responses to increase the responsivity of the individual to changes in their
environment, and to increase the likelihood that the appropriate behavior is elicited in
speciﬁc contexts. Thus a feed-back system exists in which hormones can inﬂuence the
occurrence of a behavior, and in which that same behavior may also bring about a change
in the animal’s environment, which can then again inﬂuence the animal to affect its
endocrine physiology (Nelson, 2005). The ﬁeld of behavioral endocrinology therefore
encompasses the study of both environment—endocrine interactions, and endocrine-
behavior interactions.

In this dissertation, I present work done with my collaborators investigating
aspects of both of these interactions in spotted hyenas (Crocuta crocuta) living in their
natural environment. We investigated environmental inﬂuences on the physiology of
steroid hormones secreted by the adrenals and the gonads, and we also evaluated
behaviors likely to be mediated by these steroid hormones in this species. The majority of

this dissertation work was focused on proximate level explanations of behavior.

However, the beneﬁt of studying free-living animals is that this allows us to observe
behavior within a relevant socio-ecological framework, which provides the opportunity to
ask about the ultimate functions of the behavior. Therefore my overarching goal with this
work was to incorporate answers to both “how” and “why” questions into our
understanding of spotted hyena behavior.

Our current understanding of hormonal mediation of behavior has been developed
by investigation of a large array of species, and it has been determined that the basic
features of the endocrine system are highly conserved among all of the major vertebrate
groups (Becker, et al., 2002; Nelson, 2005; Silver and Ball, 1989). Steroid hormones are
the end products of a cascade of chemical events regulated by the central nervous system.
Secretory neurons in the hypothalamus produce specialized releasing factors, which
stimulate the release of pituitary hormones from the pituitary. These travel through the
blood stream to act on peripheral endocrine glands, where they stimulate hormone
production and release. Endocrine glands, such as the adrenals or gonads, respond by
secreting steroid hormones into the circulatory system to act on target tissues both in the
periphery and in the brain, ultimately inﬂuencing both physiology and behavior (Becker
et al., 2002; Nelson, 2005).

The hypothalamus, as the bridge between the central nervous system and the
endocrine system, integrates endogenous and exogenous information, including a vast
amount of sensory input from the individual’s environment (Kandel, et al., 2000). In this
way, the environment, both social and ecological, can have a profound effect on
endocrine physiology. This is most evident in the study of the endocrinology of stress,

which examines how an animal perceives and responds to perturbations of their

physiological balance (Sapolsky, 2002). The impact of stressors is frequently measured
as variation in circulating glucocorticoids (GC), the products of the hypothalamic-
pituitary-adrenal (HPA) system. Acute activation of the HPA axis, and short term release
of GC concentrations from the adrenals are adaptive parts of the “ﬁght or ﬂight” system;
however, chronic elevation of GC concentrations can lead to pathology, including
compromise of cardiovascular, immune, neural, and reproductive function (Sapolsky,
2002). Among group—living species, aspects of sociality may be perceived as stressors.
For instance, among several cooperatively breeding carnivores such as the African wild
dog (Lycaon pictus), dominants have higher GC concentrations than do subordinates,
which may be associated with higher rates of aggression emitted by dominates to obtain
and maintain their rank (Creel, et al., 1997; Creel, 2005; Creel, 1997). Ecological factors
can also affect stress hormone physiology, for example, there is a negative correlation
between food availability and GC concentrations among wild populations of elephants
(Loxodonta africana) (Foley, et al., 2001) and chimpanzees (Pan troglodytes) (Muller
and Wrangham, 2004). Fairly recently, researchers have been studying environmental
inﬂuences on stress physiology to investigate the effects that human disturbance might
have on wild populations (Walker, et al., 2005; Wingﬁeld, et al., 1997).

In his textbook on behavioral endocrinology, Nelson (2005) introduces hormone-
behavior interaction by pointing out that humans had an implicit understanding of the
hormonal control of behavior long before hormones had been described by science. For
instance, castration has been a useful tool to modulate animal behavior for millennia. We
now know that castration, or gonadectomy in general, removes the endocrine glands

responsible for the production of gonadal steroid hormones, and this removal, in turn,

causes the termination of certain behaviors. The results of castration represent an
example of what Phoenix et at. (1959) called the “activational” control of steroid
hormones on behavior. Yawning behavior among rhesus macaques (Macaca mulatta)
provides another, more elegant, example of this activational control (reviewed by Graves
and Wallen, 2006). Yawning is a sexually dimorphic behavior rarely exhibited by
females, and frequently seen in post-pubertal males. Castration of males abolishes the
behavior, whereas subsequent exogenous androgen administration restores the behavior
(Phoenix, et al., 1973). Finally, females can be induced to yawn when administered
exogenous androgen (Goy and Resko, 1972). This example also nicely outlines the
experimental paradigm necessary to show that a behavior is inﬂuenced by “activational”
effects of a hormone (Phoenix, et al., 1959). Whereas in this example, yawning is
mediated by androgens, ovarian hormones, like estrogen and progesterone, can also
activate behaviors, such as lordosis behavior in female rodents (Breedlove and Hampson,
2002; Nelson, 2005).

Relationships between hormones and behavior that support the activational
hypothesis can also be observed without experimental manipulation. For instance, among
seasonally breeding animals, such as the red deer (Cervus elaphus), males exhibit
aggressive behaviors during the breeding season when circulating androgens are high;
however, during the non-breeding season, the testes regress, and androgen production is
reduced, as are rates of aggression (Lincoln, et al., 1972). Female reproductive cycles
provide another predictable source of natural variation in hormone production. Maternal
behaviors are tightly coupled to reproductive state in most species displaying maternal

care, and the onset of these behaviors have been extensively studied with respect to

estrogen, progesterone, and the peptide hormone prolactin (reviewed by Lonstein and
Gammie, 2002). Menstrual and estrus cycles also inﬂuence female behavior, obviously
with respect to sexual behavior (W allen and Zehr, 2004), but variation in gonadal
hormones during these cycles has also been linked to variation in behaviors associated
with memory and cognition (Farage, et al., 2008; Sherwin, 2003).

In contrast to these impermanent activational effects, which wax and wane with
hormone titers, steroid hormones present during development may have more lasting
effects on behavior. Hormone exposure early in life, particularly exposure to androgens,
“organizes” the developing neural substrates of behavior in a permanent way. In their
seminal paper, Pheonix et a1. ( 1959) demonstrated that female guinea pigs exposed to
prenatal androgens were unable to display normal female sexual behavior despite
exogenous administration of estrogen and progesterone in adulthood. This work helped
them formulate the concept of irreversible neural modiﬁcation during development,
which has become a central tenet of behavioral endocrinology. The sex differences seen
in juvenile play in several mammalian species provide a good example of these
organizing effects. Juvenile males engage in more vigorous forms of play than do
juvenile females, and this sex difference is apparent before sexual maturity while the
gonads are still quiescent with respect to hormone production. Juvenile sex differences in
play behavior have been linked to prenatal, or in some cases neonatal, androgens in
rodents (Meaney, et al., 1983), non-human primates (Goy, et al., 1988; but see Herman,
et al., 2003), and humans (Berenbaum and Hines, 1992).

There are a growing number of experimental paradigms now in use whereby

prenatal hormone environment may be altered to test predictions pertaining to the

organization of behavior. However, as in the observational studies used to corroborate
activational effects, there is also natural variation in the prenatal hormone environment,
and this variation can lead to individual differences in behavior (Goy et al., 1988;
Groothuis, et al., 2005; Ryan and Vandenbergh, 2002; Wallen, 2005). Given the
importance of prenatal hormone exposure on fetal development, it is useful to consider
the variety of sources that can inﬂuence the prenatal steroid hormone environment. The
predominant source is of course the fetus itself; the fetal testes produce androgens soon
after gonadal differentiation, and these androgens are critical for masculinization of both
morphology and behavior (Becker et al., 2002; Nelson, 2005). In litter-bearing species,
intra-uterine position inﬂuences prenatal hormone environment, and the androgen
exposure experienced by a female fetus situated between two males has profound effects
on her behavior and fertility (Clark and Galef, 1995; Clark and Galef, 1998; Ryan and
Vandenbergh, 2002). Although the placenta usually acts as a buffer between the mother
and the fetus in mammals, maternally derived steroid hormones do inﬂuence the prenatal
environment of the fetus, and this may provide a link between the fetal environment and
the maternal environment. Maternal environmental stress is commonly studied with
respect to its pathological consequences on fetal development (W einstock, 2008).
However, studies of maternal effects in oviparous species suggest that hormonal input
from the mother may be adaptive for offspring development and survival (Groothuis et
al., 2005), and this concept is now being investigated in mammals (e. g. Dloniak, et al.,
2006b).

To summarize, the activational hypothesis predicts that hormone concentrations

and the expression of the behavior should be positively correlated, and the organizational

hypothesis predicts that exposure to steroid hormones early in development will have
long-lasting consequences with respect to behavior. Frequently, these two mechanisms
work in concert, and both organization and activation are required for the full compliment
of the behavior to be displayed (Phoenix et al., 1959). Although organizational and
activational effects of steroid hormones were originally conceptualized to explain
differences in sexual behavior, there is now a large literature using this heuristic
framework to examine mechanisms underlying a variety of sexually dimorphic behaviors.
In this dissertation, I focus on a particular behavior, aggression, which has been
studied extensively with respect to both organizational and activational effects of steroid
hormones. Males are typically viewed as the more aggressive sex among mammals
(Archer, 1988; Floody, 1983; Osterrneyer, 1983), and investigation of steroid hormone
control of aggression among males has been productive (Bouissou, 1983; Harding, 1981).
Intrasexual male aggression is often sexually driven; aggreSsion among males increases
when reproductive competition increases, be it during a breeding season, or simply in the
presence of a fertile female (Nelson, 2005), and during these sexually charged situations,
there is a strong relationship between circulating androgens and rates of the behavior
(“Challenge Hypothesis,” Wingﬁeld, et al., 1990). Activating effects of androgens on
aggressive behavior among males are well established (rodents, Buck and Barnes, 2003;
primates, Cavigelli and Pereira, 2000; Zumpe and Michael, 1996; carnivores, Creel, et
al., 1993; Creel, 2005; Dloniak, et al., 2006a; Goymann, et al., 2003; ungulates, Lincoln
et al., 1972). There is also evidence in support of organizational control of aggressive
behavior. For instance, in studies investigating effects of intrauterine position, male mice

that developed between two male fetuses were found to have higher rates of intramale

aggression than males raised between two females; although, there does appear to be
species speciﬁc variation in this mechanism of behavioral control (vom Saal, 1989). As 1
indicated previously, the investigation of organizational control of steroid hormones on
behavior in oviparous species has advanced more quickly than in mammals, primarily
because their prenatal environment, the egg, is easier to manipulate than that in placental
mammals. For example, manipulated levels of testosterOne in the eggs of the black-
headed gull (Larus ridibundus) were positively correlated with aggressive behaviors after
hatching (Groothuis et al., 2005; Muller, et al., 2006).

Despite this past research focus on aggression among males, aggression among
females is reported to be prominent in a number of species (Floody, 1983), for instance,
in species with cooperative breeding strategies, aggression is thought to play a role in
maintaining the dominant status of breeders and the suppression of reproduction in
subordinates (rodents, Bennett, 2000; carnivores, Moehlman, 1997; primates, Solomon
and French, 1997). Female aggression is also a topic at the forefront of studies
investigating species with female dominance. Although rare, the evolution of female
social dominance over males has occurred independently in several different taxonomic
groups (rodents, Clarke and Faulkes, 2001; primates, Drea, 2007; birds, Smith, 1980;
carnivores, Kruuk, 1972), and the potential for steroid hormone control of female
aggression exists among these species (Dloniak et al., 2006b; Drea, 2007; Glickman, et
al., 1993; Sannen, et al., 2003; von Engelhardt, etal., 2000). For example, in the
ringtailed lemur (Lemur catta) seasonal elevations in female aggression are positively

correlated with circulating androstenedione and estrogen concentrations among females

(Drea, 2007). This study suggests that not only androgens, but also ovarian hormones
might be mediating aggressive behavior among females.

The spotted hyena (C rocuta crocuta) is another species with well-documented
female dominance. Spotted hyena society has a dominance structure based on maternal
rank inheritance, more similar to that seen in cercopithecine primates than that in other
social carnivores (Engh, et al., 2000; Frank, 1986; Smale, et al., 1993). Offspring slot into
the social dominancy hierarchy right underneath their mothers, and the youngest sibling
outranks older siblings from prior litters (Engh et al., 2000; Holekamp and Smale, 1993).
Females are philopatric and males generally disperse between the ages of 2 and 5 years
(Boydston, et al., 2005; Smale, et al., 1997). When males immigrate into a new clan, they
slot into the lowest position in the social hierarchy, below all natal members of the group
and all other adult immigrant males that arrived before them (East and Hofer, 2001;
Engh, et al., 2002). Thus these reproductively mature males are socially subordinate to all
natal members of the group (Kruuk, 1972; Smale et al., 1993).

All female hyenas in the social group are able to breed; however, there is strong
reproductive skew, and higher-ranking females out-perform lower-ranking females on
several measures of reproductive success (Holekamp, et al., 1996). Social rank
determines priority of access to food in this species, and food availability is tightly
coupled to reproduction (Holekamp et al., 1996; Holekamp, et al., 1999). This
relationship has lead to the prevailing hypothesis to explain the evolution of female
dominance in spotted hyena society, that selection has favored large, aggressive females

who can dominate access to food (Frank, 1986; Gould, 1981).

In addition to this behavioral role-reversal, female spotted hyenas have
masculinized external genitalia (Frank, et al., 1990; Kruuk, 1972; Matthews, 1939;
Neaves, et al., 1980), which is unique even among species characterized by female
dominance (Drea, 2007; Drea, et al., 1998). We have seen that androgens play a key role
in shaping male-typical mammalian behaviors, such as aggression, but in most species
they are also largely responsible for the development of male morphology and
reproductive physiology (Jost, et al., 1973b), and when present at critical stages of
development, androgens can masculinize the morphology and behavior of females
(Beach, et al., 1982; Goy et al., 1988; Herman, etal., 2000; Jost, et al., 1973a; Phoenix et
al., 1959). When compared to males, female spotted hyenas have high concentrations of
circulating androstenedione (A4), an androgenic “prohormone” that can be converted
either to testosterone or estrogen (Glickman, et al., 1992; Goymann, et al., 2001;
Lindeque, et al., 1986). Primarily from an ovarian source, A4 concentrations rise during
pregnancy and are converted to testosterone by the placenta, bathing the fetus in
androgens during development (Licht, et al., 1998; Yalcinkaya, et al., 1993). Therefore,
androgens would appear to offer a convenient mechanism by which selection might
promote aggressive females able to dominate access to critical resources. The
development of the female phallus was long thought to be a by-product of this androgen-
mediated selection in spotted hyenas (Gould, 1981). However, investigation of urogenital
development in this species has shown that the genital tissue is committed to phallic
development even before fetal gonadal differentiation (Cunha, et al., 2005; Glickman, et
al., 2005; Licht et al., 1998), and administration of anti-androgens during gestation does

not prevent the development of the phallus in female fetuses (Drea et al., 1998). On the

10

other hand, there are important sex differences in the structure of the phallus (Frank et al.,
1990; Glickman et al., 1992); for instance, the female phallus is much more elastic than
the penis, and experimental administration of anti-androgens has shown that these sex-
differences are dependent on androgen exposure during early development (Drea, et al.,
2002; Drea et al., 1998). Researchers are now expanding their focus toward steroid-
independent mechanisms to enhance our understanding of urogenital development among
spotted hyenas.

Although it is apparent that androgens are not solely responsible for the
masculinization of the external genitalia of female spotted hyenas, there is clear evidence
that prenatal androgen exposure inﬂuences the behavior of spotted hyenas. Maternal
androgen concentrations during pregnancy, measured in feces, are correlated with rates of
mounting and aggressive behaviors in both male and female offspring when they are
juveniles (Dloniak et al., 2006b). Offspring from mothers treated with anti-androgens
during pregnancy exhibit a reduction in the postnatal sibling aggression typically seen in
this species (C. Drea and SE. Glickman, pers. communication). Taken together, these
results suggest that prenatal androgen exposure may be organizing the neural structures
underlying aggressive behaviors in both male and female spotted hyenas. However, little
is known about the factors mediating aggressive behavior among adult hyenas.

There is evidence to suggest that aggressive behavior among spotted hyenas is
under activational control of steroid hormones. Perhaps not surprisingly, circulating
androgen concentrations among adult males are tightly correlated with aggression related
to courtship and mate defense (Dloniak et al., 2006a; Goymann et al., 2003). The

evidence suggesting that gonadal steroid hormones activate female aggression is less

11

straightforward. Among captive hyenas, juvenile ovariectomy appeared to reduce rates of
inter-sexual aggression emitted by captive female hyenas as adults (Baker, 1990).
However, gonadectomy had no effect on rates of intrasexual aggression among juveniles
when compared to controls (Frank, et al., 1989). In this dissertation, I address the
possibility that steroid hormones are mediating aggression in adult spotted hyenas living
in the wild, and I investigate predictions of both the organizational and activational

hypotheses of steroid hormone control of aggressive behavior in this species.

DESCRIPTION OF CHAPTERS

This work predominantly draws on data collected as part of a collaborative effort
to monitor a single, large social group of hyenas living in the Masai Mara National
Reserve. Begun in 1988 by Drs. Kay Holekamp and Laura Smale, this study has
produced almost 20 years of continuous behavioral and ecological data, and over ten
years of fecal hormone sampling. This longitudinal project was supervised by Dr.
Holekamp, and data were collected by numerous graduate students and lab assistants
under her training. It is only now, with 20 years of data, that I might begin to ask the sorts
of questions I explore in the following chapters of my dissertation, and thus I will be
using the ﬁrst person plural as I write the data chapters ahead to indicate that this work
was only possible as a collaborative effort.

In Chapter 1, I ﬁrst present a validation for the measurement of estrogens excreted
in spotted hyena fecal matter. Endocrine studies of free-living animals are plagued by
difﬁculties associated with obtaining sufﬁcient samples for analysis of hormones in

blood. Mammalian carnivores, in particular, represent a taxonomic group in which

12

endocrine research advanced slowly until the advent of non-invasive hormone sampling
(Brown, et al., 1996; Young, et al., 2004). Advances in non-invasive techniques, such as
fecal steroid hormone analysis, offer an economical alternative to blood sampling for
frequent collection of samples without disrupting the normal behavior of individuals or
groups. Many species excrete multiple steroid metabolites that can be measured with
immunoassays like those used to measure steroid hormones in blood.

Drs. Stephanie Dloniak and Jeffery French have already published similar
validations for non-invasive measurement of both fecal androgens and fecal
glucocorticoids in the spotted hyena (Dloniak et al., 2006a; Dloniak et al., 2006b;
Dloniak, et al., 2003). The validation of a method for fecal estrogen analysis, presented
here, adds to this arsenal of tools available for use in the investigation of hormonal
mediation of behavior among spotted hyenas. In addition, having access to estrogen
measures from females increases our ability to evaluate their reproductive condition. This
work was published in General and Comparative Endocrinology (Van Meter, et al.,
2008).

As a demonstration of the responsivity of hormone physiology to environment
variability, in Chapter 2 we investigated both naturally occurring and anthropogenic
inﬂuences on stress physiology in spotted hyenas. In this chapter we used both
longitudinal data from a single social group, and cross-sectional data from multiple social
groups to examine social, ecological, and anthropogenic factors as possible stressors in
the lives of wild spotted hyenas. From our longitudinal study, we learned that spotted
hyenas are well adapted to variation in their natural ecology, including seasonal

ﬂuctuations in prey and rainfall. However, unpredictable events, such as sudden periods

13

of instability in the social hierarchy do inﬂuence spotted hyena stress physiology. Finally,
among adult male hyenas, we saw an impact of increasing human disturbance on stress
physiology. This result was strengthened by our cross-sectional comparison, which
demonstrated that this population of hyenas, which is now exposed to high levels of
human disturbance, had elevated stress hormone concentrations when compared to
hyenas exposed to relatively low levels of human disturbance. This cross—population
study was made possible by the work of two students who received their Ph.D.’s under
Dr. Holekamp’s supervision. Dr. Joseph Kolowski expanded our understanding of hyenas
living in the Masai Mara by intensively studying a second group living in the Mara. Also,
Dr. Heather Watts began a parallel study of hyenas living in Amboseli National Park for
her dissertation work. It was their effort and foresight that permitted the comparison
presented in this chapter, and thus they are co-authors on this manuscript, which was
published in Hormones and Behavior (Van Meter, et al., 2009).

The last two chapters in this dissertation primarily focus on aggressive behavior
among adult spotted hyenas. In Chapter 3, I had two main goals. First, using 17 years of
behavioral data collected from our longitudinal project, I endeavored to provide a
comprehensive description of inter— and intrasexual aggression among adult spotted
hyenas in a variety of social contexts. Second, I used these data along with 12 years of
hormone measurements collected non-invasively, to test predictions stemming from both
the organizational and activational hypotheses of steroid hormone action. Using careful
behavioral rate calculations that controlled for variation in opportunity to behave
aggressively, we show that rates of aggression emitted by adult females are clearly higher

than those emitted by adult males, and that females are more aggressive to males than

14

males are towards females. While it appears that rates of intrasexual aggression do not
differ signiﬁcantly between the two sexes, aggression among females is of higher-
intensity that that among males. I also showed that rates of intersexual aggression emitted
by females as adults, were correlated with their prenatal androgen exposure, supporting
the organizational hypothesis of steroid hormone control of behavior.

In the ﬁnal data chapter, I examined a particular type of aggressive behavior
exhibited by spotted hyenas, unprovoked aggression. I tested two functional hypotheses
as possible explanations for this behavior among adult females. First, I investigated
unprovoked aggression as a possible mechanism for reproductive suppression, like that
seen in certain cooperatively breeding species (Solomon and French, 1997). The
prediction here was that females receive higher rates of unprovoked aggression when
they were reproductively vulnerable. Second, I hypothesized that females use unprovoked
aggression to test the strength of rank relationships. There was no supporting evidence
that aggression is preferentially directed toward females to suppress their reproductive
efforts, as females did not receive higher rates of unprovoked aggression during early
pregnancy or around the time of conception relative to when they were in other
reproductive states. Females appear to use unprovoked aggression as a means for testing

rank relationships among closely ranked individuals.

15

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23

CHAPTER 1

Van Meter, P. E., French, J. A., Bidali, K., Weldele, M. L., Brown, J. L., Holekamp, K.
E., 2008. Non-invasive measurement of fecal estrogens in the spotted hyena (Crocuta
crocuta). General and Comparative Endocrinology 155(2), 464-471.

24

CHAPTER 1
NON-INVASIVE MEASUREMENT OF FECAL ESTROGENS IN THE SPOTTED
HYENA (CROC UTA CROC UTA)
INTRODUCTION

Knowledge about a female’s reproductive state is essential to understanding
aspects of her basic biology, including her fertility, behavior (Altmann et al., 2004;
Maestripieri, 1999; Ramirez et al., 2004; Wise, 1974), nutritional requirements (Lambert
et al., 2005; Schneider et al., 2000), and immune function (Gu et al., 2005; Hemandez-
Gonzalez et al., 2006). Despite the importance of understanding reproductive cycles in
females, there are many species for which we know virtually nothing about variation in
reproductive status, particularly within free-living populations of large predatory
mammals. Estrogen concentrations often vary predictably during female reproductive
cycles, so the ability to measure estrogens offers one potential mechanism for monitoring
ovarian status. Here our goal was to develop methods for non-invasive measurement of
estrogens in free-living spotted hyenas (Crocuta crocuta).

Studies of the endocrinology of spotted hyenas to date have focused on
androgenic hormones, so a great deal is known about circulating and excreted androgens
in Crocuta (Dloniak et al., 2006; Dloniak et al., 2003; Glickman et al., 1998; Glickman et
al., 1992; Goymann et al., 2001; Licht et al., 1992; Licht et al., 1998; Van Jaarsveld and
Skinner, 1991). By contrast, little is known about estrogenic hormones in this species. In
a study of captive spotted hyenas between infancy and sexual maturity, Glickman et a1.
(1992) found that plasma estrogen concentrations begin to rise in females by 23 months

of age, which is also when they may conceive their ﬁrst litters in nature (Holekamp et al.,

25

5'11

1996). It was further shown that plasma estrogen concentrations are higher in adult
females than in adult males, and that mean plasma estrogen concentrations are higher in
adult than sub-adult females (Glickman et al., 1992). Additionally, pregnant females have
higher plasma estrogen concentrations than non-pregnant females, and plasma estrogen
concentrations peak during late pregnancy (Licht et al., 1992). However, we know very
little about the female’s estrous cycle in this species, including the cycle length, and
whether ovulation is induced or spontaneous, nor is anything known about patterns of
estrogen secretion among wild hyenas.

Gaining information about the reproductive condition of wild female hyenas is
difﬁcult. There are no external indicators of changing reproductive state, such as genital
swelling, color change, or other visual signs of ovulation often used by researchers
studying free-living mammals. Even late pregnancy is difﬁcult or impossible to detect by
visual inspection in this species because female body mass can change by up to 18 kg at a
single meal (e. g. Henschel and Tilson, 1988). Sampling blood from hyenas for
measurement of steroid hormone concentrations requires invasive and potentially
dangerous procedures, including isolation and anesthesia (Glickman et al., 1992), so
multiple samples from the same individual are difﬁcult to obtain in both wild and captive
settings. Recent advances in non-invasive sampling methods for hormone analysis offer
opportunities for more rigorous investigation of the endocrine correlates and control of
reproduction in Crocuta. Fecal steroid analysis, in particular, offers an attractive
alternative to blood sampling in both wild and captive animals. For collecting samples
frequently, non-invasively, and without disrupting normal behavior of individuals or

groups, techniques have been developed to assay fecal estrogen concentrations in a

26

number of other mammalian carnivores (Brown et al., 1994; Monfort et al., 1997; Onuma
et al., 2002; Shille et al., 1984; Young et al., 2001). A preliminary study in captive
Crocuta detected measurable amounts of radiolabeled estrogen in fecal matter, and
indicated that feces, rather than urine, should prove useful for sampling of excreted
steroid hormones (Koretz, 1992).

Our aim here was to develop and validate an enzyme-immunoassay (EIA) for
measurement of fecal estrogens (fE) in wild and captive spotted hyenas. First, we
identiﬁed the immunoreactive estrogen metabolites in hyena feces using high-
performance liquid chromatography (HPLC). We then investigated assay parallelism,
examined possible procedural covariates of estrogen concentrations, and assessed the
biological validity of our assay by monitoring changes in estrogen concentrations
following treatment with luteinizing-hormone releasing hormone (LHRH) of wild and
captive hyenas. Finally, we compared patterns of estrogen concentration between feces
and plasma among classes of hyenas that were expected to exhibit signiﬁcant variation in

estrogen (i.e., pregnant vs. non-pregnant females, mature vs. immature females).

METHODS
Field study site, subjects and sample collection
Field data were collected in the Talek region of the Masai Mara National Reserve,

Kenya. Subjects were members of one large, stable social group (“clan”) that defended a

territory of 62 km2 (Boydston et al., 2001). Talek hyenas have been closely monitored

since 1988, and clan members are usually observed intensively on at least 23 days each

month. All clan members were individually recognized by their unique spot patterns, and

27

sex was determined by the dimorphic glans morphology of the erect phallus (Frank et al.,
1990). Ages of all individuals born into the clan since 1988 were known, and mother-
offspring relationships were established on the basis of nursing associations. Age at ﬁrst
reproduction and full reproductive histories were known for all female members of the
clan from 1988-2004 based on known dates of birth and weaning of cubs. Each female
was assigned to one of the following three reproductive states each time she was
observed:

Immature-Females were considered reproductively immature from birth until
ﬁrst conception. In the wild, we can conﬁdently assign a date to each female’s ﬁrst
parturition because of the extensive tearing and scarring of her phallus during the birth
process (Frank and Glickman, 1994). Gestation in spotted hyenas lasts 110 days (Kruuk,
1972), so we could accurately estimate the date of ﬁrst conception by counting
backwards 110 days from the known date of ﬁrst parturition.

Pregnant-Females were considered pregnant during the period between the birth
of each litter and its estimated conception date. Birth dates were assigned based on the
size, pelage, and behavior of cubs when they were ﬁrst seen above ground, and were
accurate to within i 7 days (Holekamp et al., 1996). Pregnancy was further divided into
trimesters to analyze variation in hormone concentrations among trimesters. Early
pregnancy was the period between conception and day 37 of gestation. Middle pregnancy
was day 38 through day 74, and late pregnancy was day 75 though the day of parturition.

Lactating-The lactation interval in Crocuta is highly variable in length (from 7 to
24 months), as it is affected by food availability, social rank, and litter composition (twin

vs. singleton litters) (Holekamp et al., 1996; Holekamp et al., 1999). Termination of

28

lactation was determined here either by conception of the next litter, or by weaning or
disappearance of the current litter, whichever came ﬁrst. Wean dates were determined
using behavioral observations of mother-infant conﬂicts over nursing, as described by
(Holekamp et al., 1996). Wean dates for which mother-infant conﬂicts were not observed
were calculated as the day halfway between the last time the cubs were seen nursing and
the next time dam and cubs were seen together, if these could be estimated to :t 10 days.
Starting in 1990, each clan member was immobilized at least once for collection

of physiological and morphological data. Individuals were anesthetized with Telazol (6.5

mg/kg body mass) administered in a plastic dart ﬁred from a C02 riﬂe. All

immobilizations took place between 0630 and 0900 hours, and were performed in
accordance with Kenyan law and NIH Guide for the Care and Use of Laboratory
Animals. Within 17 minutes of Telazol administration, a blood sample (10 ml) was taken
from the jugular vein in a heparinized vacutainer tube. Blood samples were centrifuged,

and plasma was stored in liquid nitrogen until it could be shipped on dry ice to Michigan

State University (MSU), where it was stored at —80°C until radioimmunoassay.

Beginning in 1993, fecal samples were collected whenever a known hyena was
observed to defecate. Fecal samples were collected either in the morning (0600-0900) or
evening (1700-2000) into plastic bags within 30 minutes of excretion. Samples were
maintained at room temperature after collection for up to 12 h, after which they were
mixed and stored in liquid nitrogen until they were shipped on dry ice to MSU. (Dloniak,
2004). Dloniak (2004) found that variation in time elapsed between collection and

freezing of fecal samples does not signiﬁcantly affect concentrations of other steroid

29

hormones in the samples, and we assumed here this applied to fE as well. Samples were
stored at MSU at minus 20°C until extraction and enzyme immunoassay.

Administration of exogenous LHRH, acting via intermediate effects on the
pituitary, stimulates secretion of steroid hormones from the gonads of hyenas (Ensley et
al., 1982; Holekamp and Sisk, 2003; Place et al., 2002). Nine parous wild females were
tested during late lactation (7.9 - 13.3 months after parturition) for steroid production in
response to LHRH injection. Additionally, three wild adult females were administered
saline to serve as handling and injection controls. After immobilization, blood samples
were taken at 5-minute intervals from the jugular vein for 45 minutes. Then, females
were injected intravenously with LHRH (1 ug/kg LHRH, L-7134 Sigma Chemical CO.,
St. Louis, MO). Blood sampling then continued at 5-minute intervals for 120 minutes.
Supplementary doses of Telazol were administered as necessary throughout sampling to
maintain deep anesthesia.

Captive study site, subjects, and sample collection

Captive data were collected from C rocuta in the colony maintained at the Field
Station for Behavioral Research of the University of California, Berkeley. Subjects were
housed individually or in small groups, and fed a standard zoo carnivore diet

supplemented with bone. All fecal samples were collected between 0800 and 1200 hours,
mixed, and stored at -80°C until extraction and enzyme immunoassay.

Five gonadally intact adult captive hyenas (3 males and 2 females) were injected
with LHRH to induce the release of gonadal steroids. On the day of LHRH challenge,
subjects were isolated, and then immobilized with ketamine (6.0 mg/kg body mass) and

xylazine (1 mg/kg body mass) administered by blow dart. Each subject was administered

30

an intravenous injection of LHRH, allowed to recover from anesthesia and released back
into its home enclosure. Fecal samples were collected daily for 7—8 days prior to LHRH
injection, on the day of injection, and then for 7—8 days following injection.
Extraction and immunoassay of fecal samples

A total of 871 fecal samples were collected from known female Talek hyenas.
Extraction of steroid hormones has been described elsewhere (Dloniak et al., 2006;
Dloniak et al., 2003). Brieﬂy, frozen samples were lyophilized (Labconco Freeze-Dry
System 10-269), ground to a ﬁne powder, and shaken overnight in 100% ethanol.
Samples were then boiled and centrifuged, and the resulting supernatant was poured off
into culture tubes while the remaining fecal pellet was discarded. Finally, the supernatant

was allowed to evaporate. Extracted samples were then reconstituted in 1.0 ml phosphate
buffered saline (PBS; pH 5.0), sealed, and stored frozen at -20°C until assay.

The number and proportion of estrogen metabolites in C rocuta feces were
determined by HPLC analysis of fecal extracts from one pregnant and one non-pregnant

wild female hyena using modiﬁcations of methods described in Brown et al. (1994).

Extracted samples were spiked with 7000 cpm of 3H-estrone (E1) and 3H-estrone sulfate

(ES), and air-dried. Samples were then reconstituted. in 500 pl of PBS and vortexed. A

C18 sample preparation cartridge (SpiceTM Cartridge; Analtech, Inc., Newark, DE), was

primed with methanol (MeOH) and distilled water, and loaded with the total volume of
sample in PBS. Distilled water and MeOH were pushed through the cartridge and
collected into test tubes. The MeOH portion was allowed to evaporate and the residue

was resuspended in MeOH. HPLC was then conducted by injecting 0.05 ml of the

31

lt‘S'

10.“

ES

lift

Eli

la"

‘I .3
343'»

pm

End

read

resuspended residue onto the column (Reverse Phase MicrosorbTM MV 100 C18, Sum

diameter particle size, Varian Analytical Instruments, Woburn, MA). Fecal extracts were
recovered by a mobile phase gradient of 20% increasing to 80% MeOH in water over 80

minutes (1 ml/mm) at room temperature. A portion of each fraction (0.1 ml) was assayed

for radioactivity to determine the retention times for the radiolabelled standards (E1 and
ES). The remainder of the fractions (0.9 ml) was allowed to evaporate until dry and
reconstituted in 0.25 ml PBS. These samples were then assayed with an estradiol (E2)

EIA and an estrone conjugate (ElC) EIA (French et al., 1996) to evaluate the
immunoreactivity of the fractions.

Based on the results of these two assays (see Results), the E2 EIA was chosen for
use here, and all fecal samples were assayed for estrogens using a modiﬁed protocol
previously described (Nunes et al., 2000). We used an E2 antibody (R4972; Clinical
Endocrinology Laboratory, University of California, Davis); other compounds cross-
reactive with this E2 antibody were estrone (3.3%), progesterone (0.8%), testosterone
(1.0%), and androstenedione (1.0%). Absorbance was measured with a Dynex plate
reader when optical density in B0 wells reached 1.0. Precision of the assay was monitored

by measuring two sets of hyena fecal extract pools. The intra-assay coefﬁcients of
variation for high and low pools were 3.66 and 3.30%, respectively. The interassay
coefﬁcients of variation were 11.96 and 14.71%, respectively (n = 25 plates). The 871
fecal samples were assayed over the course of two years. The correlation between 13
randomly chosen samples assayed repeatedly in each year was r = 0.98, demonstrating

low inter-year variation.

32

Assay parallelism was tested by measuring fecal estrogen concentrations in serial
dilutions of samples from three different individuals. The slopes of the resulting
displacement curves were compared to that of the standard curve using a test of slopes
available in the program Prism (GraphPad Software Inc.).

Immunoassay of plasma samples

Plasma samples collected from free-ranging hyenas .in the Talek clan were

. . . . 125 . . .
assayed for estradiol in duplicate us1ng coated tube I radioimmunoassay (RIA) kits

(Diagnostic Products Corp; Los Angeles, CA). Kit validation for use with Crocuta was
accomplished by demonstrating parallelism between curves representing serial dilutions
of pooled hyena samples, spiked samples, and standard curve calibrators included with
the kit. The minimum detection limit for this assay was 7.4 pg/ml. The antibody is highly
speciﬁc for estradiol and has minor cross-reactivity with other compounds: estriol 0.32%,
estrone 10%, estrone-3-sulfate 0.58%, estradiol monosulfate 0.29%, estradiol propionate
0.70%, and DHEA, androstenedione, Sa-dihydrotestosterone, and testosterone all at less
than 0.001%. The mean intra-assay coefﬁcient of variation was 7.85% and interassay
coefﬁcient of variation was 12.14% (11 = 23 assays).
Statistical analysis

Data were log-transformed before analysis if not normally distributed. Procedural
covariates (sample collection time and hyena identity) were analyzed with a step-wise
regression allowing for each predictor variable to be measured while controlling for
reproductive state. Signiﬁcant predictors were then further investigated using within
subjects analyses. Differences among reproductive states (immature, pregnant, and

lactating) were evaluated using one-way analysis of variance (ANOVA). Along with test

33

statistics, means and standard errors are presented. For further investigation of signiﬁcant
differences, two-tailed t-tests were used except when testing speciﬁc directional
hypotheses based on expected differences in hormone concentrations between
reproductive states, as when comparing reproductively immature and mature females, or
pregnant and lactating females; in these cases one-tailed tests were used. To examine
differences among trimesters of pregnancy, a post-hoc Tukey’s analysis was performed
for both plasma and fecal samples. When log-transforrned data were not normally
distributed, or sample size was very small (as in the case of the LHRH challenges), non-
parametric tests were used. Signiﬁcant differences were identiﬁed using a = .05. All

analyses were performed using SPSS for Windows (Version 12.0.0, SPSS Inc.).

RESULTS
High-performance liquid chromatography

Immunoreactive E2 was detected in fecal extracts at levels three- to four-fold
higher than immunoreactive EC in samples from both pregnant and non-pregnant

females. We observed differences in immunoreactivity between samples for pregnant and

non-pregnant females of roughly an order of magnitude (Figure 1.1). The highest
concentrations of immunoreactive estrogen metabolites co-eluted with the 3H-estrone
standard (fractions 60-65), accounting for more than 74% of total immunoreactive
estrogens. Also, a smaller peak in E2 immunoreactivity was present in fractions 65-70,
accounting for 14% of the total immunoreactivity. Thus, the E2 antibody appears to

detect multiple estrogen metabolites in the feces of female spotted hyenas, and may

therefore be used as a measure of total fecal estrogens (fE).

34

(I.

lh'

Fecal EIA parallelism

Serial dilutions of three hyena samples were plotted along with standards as
relative dose vs. percent bound. Fecal extract curves did not differ signiﬁcantly from the
standard displacement curve within the range of 20 — 80% antibody binding (F 3, g = 2.06,
P = 0.18; Figure 1.2). Of our assayed fecal samples (n = 871), 83% fell within these
binding parameters, and samples falling outside this range were diluted accordingly and
re-analyzed.
LHRH challenge experiments

Estradiol was measured in repeated plasma samples drawn from 9 free-living
females challenged with LHRH administration during late lactation, and from 3 saline-

treated control females. For each challenged individual, the sample with the highest
concentration of E2 prior to LHRH administration (pre-injection peak, 17.76 i 2.44
pg/ml) was compared to its sample with the highest E2 concentration after LHRH
administration (post-injection peak, 21.71 :t 2.21 pg/ml). Peak E2 concentrations were
signiﬁcantly higher after than before LHRH administration (paired t8 = -6. 19, P < 0.001;
Table 1.1). By contrast, the three females injected with saline demonstrated no rise in
plasma E2 (Wilcoxon matched pairs test: Z3 = -5.35, P = 0.59). Thus, treatment with
LHRH was followed by a signiﬁcant rise in circulating E2 in lactating female spotted

hyenas.
Five captive hyenas (Table 1.2) were also challenged with LHRH. For each

individual, the sample with the highest fE prior to LHRH administration (484.18 x

35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A 4000 3000 - 800
-O- Hormone Standards (CPM) E
-O- Immunoreactive Estradiol 1 l
-O— Immunoreactive Estrone Conjugates ‘i‘ ' 2500 __
, cu
.1 _ 1—
3000 BIS 3 600 i
. - 2000 g) a,
a a:
-' OD
Ezooo- 45005-40053,
D g g
'1 63 U
"s — 1000 8
O
1000‘ I ‘ ~ 200 E
I 9 ~ 500
9‘) ' ,
1! ‘ e 3,:
O > it - “we. .toﬁ'j: "- b O ‘ O
0 20 40 60 80
Fraction
5000 300 ‘ 70
B —o— Hormone Standards (CPM)
-O- Irnmunoreactive Estradiol _ 60
4000‘ —Q— Immunoreactive Estrone Conjugates - 250 _
E1 g
— 50
- 200 73) E?
a cu
3000 E13 3) t 40 ‘5
E G- 2?
0 =5 _ 30 o
g 3
(I)
m - 8
20 3:
L3
- 10
- 0

Fraction

 

 

 

Figure 1.1. HPLC results from A) pregnant and B) non-pregnant female hyenas.
Fractions were assayed for immunoreactivity with both estradiol (open circles) and
estrone conjugate (gray circles) enzyme immunoassays, and immunoreactivity is
expressed on the right-hand axes. Elution of radiolabeled standards (black circles),

estrone (E1) and estrone sulfate (E1S), are expressed as counts-per-minute (CPM) per
fraction on the left-hand axis.

36

 

 
   

Standard 0 I

80 - Female Hyena 1 O
A . Female Hyena 2 v
EC 70 4 Female Hyena 3 v
99, 60 -
DD
.5 50—
"D
.E 40 -
m
5° 30 -

20 ‘

 

 

 

I T I T

19 40 78 156
pg Estradiol/g Fecal Mass

Figure 1.2. Relative dose vs. percent of antibody bound for standards and serial dilutions
of samples from three hyenas. Slopes of all curves are parallel in this range (20—80%
binding; P = 0.18).

37

Table 1.1.

Free-living hyena plasma estradiol (pg/ml): Individual and mean (1 standard errors)
response patterns to LHRH challenge

 

 

Hyena ID Pre-Injection Post-Injection % Pre-Injection Minutes to
Peak Peak Peak Maximum

BM 7.8 14.23 182.40 58

COCH 26.3 29.51 109.70 24

DJ95 15.4 21.66 140.65 64

JAB 22.7 28.52 125.64 114

PT 18.4 19.27 108.42 67

SX 18.5 22.59 122.11 19

UA94 13.4 15.35 114.55 109

UA96 8.5 13.7 161.18 60

WHO 28.2 30.58 108.44 19

Mean 17.76 :1: 2.44 21.71 :1.- 2.21 129.93 i 8.87 59.33 1 11.81

 

38

Table 1.2.

Captive hyena fecal estrogen (ng/g feces): Individual and mean (:t standard errors)
response patterns to LHRH challenge

 

 

Hyena ID Pre-Injection Post-Injection % Pre-Injection Days to
Peak Peak Peak Maximum

Female 35 129.16 174.99 135.49 2

Female 49 1935.52 3480.34 179.81 2

Male 13 226.69 3175.10 1400.66 2

Male 45 84.06 191.82 228.18 3

Male 51 45.49 164.07 360.64 1

Mean 484.18 :t 364.09 1437.26 :1: 773.30 460.96 :1: 237.94 2.0 :t 0.31

 

39

364.09 ng/ g) was compared to peak fE concentrations after LHRH administration

(1437.26 :1: 773.33 ng/g). Peak 1E concentrations were signiﬁcantly higher after than

before LHRH administration (Wilcoxon matched pairs test: z5 = 2.02, P = 0.04). Mean

latency to peak fE post-challenge was 2.0 i 0.31 days. Thus, as with plasma E2, LHRH

administration was followed by a measurable increase in fE, but with an average time lag
of two days.
Comparison of plasma and fecal estrogen concentrations

Procedural covariates

To evaluate procedural covariates associated with our sampling technique and to
avoid pseudoreplication, a step-wise regression was used to determine how much
variation in fE concentrations could be explained by collection time (morning, 0600-

0900, or evening, 1700-2000) and individual identity, after reproductive condition had

2
been controlled. The full model yielded an R = .07, F 3,536 = 14.09, P < 0.001.

Reproductive state (immature, pregnant, and lactating) explained a signiﬁcant portion of
the variance in fE (P < .001). Collection time was a signiﬁcant predictor over and above
the variance explained by reproductive state (P = 0.009), whereas hyena identity was not
(P = 0.22). The effect of collection time was further examined by pairing morning and

evening samples for 35 individuals. Concentrations of fE were signiﬁcantly higher in
morning than evening samples 034 = 4.19, P < .001). Therefore all subsequent analyses

utilized only morning samples.
Age
To examine how fE varies with age between 11 — 35 months, we assigned females

that had not yet given birth to a speciﬁc four-month age interval, and calculated the

40

 

80

 

 

 

 

 

 

 

 

 

 

 

OD
F0 60 - 7
s:
o
o
1—4
A
m 40 -
d)
F-l
Cd
E
:13
,H 20 -
0
8°
0
11 V 19 23 27 31 35
Age in Months

Figure 1.3 Percentage of nulliparous females sampled in each 4-month age interval
whose mean fecal estrogen concentrations exceeded 100an feces. Numbers over bars
represent females sampled in each age group. Multiple samples collected from individual
females within age interval were averaged.

41

percent of these females whose feces contained more than 100 ng/ g fE within each
interval (Figure 1.3). The threshold of 100 ng/g was chosen because it exceeded average
fE concentrations from pre-reproductive females. Concentrations of fE were averaged for
females sampled multiple times within a given age interval. These data suggest that fE
begins to rise at approximately 23 months of age in free-ranging females.

Reproductive state

During immobilizations conducted in the wild from 1990-2004, we collected 136
blood samples from 89 females in known reproductive condition. Plasma E2

concentrations were averaged for females sampled multiple times when they were in a

single reproductive state. A one-way between subjects AN OVA indicated a signiﬁcant
effect of reproductive state (immature, pregnant, or lactating) on plasma E2 (F 2,1 18 =
138.88, P < 0.001). Plasma E2 concentrations were low in immature females (5.29 i 0.42 _

pg/ml) and lactating females (6.44 a: 0.77 pg/ml), and highest in pregnant females
(137.79 :1: 36.88 pg/ml). We next looked within individuals by conducting paired t-tests

on samples from females that were sampled multiple times when they were in different

reproductive states. Plasma E2 concentrations were signiﬁcantly higher in females
sampled after than before reproductive maturity (t23 = -3.53, P < 0.001; Figure 1.4.A),
and concentrations were also higher during pregnancy than lactation (t7 = 4.67, P <

0.001; Figure 1.4.B). Within pregnancy, plasma E2 increased across trimesters (F 2, 21 =
15.53, P < 0.001) (Figure 1.5). A post-hoe Tukey’s test (HSD = 0.20) showed that plasma
E2 concentrations were signiﬁcantly higher in late pregnancy than during either the early

or middle trimesters.

42

 

_ Plasma Estradiol
Fecal Estrogen

n=24,p=.001

 

m
C
1

Plasma Estradiol (pg/m1) a)
3

 

 

Immature Mature

 

n=22,p=.002

 

 

      
    
 
 

‘I—c

 
 

i._1

. .
AAIu'ALhmkni '

I
. I
”filo

o-‘nn 'I I T
. 111""..121'
. t

A" n
1 " 'I . . -r ‘ I -
.i .l‘s’ghnihiltuw

Immature Mature

 

— Plasma Estradiol
[:23 Fecal Estrogen

§

n=8,p=.001

 

“8’

Plasma Estradiol (pg/ml) w
é

 

 

Pregnancy Lactation

n=20,p=.001

 

 

 

 

 

 

~900

r600

r300

 

L 1200

~800

~400

 

 

Pregnancy Lactation

Fecal Estrogen (ng/g feces)

Fecal Estrogen (ng/g feces)

Figure 1.4 Within-female comparisons between A) reproductively immature and mature
states, and B) pregnant and lactating states for plasma estradiol (black) and fecal
estrogens (gray). Means and standard errors are presented, and signiﬁcance was tested

using paired t-tests. Note that the scales represented on the two y-axes differ between

sampling techniques.

43

 

 

 

 

 

 

_ Plasma Estradiol
(g 300 ‘ Fecal Estrogen b d g
o
3.0 L 1500 19;.)
E: in
a 52°
a) 200 - v
e - 1000 5
‘53 8°
LL} 1:»
a: m
- 1::
co cc:
5 8
LL

0 .. r 0

Early Middle Late Early Middle Late

Trimester of Pregnancy

Figure 1.5 Estrogen concentrations in plasma (black) and fecal material (gray) across
trimesters of pregnancy, averaged across females. Signiﬁcant differences among mean
(:tSEM) plasma estradiol and fecal estrogen concentrations are indicated by different
letters, and were tested using Tukey’s post-hoc test. Note that the scales represented on
the two y-axes differ between sampling techniques.

Variation in fE among reproductive states resembled that seen in plasma E2. From

1993-2005, 295 morning fecal samples were collected from 56 females. A one-way
between subjects ANOVA indicated a signiﬁcant effect of reproductive state on fE
(F 2,294 = 56.57, P < 0.001). Fecal estrogen concentrations were lowest in immature
females (150.27 1 32.95 ng/g), higher in lactating females (368.91 1 37.25 ng/g), and

highest in pregnant females (1235.73 :1: 229.24 ng/g). This result was conﬁrmed by paired

t-tests within females sampled in multiple reproductive states. Females had signiﬁcantly
higher fE concentrations after than before reaching reproductive maturity (t21 = -6.49, P
< 0.001; Figure 4a), and fE concentrations within females were higher during pregnancy

than lactation (t19 = 3.23, P = 0.002; Figure 4b). Finally, fE rose during the course of
gestation (F 2,75 = 3.22, P = 0.045; Figure 5). A post-hoe Tukey test (HSD = 362.13)

comparing trimesters revealed that late trimester fE concentrations were signiﬁcantly
higher than early fE concentrations, but middle trimester fE concentrations were not

signiﬁcantly different from either early or late trimester concentrations.

DISCUSSION
The aim of this study was to validate a non-invasive technique for measuring

estrogens in spotted hyenas. Our results show that biologically signiﬁcant variation in
fecal concentrations of estrogen metabolites can be assessed using the E2 EIA in both

wild and captive hyenas. Furthermore, estrogen concentrations in feces show the same
patterns of variation as those in plasma. Fecal hormone analysis offers a means for

collecting samples repeatedly and frequently from individuals without disturbance of

45

normal behavior, and should therefore help future researchers to answer a number of

questions about the developmental and reproductive biology of this species.

For the EIA developed here, we chose an antibody speciﬁc for E2 because
immunoreactivity of fractions from female hyenas was three- to four-fold higher when
assayed with the E2 antibody than with an antibody speciﬁc for EC. Use of the EC
antibody in the immunoassay would have rendered a large proportion of our samples

below detectable limits, whereas all of our samples run with the E2 EIA yielded

measurable fE. Although E2 was not run as a radiolabeled standard in our HPLC analysis,

HPLC results from several felids, the taxonomic group closest to the hyenids (Bininda-

Emonds et al., 1999) from which we have HPLC data from feces, reveal that elution of
E2 often overlaps with that of E1 (Brown et al., 1994), the largest immunoreactive peak in
fecal samples from both pregnant and non-pregnant hyenas seen here. Additionally,

radiolabeled estrogens in the domestic cat are predominantly excreted as unconjugated E2

and E1 (Brown et al., 1994; Shille et al., 1984), and we presume because of their close
taxonomic relationship to hyenas, these particular estrogens may also be important
immunoreactive metabolites in hyena feces. All of these factors suggest that an E2-

speciﬁc antibody represents an acceptable choice for measuring estrogen in hyena feces.
With the understanding that other estrogens, and perhaps also novel metabolites, may be
cross-reacting with this antibody in Crocuta samples, our assay permits measurement of
total fecal estrogens, rather than fecal estradiol per se.

We assessed parallelism by comparing the slopes of curves generated by several

diluted samples to the slope of the curve generated by the E2 standards. Although these

46

slopes were parallel only when antibody binding was between 20 and 80%, this assay
should nevertheless be informative as long as this limitation is considered. The large
majority of our samples fell within this range, and samples outside of this range could be
assayed after appropriate dilution.

Circadian rhythms in fecal and urinary steroid hormones have been documented
in many marmnals (Bos et al., 1993; Sousa and Ziegler, 1998), so we examined time of
day here as a procedural covariate. We found that, like fecal glucocorticoids (Dloniak,
2004), but unlike fecal androgens (Dloniak et al., 2003), concentrations of fecal estrogens
in C rocuta samples collected in the morning are higher than in samples collected in the
evening. This source of variation, once identiﬁed, was easily controlled by restricting
analysis to morning samples.

The biological tests we conducted in vivo further validated our assay. After
excluding evening fecal samples from our analyses, the same variation seen in plasma
estradiol among age classes and reproductive states was observed in fE values. As in
plasma, fE concentrations remained low until reproductive maturity, were highest during
the ﬁnal trimester of gestation, and were relatively low during lactation. Furthermore,
although this requires further testing, our fE data offer a preliminary indication that
estrogen concentrations in wild spotted hyenas began to rise late in the second year of
life, as is also true in captive females, based on measurements of plasma estrogen
(Glickman et al., 1992).

Our data suggest that, like plasma estrogens, fE concentrations rise in response to
LHRH challenge, but not after saline injection. A previous study had shown that plasma

LH, testosterone, and androstenedione concentrations in both males and females rise in

47

response to LHRH administration (Place et al., 2002), but estrogen was not among the
steroid hormones measured in that work. In the brown hyena (Parahyaena brunnea), a
species closely related to the spotted hyena, non-invasively measured estrogens similarly
appeared to rise after LHRH challenge in females (Ensley et al., 1982). Here captive
hyenas of both sexes showed signiﬁcant increases in fE concentrations after LHRH
challenge, despite having highly variable baseline values. The variable responses
observed in the two parous females may have been a consequence of differing stages of
the ovulatory cycle at the time of the LHRH challenge. Endogenous hormone levels and
ovarian status can greatly affect responses of the HPG axis to LHRH (Edwards et al.,
1963; Hamilton and Armstrong, 1991; Jeffcoate, 1992), and Place et al. (2002) showed
that the response of plasma LH to LHRH challenge was more variable in female than
male Crocuta. Here, the three captive males, whose average baseline fE concentrations
were lower than those in females, had post injection fE concentrations roughly equal to
those in the females. Estrogens in male mammals are produced in small amounts by the
testes, and may also be aromatized from circulating testosterone in various other
peripheral tissue (Nitta et al., 1993). However, the source of estrogens is not known for
male spotted hyenas.

Use of the assay developed here for analysis of fecal estrogen should contribute to
our understanding of reproduction and genital development in the female spotted hyena.
There has been a keen interest in the endocrinology of this species and its ties to the
unique characteristics of female dominance and genital masculinization; however,
conventional methods of blood sampling for hormone analysis cannot be repeated with

the frequency needed to explore these issues. Glickman et al. (unpublished data) have

48

obtained preliminary data suggesting that estrogens may be involved in formation and
development of the phallus in this species. Furthermore, we currently know virtually
nothing about estrous cyclicity in any hyena species. By allowing for repetitive sampling
from individuals in a non-invasive manner, it is our hope that availability of an assay for
fecal estrogens will help answer these and many other questions about these fascinating

animals.

49

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52

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53

CHAPTER 2

Van Meter, P. E., French, J. A., Dloniak, S. M., Watts, H. E., Kolowski, J. M.,
Holekamp, K. E., 2009. Fecal glucocorticoids reﬂect socio-ecological and anthropogenic
stresors in the lives of wild spotted hyenas. Hormones and Behavior 55(2): 329-337.

54

CHAPTER 2

FECAL GLUCOCORTICOIDS REFLECT SOCIO-ECOLOGICAL AND
ANT HROPOGENIC STRESSORS IN THE LIVES OF WILD SPOTTED HYENAS

INTRODUCTION

Whereas endocrine studies have traditionally been used in conservation to
monitor reproduction in rare or threatened species, hormones are now being used to
monitor stress physiology as an indicator of how animals cope with social and ecological
challenges in their daily lives (Millspaugh and Washbum, 2004; Walker, et al., 2005a;
Wingﬁeld, et al., 1997). In this context, ﬂuctuating glucocorticoid (GC) concentrations,
released from the adrenal glands in response to stressful stimuli, are often measured as an
expression of how animals perceive and respond to both predictable (e. g. seasonal
changes in food availability) and unpredictable (e. g. encounters with predators) changes
in their environment (Walker et al., 2005a; Wingﬁeld et al., 1997). In the short term,
circulating GC concentrations released in response to an acute stressor help mobilize
energy reserves, and suppress non-essential processes like growth and digestion.
However, chronic elevation of GC concentrations can lead to pathology, including
compromise of cardiovascular, immune, neural, and reproductive function (Sapolsky,
2002).

In recent years, our understanding of responses by free-living animals to potential
stressors has been greatly enhanced by the development of methods for non-invasive
hormone sampling (Keay, et al., 2006; Millspaugh and Washbum, 2004; Walker et al.,
2005a; Wasser, et al., 2000), mainly because more traditional methods of blood sampling
can themselves be stressful (e.g. Sapolsky, 1985). Fecal steroid analysis, in particular,

offers an economical alternative to drawing blood for frequent collection of samples

55

without disrupting the normal behavior of individuals or groups. Many species excrete
multiple steroid metabolites, which can be extracted and measured with immunoassays
like those used to measure circulating steroid hormones (Millspaugh and Washbum,
2004; Wasser et al., 2000).

Here we investigated endocrine responses to a number of potential stressors
experienced by wild African carnivores, using spotted hyenas (C rocuta crocuta) as model
animals. Spotted hyenas offer an excellent model because of their behavioral plasticity:
they occur in a wide array of ecosystems throughout sub-Saharan Africa, can be active
night or day, breed year round, and can survive on foods ranging from termites to
elephants. As this plasticity far exceeds that observed in other large African carnivores,
responses observed in spotted hyenas may represent conservative indicators of how more
specialized carnivores might respond to the same potential stressors.

We inquired about likely sources of stress experienced by spotted hyenas. First,
seasonal variation in ecological factors, including local prey abundance and rainfall,
might affect their stress physiology. Second, competition and intra-guild predation occur
among carnivores exploiting similar ecological niches (Caro and Stoner, 2003). In most
African ecosystems, lions (Panthera leo) are the most important interspeciﬁc competitors
for spotted hyenas; lions not only compete with hyenas for access to live prey and
carcasses, but they also represent a signiﬁcant source of hyena mortality (Honer, et al.,
2002; Kruuk, 1972; Watts and Holekamp, 2009). Thus, lions may represent important
stressors for hyenas. Third, many gregarious carnivores, including spotted hyenas, also
experience intra-speciﬁc social stress (Creel, 2005 ; Goymann, et al., 2001; Young, et al.,

2006). Unlike other gregarious carnivores, which are predominantly cooperative

56

breeders, all adult spotted hyenas have the opportunity to breed despite status-related
reproductive skew occurring in both sexes (Holekamp, et al., 1996; Engh et al., 2002).
Spotted hyena society is structured by strict hierarchical rank relationships like those
found in troops of cercopithecine primates, and it appears that, as in these primate
societies (Abbott, et al., 2003; Creel, 2001), there is no relationship between rank and
fecal GC concentrations among male hyenas (Dloniak, 2004; Goymann, et al., 2003).
Although one study demonstrated a weak relationship between rank and fecal GC
concentrations among female hyenas that were neither pregnant nor lactating (Goymann
et al., 2001), these results were not conﬁrmed in our study population (Dloniak, 2004).
Rare events such as the overthrow of the alpha matriline have been found to increase GC
concentrations among wild baboons (Engh, et al., 2006; Sapolsky, 1983). Such events,
and the ensuing periods of social instability, could potentially affect animals in any
hierarchical society, so we asked whether social instability might affect stress hormone
concentrations among spotted hyenas.

Finally, burgeoning human populations near many African wildlife reserves have
put increasing pressure on protected lands and resources in recent decades (W ittemyer, et
al., 2008). Humans now represent an important mortality source for hyenas and other
carnivores living inside protected areas (Kolowski and Holekamp, 2006; Watts and
Holekamp, 2009; Woodroffe, 2000; Woodroffe and Ginsberg, 1998). In addition, recent
work has suggested that anthropogenic activity, particularly that occurring in the form of
pastoralists and grazing livestock within the boundaries of protected areas, alters the
behavior of spotted hyenas and forces them to make energetic compromises not observed

where pastoralists are absent (Boydston, et al., 2003b; Kolowski and Holekamp, 2008).

57

Measures of tourist activity have also been shown to inﬂuence stress physiology in
vertebrates (e.g. Creel, et al., 2002; Pereira, et al., 2006; Walker, et al., 2005b).
Therefore, pastoralist activity, and perhaps also seasonal variation in visitation by
tourists, might be expected to affect hyena stress physiology.

We analyzed both longitudinal data from a single large hyena group monitored
continuously for many years, and cross-sectional data collected from shorter-term studies
of multiple groups in two different protected areas, to inquire about these potential
stressors in the lives of wild hyenas. We utilized non-invasive sampling of fecal
glucocorticoid (fGC) concentrations, which have previously been shown to accurately
reﬂect stress responses in spotted hyenas (Dloniak, 2004; Dloniak et al., 2006b;
Goymann et al., 2003; Goymann et al., 2001; Goymann, et al., 1999). We used the
longitudinal dataset to determine whether variation in fGC concentrations among spotted
hyenas was associated with seasonal variation in rainfall and prey abundance, temporal
variation in the presence of lions, periods of social instability, and tourism and pastoralist
activity as two forms of anthropogenic disturbance. The study groups used for the cross-
sectional study were selected to take advantage of natural variation among the study areas
relating to ecological and anthropogenic factors. Speciﬁcally, these study groups varied
markedly with respect to climate, prey abundance, local lion density, and anthropogenic
disturbance, thus allowing us to test speciﬁc hypotheses about the effects of these

variables on fGC concentrations.

58

MATERIALS AND METHODS
Study populations and subjects

Four social groups, or “clans,” of free-living spotted hyenas were monitored, two
in each of two different national parks in southern Kenya, the Masai Mara National
Reserve (MMNR), and Amboseli National Park (ANP) (Figure 1). Both the MMNR and
ANP are areas of open grassland grazed year-round by several different ungulate species
(Kolowski, et al., 2007; Western, 2007). Each of the four study clans contained multiple
matrilines of adult females, their offspring, and several immigrant males that had joined
the clan as adults. All clan members were individually recognized by their unique spot
patterns, and sex was determined by the dimorphic glans morphology of the erect phallus
(Frank, etal., 1990). Members of each clan defended a stable group territory. Clans were
observed daily in the morning from 0600 to 1000 and in the evening from 1600 to 2000
hours.

We monitored fGC concentrations among adults of both sexes. Whereas female
hyenas remain with their natal clan throughout their lives, most males disperse at 2 to 5
years of age and immigrate into a new clan (Henschel and Skinner, 1987; Smale, et al.,
1997). As adult males who have not yet dispersed differ from immigrant males with
respect to both their behavior and physiology (Dloniak, et al., 2006a; Holekamp and Sisk,
2003; Smale et al., 1997), here we included only immigrant males in our analyses.

Female spotted hyenas are promiscuous and breed throughout the year
(Holekamp, et al., 1999; Kruuk, 1972). Reproductive histories were known here for
female members of all study clans. Ages of all individuals born into each clan during the

study period were known, and mother-offspring relationships were established on the

59

basis of nursing associations. Birth dates (1 7 days) were assigned based on appearance
and behavior of cubs when they were ﬁrst seen (Holekamp et al., 1996). Female spotted
hyenas reach reproductive maturity at around 2 years of age (Glickman, et al., 1992;
Holekamp et al., 1996; Van Meter, et al., 2008), and were considered here to be adults at
24 months. Duration of gestation in spotted hyenas is 110 days (Kruuk, 1972), so,
counting backward from parturition dates, we could determine conception dates. We
classiﬁed adult females as “nulliparous” from 24 months until their ﬁrst known
conception; ﬁrst parturition could be detected by the extensive tearing of the phallus that
occurs then, even when the cubs do not survive (Frank and Glickman, 1994).
Glucocorticoid concentrations vary with reproductive state among female hyenas
(Goymann et al., 2001), and are highest during pregnancy (Dloniak et al., 2006b);
therefore, we treated pregnant and non-pregnant females separately throughout the study.
Females were categorized as “pregnant” for the 110 days prior to the known birth of a
litter, and were considered to be “non-pregnant” from the day they gave birth until their
next litter was conceived. We further assigned our non-pregnant females as either
lactating or non-lactating. Females were considered to be lactating from the day they
gave birth until weaning of their litter or disappearance of the cubs, whichever came ﬁrst.
Weaning dates were assigned as described by Holekamp et al. (1996). Females were
classiﬁed as non-lactating during the gap between weaning one litter and conceiving the
next. Little is known about the timing or length of the estrus cycle in spotted hyenas, so

females that were neither pregnant nor lactating here may have been cycling.

60

Longitudinal study of the Talek clan

Longitudinal data were collected from a single clan that defended a large territory
along the northeastern border of the MMNR in the Talek region (Figure 1). The Talek
clan had been intensively monitored since 1988, but data analyzed here were collected
between January 1993, when fecal sample collection began, and December 2005 .

Socio-ecological variables: We investigated two ecological variables that vary
seasonally in the MMNR. Daily rainfall was measured (m) using a standard plastic rain
gauge. We calculated the mean rainfall during the 30 days before collection of each fecal
sample. Abundance of prey animals within the Talek home range was assessed biweekly
by counting all ungulates within 100 m of two 4 km transects. These transect counts were
used to calculate the mean prey availability during the 30 days before collection of each
fecal sample.

To examine the effect of lions on spotted hyena fGC concentrations, the presence
of lions was recorded when one or more lions were present within 200 m of any Talek
hyena. Fecal samples reﬂect a general pattern of circulating hormone concentrations
depending on the internal clearance time of the hormone (Brown, et al., 1994; Wasser et
al., 2000). Captive spotted hyenas administered an adrenocorticotrophic hormone
(ACTH) challenge, which increases circulating GC, exhibited elevated fGC
concentrations from 1 to 8 days after the challenge (Dloniak, 2004; Goymann et al.,
1999). Acute stressors, such as caged transport, also produced measurable elevations in
fGC concentrations for several days after the incident (Goymann et al., 1999). Therefore,
we identiﬁed fecal samples from individuals present with lions up to one week prior to

sample collection. We coded lion presence as a binary predictor variable such that each

61

fecal sample was or was not associated with the presence of a lion during the preceding
week.

Whereas the social hierarchy of the Talek clan was generally stable and
unchanging over time (Frank, 1986; Holekamp, et al., 1993), during the course of our
longitudinal study two events occurred that resulted in brief periods of social instability
within the clan. First, in May of 1999, the highest-ranking (alpha) female died after
having occupied that rank since the mid 19805 (Holekamp et al., 1993). For some months
after her death, the daughters of the alpha female fought vigorously for position, and
observers noted an increase in serious wounding among them. Here we considered the six
months following the alpha’s death to be a period of social instability. Second, in 2000,
the Talek clan split into two smaller clans; by 2001, contact between the two groups
resulting from this ﬁssion event was rare, and restricted to only a single individual (J .E.
Smith, unpublished data). Therefore we considered the last six months of 2000 to
represent a second period of social instability. For the purpose of analysis we coded
social stability as a binary predictor variable indicating stable or unstable monthly
periods.

Anthropogenic disturbance: Since the mid-19905, increasing anthropogenic
activity in the Talek area has led to altered use of dens and territory, altered temporal
patterning of activity, and frequent human-hyena conﬂict (Boydston et al., 2003b;
Kolowski and Holekamp, 2006, 2008; Kolowski et al., 2007). Although illegal, the once
rare presence of livestock and herders inside the reserve was a daily occurrence by 2005
(Kolowski and Holekamp, 2006, 2008). The Talek region now supports a high density of

pastoralist settlements along the northern border of the MMNR (Reid, et al., 2003), and

62

thus along the northern edge of the Talek clan territory. Kenyan census data indicated
that the human population in the Talek area has approximately doubled every 15 years
since 1950 (Lamprey and Reid, 2004). To investigate the effect of this burgeoning human
population along the border of the home range of the Talek clan, we asked whether or not
fGC concentrations increased over the 12-year duration of our study, measured in
samples collected during twenty-six 6-month intervals.

Tourism also brings spotted hyenas into contact with humans, although tourists
travel in vehicles whereas pastoralists travel on foot. Since the 19805, the MMNR has
been one of the top safari destinations in the world; tourist visits to the MMNR peaked in
the early 19905 and have remained high over the past decade (Okello, et al., 2005).
Tourist visitation to the MMNR is highly seasonal, with peaks between June and
October, and also in December (Heath, 2008), collectively treated here as the “high
season” for tourism. The remaining six months of the year were considered the “low
season” for tourism. To evaluate seasonal tourism as a possible stressor among spotted
hyenas, we coded the high and low seasons as a binary predictor variable.
Cross-sectional comparison among clans

To compliment our longitudinal data, we investigated variation in fGC
concentrations among several clans in a cross-sectional study. Starting in September
2002, a second clan within the MMNR was monitored, the Mara River clan. The territory
of the Mara River clan was located deep within the Reserve (Figure 2.1), and too far from
pastoralist settlements for livestock grazing. However, the closest border of the territory
of the Mara River clan was only 6 km from the Talek clan’s territory. Also, from October

2003 through July 2005, two hyena clans were monitored that occupied adjacent

63

  

AMBOSELI NATIONAL PARK

 
 

7!

2% Mara River Territory

I Talek Territory

0 12345 10 15
KILOME'I'ERS

Figure 2.1 Map of parks and clan territories.

Table 2.1. Ecological features of parks and clans.

 

 

Park Masai Mara (MMNR) Amboseli (ANP)
a
Mean monthly rainfall 84-9 mm 23-0 mm
, 6 Low: 13.8 °C Low: 15.8 °c
Mean da‘ly temperature High: 28.3 °c High: 31.8 °C
Habitat types (in order of 1) Grassland 1) Grassland
c 2) Scrub/bush 2) Scrub/bush
abundance) 3) Woodland 3) Woodland
Mean prey densityb 210.8 animals/km2 90.5 animals/km2
.d . .
Lion density (adults) a 0.15 lrons/kmz 0.06 lrons/km2

 

 

 

Hyena density 8 1.21 hyenas/km2 1.65 hyenas/km2
Annual tourist densityf 92.75 tourists/kmz 361.18 tourists/km2
Clans Talek Mara River Airstrip OI Tukai
Territory size," 28.4 km2 31.0 km2 28.0 km2 26.4 km2 ‘
Mean clan size (range) 46 (38 — 57) 27 (24 - 31) 51 (42 — 64) 39 (32 — 48)
Mean livestock per count 1386 head g ea (1 1.7 head 0 head
(range) (106—3160) (0-75)

a Watts and Holekamp (2008).

b For MMNR, Kolowski and Holekamp (2008), and for ANP, Watts and Holekamp
(2008).

C For MMNR, Kolowski and Holekamp (2008), and for AN P, Western (2007).

d Lion density values represent adults older than 2 years of age and were calculated for
both the MMNR and ANP for 2005.

e Hyena densities are based on monthly mean clan sizes and territory sizes, and were
calculated for 2003-2005 for the MMNR; AN P data are from Watts and Holekamp
(2008).

f Okello et al. (2005).
8 Kolowski and Holekamp (2008).

65

territories in ANP, the 01 Tukai clan and the Airstrip clan (Figure 2.1). In our cross-
sectional study we restricted the study period in Talek to correspond with the onset and
duration of the studies conducted on the other three clans. Therefore, Talek data in this
analysis were limited to 2002 through 2005. In all clans we used identical methods for
continuous collection of demographic, behavioral, and ecological data.

The population densities of hyenas living in the MMNR and AN P during the
period of the cross-sectional study were similar (Kolowski et al., 2007; Watts and
Holekamp, 2008) (Table 2.1); however, other ecological differences between ANP and
the MMNR might potentially affect the stress physiology of resident hyenas. First,
abundance of prey did not differ signiﬁcantly between the territories of Talek and Mara
River clans (Kolowski et al., 2007), but prey density in the MMN R was over twice that in
ANP (Table 2.1). Second, the MMNR typically received more rain on average than ANP,
and had slightly lower average daily temperatures during the study period than did ANP
(Table 2.1). Finally, the lion density in AN P was less than half that in the MMNR (Watts
and Holekamp, 2008).

The four clans also varied in their exposure to anthropogenic disturbance.
Similarly large numbers of tourists visit both ANP (141581 :1: 1331 tourists) and the
MMNR (139127 1 7864 tourists) each year (Okello et al., 2005), although the smaller
size of ANP generates a much higher tourist density than that in the MMN R (Table 2.1).
Within the MMNR, researchers encountered tour vehicles 4.8 times more frequently
while observing hyenas in Talek than in the Mara River territory (Kolowski et al. 2007).

Although comparable data were not available from the ANP clans, the territories of both

66

of these clans were located close to tourist lodges, and tourist visitation to both ANP
territories more closely approximated that in Talek than that in the Mara River territory.

In contrast to the Talek clan, none of the other three study clans experienced any
signiﬁcant exposure to pastoralist activity within their territories (Table 2.1). In ANP,
livestock and herders were seen only once during this study, within the Airstrip clan’s
territory (herd size = 75 head), and were never seen within the 01 Tukai territory (n = 22
months). Likewise, livestock were never seen in the territory of the Mara River clan (n =
25 months) (Kolowski et al., 2007). In the Talek territory, we monitored the presence 'of
livestock accompanied by herders and conducted detailed head counts of livestock. The
mean number of livestock counted in the Talek territory was 1386 :1: 181 head (Kolowski
and Holekamp, 2008). Thus the Talek clan had a much greater exposure to pastoralist
activity than did either Mara River or ANP hyenas.

The similarities and differences among our four study clans permitted us to
develop the four sets of predictions shown in Table 2.2. If ecological variables, such as
prey availability or rainfall, act as stressors (H1), then we expected to see signiﬁcant
differences in fGC concentrations between the two parks, with higher fGC concentrations
among hyenas living in the harsher AN P conditions. However, if local lion density
affected the stress physiology of spotted hyenas (H2), we predicted fGC concentrations
would be higher in both clans in the MMN R than those in ANP, because MMNR has a
higher lion density. If hyenas ﬁnd tourist visitation stressful (H3), then fGC
concentrations among hyenas in the Mara River clan should be markedly lower than in
any of the other three clans. Finally, if anthropogenic disturbance in the form of

pastoralist activity represents an important stressor to wild hyenas (H4), then we expected

67

Talek hyenas to have higher fGC concentrations than hyenas in any of the other three
study clans, regardless of the park in which they were located.
Fecal sample collection, extraction, and immunoassay

A fecal sample was collected whenever a known hyena was observed to defecate.
Samples were collected into plastic bags within 30 minutes of excretion. Within 12 hours
of collection, samples were mixed and frozen in liquid nitrogen for storage until they
could be shipped to Michigan State University (MSU) for analysis. In an analysis of
methodological covariates Dloniak (2004) examined sample collection time, time
between collection and freezing, and time between collection and assay, to show that
only sample collection time inﬂuences fGC concentrations among spotted hyena fecal
samples. We also analyzed these covariates with our expanded dataset. '

Frozen fecal samples were lyophilized and extracted in ethanol as described
previously (Dloniak, et al., 2003; Van Meter et al., 2008). Extracts were further diluted

1:20 in steroid diluent, and assayed with a corticosterone radioimmunoassay kit

(ImmuChem Double Antibody Corticosterone 125I RIA Kit, MP Biomedicals (formerly

ICN), 07-120102). The corticosterone antibody displays a high afﬁnity to the major
glucocorticoid metabolites present in the feces of a wide variety of vertebrates (W asser et
al., 2000), including spotted hyenas (Goymann et al., 1999). The minimum detection
limit for this assay was 10 ng of glucocorticoid per gram hyena feces. Precision of the
assay was monitored by assaying serial dilutions of pooled hyena fecal extracts. The
intra-assay coefﬁcient of variation was 4.79 i 2.76%, and the interassay coefﬁcient of

variation was 10.12% (n = 17 assays). Serial dilutions of pooled samples produced

68

displacement curves parallel to that produced by the corticosterone standards from the
kit.
Statistical analysis

Analyses were performed with SPSS 15.0.0 (SPSS) and Statistica 6.1 (StatSoft).
Analysis of quantile plots and frequency histograms of the distribution of our data
indicated that fecal glucocorticoid data were normally distributed after log transformation
(base 10). Although transformed values were used in data analysis, we used
untransformed fGC values for graphical representation of the data. A signiﬁcant
difference was identiﬁed when a < .05.

Longitudinal analysis of the Talek clan: We used general linear mixed models
(GLMM) to determine whether our independent variables explained variation in fGC
concentrations in the longitudinal data. Hyena identity was entered into all models as a
random factor to control for the unequal numbers of samples obtained from different
individuals, and models were ﬁtted with a heterogeneous co-variance structure. First, we
investigated whether methodological factors explained any variation among our samples.
We tested males and females separately for sample collection time (morning or evening),
time from collection to freezing (minutes), and time from collection to assay (years); in
the female model we also included reproductive state (nulliparous, pregnant, or non-
pregnant).

Based on the results of our methodological models, we further separated models
into groups based on sex and reproductive condition (immigrant males, nulliparous
females, non-pregnant females, and pregnant females) to test our variables of interest

along with group-speciﬁc covariates previously shown to inﬂuence fGC concentrations.

69

Table 2.2. Summary table showing relative ecological and anthropogenic differences
among parks and clans, and the respective sets of predictions generated for our nested
cross-sectional analysis. The ﬁrst prediction was based on differences in the ecological
conditions experienced by hyenas in each clan and park (H1), the second was based on
variation in lion density among clan territories (H2), the third was based upon variation in
tourism (H3), and the fourth was based on variation in pastoralist activity (H4). Only the
last of these predictions was conﬁrmed, as shown in Figure 2.

 

 

 

 

 

 

 

 

 

 

 

MMN R
Talek Mara River 01 Tukai Airstrip
Rainfall High High Low Low
H1 Prey High High Low Low
Prediction 1: fGC Low Low High High
Lions High High Low Low
H2 Prediction 2: fGC High High Low Low
Tourism High Low High High
H3 Prediction 3: fGC High Low High High
Pastoralist Activity High Low Low Low
H4 Prediction 4: fGC High Low Low Low

 

 

 

 

 

 

 

 

70

We controlled for lactation state among non-pregnant females (lactating or non-lactating)
(Dloniak, 2004; Goymann et al., 2001), and day of gestation for pregnant females
(Dloniak et al., 2006b). We examined the main-effects of each parameter as well as
interactions with covariates.

We used both multimodal inference (Bumham and Anderson, 2002) and
hypothesis testing to draw conclusions about the relative importance of our variables of
interest. All terms and interactions were included in the initial model and were
sequentially removed to minimize the Akaike’s information criterion, adjusted for small
sample sizes (AICc). Following Bumham and Anderson (2002) we considered all models
within 3 AICc units of the model with the lowest AICc score to be equally parsimonious,
and we included a discussion of these models in our results. We then chose the most
inclusive model that contained all terms from among the set of equally parsimonious
models to use for hypothesis testing. We used the restricted maximum likelihood method
of estimation to evaluate the parameters in our models; for our chosen models we present
the parameter estimates, degrees of freedom, Sattherwaite’s F test, and the corresponding
p-value.

Cross-sectional comparison among clans: For the cross-sectional comparison, a
nested ANOVA was used to test for signiﬁcant differences among clans in fGC
concentrations. Before performing the nested AN OVA we investigated the procedural
variables that were found to have signiﬁcant effects in the longitudinal dataset. Males and
females, and morning and evening samples were equally represented in each study group.
However, we had an unequal distribution of pregnant females among clans, so samples

from pregnant females were excluded from the nested analysis. For the nested AN OVA,

71

clan (Talek, Mara River, 01 Tukai, or Airstrip) was nested within park (MMNR or ANP)
to assess the main effects of park and clan. A post hoc analysis was performed using

Fisher’s LSD test; means and standard errors are presented.

RESULTS
Longitudinal analysis of the Talek clan

A total of 811 fecal samples were collected from adult hyenas of known identity
and reproductive condition; we collected 232 samples from 39 immigrant males, 59
samples from 26 nulliparous females, 83 samples from 34 pregnant females, and 437
samples from 44 non-pregnant adult females; of these last 437 samples, 378 came from
lactating females and 59 came from females that were neither pregnant nor lactating.

Methodological variables: Table 2.3 shows the results of the methodological
analysis. For immigrant males, only sample collection time explained a signiﬁcant
portion of the variation among fGC concentrations, indicating that samples collected in
the morning were higher than those collected in the evening. The same was true in the
model for adult females; here reproductive state also signiﬁcantly affected fGC
concentrations. We controlled for these variables in all subsequent models.

Immigrant males: Terms included in the model for immigrant males were sample
collection time, study duration time, tourism, and social instability (AICc = 230). The
further removal of study duration time (AICc = 227), tourism (AICc = 227), or social
instability (AICc = 227) generated equally parsimonious models, so we present the

parameter estimates for the model containing all terms together (Table 2.4). Whereas

72

Table 2.3. Models (GLMM) investigating methodological sources of variation in fGC

concentrations. Males and females were tested in separate models; all variables included

in the models are shown. Individual identity was entered as a random variable.

 

 

Estimate df F p-value

Immigrant males Waldz= l.41,p=0.16
Intercept 1.93 68.95 329.90 <0.001
Collection time -0. 178 112.64 21.56 <0.001
Time collection to assay (years) -0.031 100.22 0.56 0.46
Time collection to freezing (mins.) 0.000 101.46 0.72 0.40

Adult females Wald z = 2.61, p = 0.01
Intercept 1.71 276.63 631.99 <0.001
Collection time -0.1 15 482.16 40.22 <0.001
Time collection to assay (years) -0.000 261.12 0.00 0.98
Time collection to freezing (mins.) 0.000 496.91 2.54 0.11
Reproductive state 0.1 19 492.47 26.51 <0.001

 

73

Table 2.4. Parameter estimates from the selected models (GLMM) explaining fGC
concentrations among each group. Individual identity was entered as a random factor in
all models.

 

 

 

 

 

Estimate df F p-value
Immigrant males Wald z = 2.31, p = 0.02
Intercept 1.68 108.66 216.386 <0.001
Collection time -018" 183.82 46.22 <0.001
Duration of study 002*) 156.23 3.86 0.05
Tourism (104° 177.95 2.14 0.14
Instability -0,05d 184.26 1.35 0.23
Nulliparous females Wald z = 1.76, p = 0.07
Intercept 1.82 36.92 468.96 <0.001
Collection time -0.07 46.51 2.34 0.13
Instability -0. 19 47.82 5.69 0.02
Non-pregnant females Wald z = 2.27, p = 0.02
Intercept 2.23 368.72 263.89 <0.001
Collection time -0.12 368.83 34.53 <0.001
Lactation status (LS) -014" 378.99 3.60 0.06
Instability -0.20 372.92 2.41 0.12
LS X instability 0.01 374.08 1.67 0.20
Pregnant females Wald z = 0.29, p = 0.77
Intercept 2.00 50.19 359.75 <0.001
Collection time -0.09 76.92 5.09 0.03
Day of gestation 0004f 63.94 7.86 0.01
Instability -0. 1 1 69.43 2.70 0.12

 

a The negative estimate for collection time indicates that morning samples had higher
fGC concentrations than evening samples.

b The positive estimate for duration of study indicates fGC concentrations increased as
our study progressed.

c The positive estimate for tourism indicates that fGC concentrations were higher during
months of peak tourism.

(1 The negative estimate for instability indicates that samples from periods of instability
had higher fGC concentrations than those from stable periods.

e The negative estimate for lactation status indicates that samples from non-lactating
females had higher fGC concentrations than those from lactating females.

f The positive estimate for day of gestation indicates that fGC concentrations increased
during gestation.

74

only sample collection time and study duration were signiﬁcant variables in this model,

the model estimates also indicated that fGC concentrations increased among immigrant

males during months of heavy tourism, increased over the course of our study, and were
relatively high during periods of social instability.

Nulliparous females: Only sample collection time and social instability remained
in the model for nulliparous adult females (Table 2.4). Here, although the parameter
estimate for sample collection time indicated that morning samples contained higher fGC
concentrations than evening samples, the estimate was not signiﬁcant in the model. Fecal
samples collected during periods of social instability contained signiﬁcantly higher fGC
concentrations than those collected during stable periods.

Non-pregnant femies: Terms included in the model for non-pregnant females
were sample collection time, lactation status, social instability, and an interaction
between lactation status and instability (AICc = 395). The removal of lactation status
(AICc = 395), stability (AICc = 395), or the interaction between these two variables
(AICc = 393) generated equally parsimonious models, so we present the estimates for the
model containing all terms together, although only sample collection time was signiﬁcant
in this model (Table 4). Model estimates indicated that females tended to have higher
fGC concentrations when not lactating than during lactation. Samples collected during
periods of social instability generally had higher fGC concentrations than those collected
during stable periods, and the interaction term indicated that this difference was more
pronounced among non—lactating females.

Pregnant females: The model including sample collection time and day of

gestation (AICc = 86) was equally parsimonious with the model that included these two

75

terms plus social instability (AICc = 87; Table 4). Signiﬁcant terms in the model included
sample collection time and day of gestation, which indicated that fGC concentrations
increased during the course of gestation. Samples collected during periods of social
instability had higher fGC concentrations than those collected during stable periods,
although this term was not signiﬁcant in the model.
Cross-sectional comparison among clans

In this dataset, we found no effect of sample collection time, sex, or reproductive

state after pregnant females were excluded. Results from our nested ANOVA revealed an

overall signiﬁcant model (F 1,152 = 2744.84, p < 0.001). There was no effect of park

(F1,152 = 0.70, p = 0.40), but there was an effect of clan (F1152 = 3.86, p = 0.02). Further

investigation of this clan effect with a post hoc Fisher’s LSD test (Fig. 2) showed that
there was no signiﬁcant difference in mean fGC concentrations between the two AN P
clans (Ol Tukai, 103.46 i 14.31 ng/g; Airstrip, 123.46 :1; 21.60 ng/g) and that fGC
concentrations in these clans did not differ from those in the Mara River clan ( 105.91 i
25.08 ng/g). Hyenas in the Talek clan had the highest mean fGC concentrations (185.30 :t
19.13 ng/g), and these were signiﬁcantly higher than those of Mara River (p = 0.01) or 01
Tukai hyenas (p = 0.01). The Airstrip clan had mean fGC concentrations that were lower
than those in the highly disturbed Talek clan, but higher than those in the other two
undisturbed clans, though these differences were not statistically signiﬁcant. These
results reﬂect the pattern of predictions made under H4 in Table 2, and support the
hypothesis that anthropogenic disturbance, in the form of pastoralist activity, increases

fGC concentrations.

76

 

 

 

 

 

 

12
d)
0
.2
$200-
5
U)
32
01501
.2
1:
0
° 1
8100
.2
CD
To
3;. 50-
:1
Cd
0)
2 0

O] Tukai Airstrip Mara River Talek
AN P MMN R

Figure 2.2 Mean (:1: SEM) morning fGC concentrations for adults from four hyena clans.
A nested AN OVA indicated a signiﬁcant effect of clan. Different letters above bars
reﬂect signiﬁcant differences indicated by the Fisher’s LSD analysis.

77

DISCUSSION

Our aim here was to identify natural and anthropogenic inﬂuences on the stress
physiology of wild spotted hyenas using non-invasive hormone sampling techniques.
Fecal hormone sampling has proven to be a valuable tool for monitoring wild populations
with regard to their physiology, and for evaluating variation in physiological factors in
response to variable environmental conditions. The value of fecal hormone analysis as
such a tool increases when we evaluate potential variation in hormone concentrations
introduced by collection and storage conditions in the ﬁeld. In our study, samples were
frozen fresh in the ﬁeld, which is the best option for reducing the inﬂuence of handling
and storage on hormone concentrations (Millspaugh and Washbum, 2004). Nevertheless,
we examined three potential sources of methodological variability among our samples:
minutes between collection and freezing, years between freezing and assay, and sample
collection time of day. Fecal glucocorticoid concentrations in our samples were robust to
slight delays between collection and freezing, and even to long-term storage. Like
Dloniak (2004), we did see a strong relationship between time of day of sample
collection and fGC concentrations; this relationship most likely reﬂects a circadian
rhythm in circulating GC concentrations.
Socioecological variables

In our longitudinal study, natural ecological variation was not found to be a
signiﬁcant stressor in the lives of spotted hyenas. Seasonal variation in prey availability
and monthly rainfall did not explain variation in fGC concentrations among hyenas. Nor
did our data support the notion that the immediate presence of lions elevates fGC

concentrations among hyenas. Even large carnivores like hyenas experience competition

78

from intra-guild predators (Caro and Stoner, 2003); lions and hyenas compete for access
to both carcasses and live prey (Cooper, 1991; Honer et al., 2002; Kruuk, 1972). Watts
and Holekamp (2009) found that lions cause the majority of known deaths among adult
hyenas in the Talek clan, but that varying levels of competition with lions could not
explain patterns of mortality among adult hyenas. Here we were unable to detect a
physiological stress response, measured as elevated fGC concentrations, among
individuals that had encountered lions 1 to 7 days before fecal sample collection.
Although lion-hyena encounters were common, violent interactions between lions and
hyenas were rare, and it may be only the latter type of interaction that elicits a stress
response measurable in hyena fecal samples. However, like our longitudinal analysis, our
cross-sectional data comparing fGC concentrations among clans exposed to dramatically
different lion densities (H2; Table 2.2) also failed to suggest that lion presence represents
a chronic stressor to hyenas. Although the lion density in the MMNR was twice that in
ANP, there was no indication that park inﬂuenced hyena fGC concentrations, and there
were no signiﬁcant differences in fGC concentrations between the AN P clans and the
Mara River clan.

In carnivore societies structured by linear dominance hierarchies, patterns of
social stress vary among dominant and subordinate members of a group (Creel, 2005),
but this does not seem to be true among hyenas living in the MMNR (Dloniak, 2004;
Dloniak et al., 2006b). However, here we examined instability in the social hierarchy as a
predictor of social stress; such instability could potentially affect members of any
hierarchical society. Our data indicate that nulliparous females experienced elevated fGC

concentrations in response to social instability, and, although not signiﬁcant, we observed

79

trends in the same direction among pregnant and non-pregnant females, and among
immigrant males. Social instability was the only predictor, other than sample collection
time, to explain any variation in fGC concentrations among nulliparous females. Juvenile
spotted hyenas gain their rank by “inheriting” their mother’s position in the dominance
hierarchy, but it is not until their second year of life that their ranks ﬁnally become
established relative to those of older clan members (Engh, et al., 2000; Smale, et al.,
1993). The recency of their rank acquisition may account for the heightened response to
social instability apparent among nulliparous females.

A study investigating acute social stress among hyenas living in the Serengeti
National Park, demonstrated that lactating females who participated in severe ﬁghting 48
hours prior to sample collection showed elevated fGC concentrations (Goymann et al.,
2001). However, we seldom observed severe ﬁghting among females in our study
populations, and we collected too few samples after major ﬁghts to perform a comparable
analysis with our own data. Goymann et al. (2001) also reported that lactating females in
the Serengeti had higher mean fGC concentrations than did non-lactating/non-pregnant
females. By contrast, fGC concentrations did not vary signiﬁcantly with lactation state in
our study. Lactating females in the Serengeti may be more energetically challenged than
those in the MMNR (Trinkel, etal., 2006) because most Serengeti females routinely
commute long distances to forage (Hofer and East, 1993) whereas MMNR females feed
near their dens (Boydston, et al., 2003a; Watts and Holekamp, 2008). Lactating females
in the Serengeti must return frequently from distant foraging sites to their communal den

in order to nurse their cubs. The difference in energy budgets between Serengeti and

80

01
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of 1.

hr}
1W3
Illlc‘;
b? llt
indie
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Will

MMNR females may account for the difference in fGC concentrations seen between
lactating and non-lactating females in our two study populations.
Anthropogenic variables

Tourists have been present in large numbers in both the MMNR and AN P for
more than 3 decades (Okello et al., 2005). Within the MMNR, the Talek and Mara River
territories differed very little except with respect to the intense daily exposure of Talek
hyenas to the presence of tour vehicles and livestock (Kolowski et al., 2007). The Mara
River territory was visited by tourists at lower rates than were the territories of any of our
other study clans. Therefore, had tourist visitation been generally stressful to hyenas, we
expected to observe lower fGC concentrations among Mara River hyenas than among
hyenas in any of the other three study clans (H3; Table 2.2). Tourists in vehicles pose no
direct threat to hyenas, and in both parks, hyenas are well habituated to vehicle presence
and do little to avoid them. However, data from our longitudinal analysis suggest that
immigrant males may not be as well habituated to tour vehicles as are the natal members
of the Talek clan. I

In contrast to tourists, pastoralist herdsmen represent a direct threat to hyenas;
they are an important source of mortality for the Talek population, second only to lions
(Watts and Holekamp, 2009). Additionally, hyenas appear to perceive herdsmen as
threats because hyenas often ﬂee from guarded cattle herds whereas cattle left unattended
by herders are not avoided (Kolowski and Holekamp, 2008). Our longitudinal data
indicate that fGC concentrations among immigrant male hyenas have increased since the
early 19905; this increase has been correlated with the increasing size of the human

population living along the border of the Talek clan’s home range (Lamprey and Reid,

81

101
SFL‘
[11:1
1101
pas
con
adj;

mil

2004). Although our study did not directly test proximate mechanisms linking human
population growth with hyena stress physiology, a recent study comparing the behavior
of Talek and Mara River hyenas indicated that it was the presence of pastoralists and
their livestock, not tourism, that accounted for altered behavior among Talek hyenas
relative to that observed in the undisturbed Mara River clan (Kolowski et al., 2007).
Variation in pastoralist activity, but not tourism or ecological variables, was also
consistent with the patterns of variation we observed among clans with respect to fGC
concentrations. A study of Serengeti hyenas living inside and outside the N gorongoro
Crater found lower fGC concentrations among females (Goymann et al., 2001) but not
males (Goymann et al., 2003) living within the Crater. The authors attributed the lower
fGC concentrations to ecological differences experienced by the two populations, and the
lower energetic demands enjoyed by females inside the Crater. In our own cross-sectional
study, we observed no effect of park on fGC concentrations in our samples, suggesting
that the differences in ecology experienced by hyenas living in ANP and the MMNR did
not inﬂuence hyena stress physiology (H1; Table 2.2). Furthermore, differences in
pastoralist activity among monitored clans (H4; Table 2.2) better predicted elevated fGC
concentrations than did any natural ecological variables. Growing human populations
adjacent to protected areas, as has been described here for the Talek region, are common
in many African and Latin American countries (Wittemyer et al., 2008). Whereas this
form of anthropogenic disturbance has clear ties to reduction in resources available to
protected carnivores, here our data suggest that growing human populations along
borders of protected areas may also have direct physiological consequences for the

animals living within these areas. Together the results of our longitudinal and cross-

82

ch;-
16f}
neg

\V j

sectional studies suggest that increasing activity of local pastoralists in the Talek region
represents a source of stress to Talek hyenas.

Whereas many studies have documented behavioral and demographic changes in
wild populations caused by anthropogenic disturbance, relatively few have measured its
effects on stress physiology (summarized by Walker et al., 2005a). Nevertheless, our
study adds to a growing number of others demonstrating an increase in mammalian stress
responses, measured as elevated GC concentrations, caused by various forms of
anthropogenic disturbance (e. g. Creel et al., 2002; Millspaugh, et al., 2001; Pereira et al.,
2006). It seems reasonable to expect that the previously documented effects of
anthropogenic disturbance on spotted hyena behavior (Boydston et al., 2003b; Kolowski
et al., 2007), and the physiological correlates demonstrated here, may presage negative
changes in demography and ﬁtness within the Talek clan. Although adaptive in the short
term, it is well documented that chronic elevation of GC concentrations can have
negative effects on fertility in both male and female vertebrates (Sapolsky, 2002;
Wingﬁeld and Sapolsky, 2003). Future work should inquire whether or not elevated fGC
concentrations predict signiﬁcant demographic change in wild hyena populations.
Spotted hyenas are unusually adaptable animals, so their responses to the natural and
human stressors assessed here might predict exacerbated stress responses in more

specialized and endangered species, such as cheetahs or African wild dogs.

83

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88

CHAPTER 3

INTER- AND INT RASEXUAL AGGRESSION
AMONG ADULT SPOTTED HYENAS

INTRODUCTION

In most mammalian species males are socially dominant to females, and direct
aggression toward conspeciﬁcs at much higher rates than do females (Archer, 1988;
Floody, 1983; Walters and Seyfarth, 1987). However, females also exhibit aggressive
behavior toward conspeciﬁcs. For instance, defense of young is one context in which
female aggression has been well studied, and much is known about both the proximate
mechanisms and ultimate functions of this behavior (Lonstein and Gammie, 2002). In
addition, females emit aggressive behavior as they compete among themselves for access
to scarce resources such as food, territory, and to a lesser extent, mates (Floody, 1983;
Koenig, 2002; Silk, 1993). There is a growing recognition that social conﬂict and
competition experienced by females has important consequences for both their
reproduction and sociality (Silk, 1993; Smuts, 1987; Walters and Seyfarth, 1987;
Wrangham, 1987).

Researchers are now examining the neural, endocrine, genetic, and environmental
control of aggressive behavior (Nelson, 2006), and steroid hormones, particularly
androgens, feature prominently in the investigation of sex differences in aggression
(Bouissou, 1983; Harding, 1981). Exposure to steroid hormones early in life may
“organize” the developing neural substrates of aggressive behavior (Beach, et al., 1982;
Clark and Galef, 1998; Ryan and Vandenbergh, 2002; vom Saal, 1989). Maternal stress

translated to the developing fetus via glucocorticoids may also inﬂuence aggressive

89

behavior in offspring (maternal stress, Kaiser and Sachser, 2005; Marchlewska-Koj, et
al., 2003). Circulating androgens among adult males can “activate” the expression of
aggressive behavior in speciﬁc contexts (W ingﬁeld, et al., 1990), and this has been well
documented in a number of mammalian taxa (rodents, Beach and H012, 1946; Buck and
Barnes, 2003; primates, Cavigelli and Pereira, 2000; carnivores, Creel, et al., 1997; Creel,
et al., 1993; Dloniak, et al., 2006a; Goymann, et al., 2003). Strong evidence
demonstrating activating effects of steroid hormones and aggression among females has
been more elusive, but some studies suggest that female aggressive behavior may covary
with concentrations of androgens (Beehner, et al., 2005; Drea, 2007) or ovarian
hormones, such as estrogen and progesterone (Davis and Marler, 2003; Kapusta, 1998).
In a small number of species, a reversal of status from mammalian norms is
evident, such that females are socially dominant to males. The evolution of female
dominance apparently has occurred independently among taxonomic groups that are only
distantly related (e. g., carnivores, Kruuk, 1972; rodents, Clarke and Faulkes, 2001; and
primates, Drea, 2007). The leading functional explanation in the literature for female
dominance is that it represents an adaptation to high reproductive costs for females
occurring in conjunction with intense intraspeciﬁc competition for food (Drea, 2007;
Frank, 1986; Jolly, 1984); therefore, selection has favored females able to out-compete
males such that females can secure access to scarce resources for themselves and their
young. There has been much attention paid to the potential for steroid hormone control of
female aggression among female-dominated species (Dloniak, et al., 2006b; Drea, 2007;
Glickman, et al., 1993; Goymann, eta1., 2001a; Sannen, et al., 2003; von Engelhardt, et

al., 2000). In this study we investigate aggression and its possible mediation by steroid

90

11

SC

ls.)

hormones in the spotted hyena, (Crocuta crocuta), a gregarious carnivore species in
which females are socially dominant to males.

Spotted hyena society is structured by a rigid linear dominance hierarchy based on
“inheritance” of maternal rank between generations (Engh, et al., 2000; Frank, 1986;
Smale, et al., 1993). Rank determines priority of access to food in this species (Frank,
1986; Kruuk, 1972; Tilson and Hamilton, 1984), and although all females breed, high
social rank confers a large reproductive advantage, as higher-ranking females out-
perform lower-ranking females on most measures of reproductive success (Hofer and
East, 2003; Holekamp, et al., 1996). Females are philopatric, but males generally disperse
between the ages of 2 and 5 years (Holekamp et al., 1996; Smale, et al., 1997). When
males immigrate into a new clan, their social rank is not determined by ﬁghting ability,
size or other common indicators of male-male competition; instead, they slot into the
lowest position in the social hierarchy, below all natal members of the group and all other
adult immigrant males that arrived before them (East and Hofer, 2001; Engh, et al.,
2002). Thus adult immigrant males are socially subordinate to all natal members of the
group (Frank, 1986; Kruuk, 1972; Smale et al., 1993; Tilson and Hamilton, 1984).

Female spotted hyenas have the reputation of being particularly aggressive;
although, whether or not this reputation is warranted remains somewhat controversial.
Although they have usually based their assessments on anecdotal rather than quantitative
information, observers of spotted hyena behavior are largely in agreement that females
attack conspeciﬁcs more frequently than do males (Boydston, et al., 2001; East, et al.,
2003; East and Hofer, 2002; Frank, 1986; Frank, et al., 1989; Glickman, et al., 1997;

Hamilton, etal., 1986; Hofer and East, 1993; Kruuk, 1972; Tilson and Hamilton, 1984),

91

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which is the opposite of mammalian norms. Many researchers have suggested that female
aggressiveness in this species is one of many traits favored by selection to promote
female dominance over males, and secure access to food in highly competitive feeding
situations (Frank, 1986; Frank, et al., 1995; Glickman et al., 1993; Hamilton et al., 1986;
Holekamp and Smale, 1999; Tilson and Hamilton, 1984). Others (East and Hofer, 2002;
Goymann et al., 2001a) have suggested that the aggressiveness exhibited by female
spotted hyenas has been overstated, and that females only appear to be highly aggressive
because rates of aggression emitted by males are very low. These authors correctly point
out that, were data available to compare aggressive acts emitted by female spotted hyenas
with those emitted by females of other species, we might ﬁnd that female spotted hyenas
are in fact not unusually aggressive (or “hyperaggressive” to use the terminology of
Goymann et al. (2001a)). Unfortunately, such comparative data are very sparse in the
existing literature. An alternative way to assess the aggressive behavior of female spotted
hyenas would be to directly compare their behavior with that of male spotted hyenas.
Szykman et al. (2003) demonstrated that while female hyenas direct aggression toward
both male and female conspeciﬁcs, males rarely direct aggression toward adult females.
Here, our goal was to expand on those results to provide a quantitative and qualitative
comparison of the aggressive behavior exhibited by adult male and female spotted
hyenas. Speciﬁcally, we directly compare rates and intensities of intrasexual aggression
among adult females with those of intrasexual aggression among adult males, and we also
compare the sexes with respect to rates and intensities of inter-sexual aggression.

In addition to their apparent role-reversed patterns of social dominance and

aggression, female spotted hyenas also differ from mammalian norms in that they have

92

Sit

516

SP

dll

masculinized external genitalia, which is unique even among species characterized by
female dominance (Drea and Weil, 2008; Frank, et al., 1990; Matthews, 1939). Our
current understanding of sexual differentiation in mammals assumes that androgens are
necessary during development for masculinizing both morphology and behavior (J ost, et
al., 1973b). When they are present at critical stages of development, androgens have even
been found to masculinize the morphology and behavior of females (Beach et al., 1982;
Goy, et al., 1988; Herman, et al., 2000; Jost, et al., 1973a; Phoenix, et al., 1959). The
spotted hyena, however, does not appear to conform to the traditional Jostian model of
steroid-dependent masculinization, and investigations into the urogenital development in
this species are now refocusing on androgen-independent mechanisms to explain the
masculinization of the female’s external genitalia (Drea, et al., 1998; Glickman, et al.,
2005). However, we are still building our understanding of the role that steroid hormones
play in shaping behavior in this species. Androgen concentrations sampled from pregnant
spotted hyenas are higher than those measured in non-pregnant females, and this
difference has been demonstrated in concentrations measured in both blood (Dloniak, et
al., 2003; Glickman, et al., 1992; Goymann et al., 2001a; Licht, et al., 1992) and fecal
samples (Dloniak et al., 2003). Several studies have shown that androgen concentrations
are highest during late gestation among females (Dloniak et al., 2006b; Licht et al.,
1992), and that these concentrations are well within the range of those measured in
reproductively mature male hyenas (Dloniak et al., 2003; Licht et al., 1992). These
androgenic hormones are produced by the maternal ovary (Glickman et al., 1992;

Lindeque, et al., 1986) and are transferred to the developing fetus by the placenta (Licht

93

et al., 1992; Yalcinkaya, et al., 1993); this prenatal androgen exposure may have lasting
effects on spotted hyena behavior.

Maternal androgen concentrations during hyena pregnancy, measured in feces, are
correlated with mounting and aggressive behavior in both male and female offspring
when they are 2 to 6 months old (Dloniak et al., 2006b). Offspring of mothers treated
with anti-androgens during pregnancy exhibit reductions in both the postnatal sibling
aggression typically seen in this species, and in the aggression directed by females in
adulthood toward adult males during feeding competition (C. Drea and SE. Glickman,
pers. communication). Taken together, these results suggest that prenatal androgen
exposure might be organizing the neural structures underlying aggressive behavior in
both male and female spotted hyenas. There is also some evidence to suggest that
aggressive behavior among spotted hyenas might be under activational control of steroid
hormones. Among adult male hyenas, circulating androgen concentrations are tightly
correlated with aggression related to courtship and mate defense (Dloniak et al., 2006a;
Goymann et al., 2003). Furthermore, juvenile ovariectomy appears to reduce rates of
inter-sexual aggression emitted by captive female hyenas as adults (Baker, 1990). On the
other hand, gonadectomy had no effect on rates of intrasexual aggression among captive
juveniles when compared to controls (Frank et al., 1989). However, aggressive behavior
among spotted hyenas has never been systematically studied when both observation time
and number of potential target animals have been controlled. Furthermore, no one has yet
investigated the relationship between aggression and steroid hormones among adult

female hyenas living in the wild.

94

Here we documented sex differences in rates and intensities of aggressive
behavior among free-living adult hyenas, and used natural variation in steroid hormone
concentrations to test predictions about organizational and activational inﬂuences on
aggressive behavior in wild spotted hyenas. We used non-invasive hormone sampling
techniques to measure fecal steroid hormones, including fecal androgens (fA), fecal

estrogens (fE), and fecal glucocorticoids (fGC).

METHODS
Study site and subjects

This study was conducted in the Masai Mara National Reserve, Kenya. We
monitored a single large social group, or clan, of spotted hyenas from 1988 through 2005.
The clan defended a large territory in the Talek region as described previously (Boydston
etal., 2001; Frank, 1986; Holekamp, et al., 1993). All clan members were individually
identiﬁed by their unique spot patterns, and their sex was determined by the dimorphic
glans morphology of the erect phallus (Frank et al., 1990). Spotted hyena clans are
ﬁssion-fusion societies, and clan members spend their time in small subgroups, the
composition of which may change several times a day (Kolowski, et al., 2007; Smith, et
al., 2008). Females are generally more gregarious than are males, and social rank affects
the gregariousness of individuals of both sexes (Smith et al., 2008; Smith, et al., 2007);
the mean subgroup in which females are found in this study population contains 3.9
individuals, while mean subgroup size for males is 3.1 (Smith et al., 2008).

Our study clan was comprised of multiple matrilines of adult females, their

offspring, and several immigrant males that had joined the clan as adults. Ages of all

95

individuals born into the clan during the study period were known (to i 7 days)
(Holekamp et al., 1996), and reproductive histories were known for all females. Birth
dates were assigned based on appearance and behavior of cubs when they were ﬁrst seen
(Holekamp et al., 1996). Female spotted hyenas reach reproductive maturity at
approximately two years of age (Glickman et al., 1992; Holekamp et al., 1996; Van
Meter, et al., 2008), and we considered females to be adults at 24 months. Gestation in
this species is 110 days (Kruuk, 1972), so counting backward from known parturition
dates, we could estimate dates of conception. To compare rates of behavior among phases
of gestation, pregnancy was divided into trimesters of 37 days each. Lactation may last
up to 24 months in this species (Holekamp et al., 1996), and each female was considered
to be lactating from the day she gave birth until the weaning of that litter or her next
conception, whichever came ﬁrst. Incomplete lactation intervals caused by litter death
were not considered here. We assigned wean dates (1 10 days) based on observed nursing
conﬂicts between mothers and cubs, and the cessation of nursing, as has been previously
described (Holekamp et al., 1996; Holekamp, et al., 1999).

Adult natal males, who have not yet dispersed, differ from immigrant males with
respect to both their behavior and their physiology (Dloniak et al., 2006a; Holekamp and
Sisk, 2003; Smale et al., 1997). Therefore we included only immigrant males in our
analyses. Immigrants queue for position in the intrasexual dominance hierarchy and only
move up in rank as males who arrived before them die or secondarily disperse (East and
Hofer, 2001; Engh et al., 2002; Van Horn, et al., 2003). Therefore, the immigrant male

with the longest tenure in the clan is the highest-ranking of the immigrants, in a rank

96

position immediately below that of the lowest ranking natal animal in the clan’s overall
dominance hierarchy.
Behavioral observations

Most adult members of the clan were ﬁtted with radio-collars that could be
tracked from our vehicle, and researchers observed clan members daily in the morning
(0530 - 0900 hours) and again in the evening ( 1700 - 2000 hours). We collected
behavioral data during observation sessions on groups of two or more hyenas that were
separated from other hyenas by at least 200 m, and identities of all hyenas in each group
were recorded. Sessions lasted at least 15 minutes (mean 1 standard error = 43.28 i
0.003 minutes), and ended when we moved away from the subgroup. We observed adult
hyenas for a total of 5051 hours during 9863 observation sessions. All aggressive
interactions among adults were recorded as critical incidents (Altmann, 1974). For each
aggressive interaction we recorded the aggressor, behavior, recipient, response, and if
possible, the context in which the aggression occurred. Aggressive interactions among
spotted hyenas can involve one actor and one recipient, termed dyadic aggression, or
multiple actors engaging in coalitionary aggression. Here we considered only dyadic
aggression. Social ranks of adult females in the clan were determined by creating a
matrix of outcomes of dyadic aggressive and submissive interactions (Frank, 1986), and
this dominance hierarchy was updated annually. Offspring joined the dominance
hierarchy below their mothers but above their older siblings (Engh et al., 2000;
Holekamp and Smale, 1991). By convention, the highest-ranking adult individual of each
sex was assigned a rank of 1. Our ethogram of aggressive behaviors included head-wave,

displacement, lunge, push, stand over, rush, chase, bite, and bite-shake. We deﬁned biting

97

contact as high intensity aggression and all other aggressive behaviors were considered to
be of low-intensity. Responses to aggression included appeasement behaviors (head bob,
carpal crawl, squeal, back-off), and counter-attacks.

Aggressive behavior by both adult males and females occurred in multiple
contexts, classiﬁed as one of the following: feeding, “pesky,” unprovoked, “other,” and
unknown. The sex-speciﬁc contexts of mating and maternal defense were collectively
demarcated as “other” in this study. The feeding context referred to aggression directly
related to a food source, and was restricted to scenes at which a carcass was present. The
“pesky” context indicated aggression directed toward conspeciﬁcs that were clearly
annoying the aggressor, often inadvertently. Unprovoked aggressions occurred when the
actor aggressed against the recipient without provocation, and usually there were no
interactions of any sort within the dyad immediately preceding the attack. “Unprovoked”
was not the same as the “unknown” context, which was assigned when the context was
simply not clear to observers.

Quantiﬁcation of aggression
An hourly rate of aggression was calculated for each individual present in a given

observation session as:

 

(number of aggressive acts emitted by that individual -2- number of targets present)

 

number of hours in that observation session

 

 

 

This rate calculation controlled for variation among sessions with respect to opportunities
for attack available to each aggressor. The aggression rates calculated for each individual

in each session were then averaged together over his/her entire lifetime in the clan as

98

adults, or over shorter periods of time, as when females were in speciﬁc reproductive
states. These mean aggression rates therefore included sessions in which each aggressor
was seen with one or more potential “targets,” but did not attack. In this way, the mean
rates of aggression for individuals who were frequently seen with targets, but did not
attack, were weighted in comparison to individuals who were seen less often.

The deﬁnition of “targets” was determined by the question being asked in each
speciﬁc analysis. First we wished to inquire about sex differences in aggression emitted
by adults toward other adults and toward juveniles. Thus lifetime aggression rates toward
adults were calculated using all sessions in which the aggressor was present with one or
more adults, and lifetime aggression rates toward juveniles were calculated using all
sessions in which the aggressor was present with one or more natal animals less than 24
months. Next, we wished to evaluate sex differences in aggression directed by adults of
each sex toward other adults of the same or opposite sex. We refer to aggression directed
by adults toward adults of the opposite sex as inter-sexual or between-sex aggression. We
had two categories of intrasexual aggression indicating whether the subject was directing
aggression toward same-sex individuals who were higher-ranking than themselves, or up
the hierarchy, or instead toward same-sex individuals who were lower-ranking than
themselves, or down the hierarchy.

Testing hypotheses suggesting steroid hormone mediation of female aggressive behavior

If steroid hormones activate female aggression, then naturally occurring steroid
hormone concentrations should be positively correlated with rates or intensities of
aggressive behavior. As concentrations of circulating (Goymann et al., 2001a; Licht et

al., 1992; Van J aarsveld and Skinner, 1991) and excreted (Dloniak et al., 2003;

99

Go

pit

the

63

0
a:

lit

IE

SE

it

Goymann, et al., 2001b; Van Meter et al., 2008; Van Meter, et al., 2009) steroid
hormones are higher during pregnancy than during lactation among spotted hyenas, we
predicted that rates of aggression should be higher when females are pregnant than when
they are lactating. Additionally, since steroid hormone concentrations are highest late in
gestation (Dloniak et al., 2006b; Licht, et al., 1998; Van Meter et al., 2008), aggression
rates should also increase as gestation progresses. We used repeated measures analyses to
investigate changes in rates of aggression within individuals during the various stages of
each female’s reproductive cycle. We examined both rates of inter- and intrasexual
aggression in this manner.

We used fecal samples collected from females to investigate whether steroid
hormone concentrations directly predicted their rates of aggression. We controlled for
reproductive condition by comparing concentrations of fA, fE, and fGC measured in
samples collected only from females during the second half of pregnancy, and evaluated
these hormone concentrations in relation to the mean rate of aggression emitted by each
female during this reproductive state.

We also tested the hypothesis that the organizational effects of androgens on
aggressive behavior, described for juvenile hyenas by Dloniak et al. (2006b), continue to
be expressed in adulthood. Here we quantiﬁed rates of aggression emitted by 14 adult
females for whom excreted steroid hormone concentrations could be measured in their
mothers while those female offspring were in utero. Like Dloniak et al. (2006b), we also
compared maternal fecal hormones measured during only the second half of gestation,
with the rates of aggression of their female offspring as adults (11 = 9 females). Prior

studies have demonstrated that fE (Van Meter et al., 2008) and fGC concentrations

100

(Dloniak, 2004; Van Meter et al., 2009) vary with the time of day (morning vs. evening)
of fecal sample collection. Therefore we investigated this factor here, but did not ﬁnd
signiﬁcant effects of sample collection time in our dataset, so we used both morning and
evening samples. From two females, we collected two samples within 10 days of each
other, and in these cases we calculated a mean maternal hormone value for that
pregnancy. We used maternal hormone concentrations as linear predictors of rates of
offspring aggression as adults toward a) all adults, b) lower-ranking females, and c)
immigrant males.

Fecal sample collection, extraction, and immunoassay

Fecal samples were collected into plastic bags within 30 minutes of excretion
whenever a known hyena was observed to defecate. Within 12 hours of collection,
samples were mixed, aliquoted, and frozen in liquid nitrogen for storage until they could
be shipped to Michigan State University for analysis.

Frozen fecal samples were lyophilized and extracted in ethanol as described
previously (Dloniak et al., 2003; Van Meter et al., 2008). Methods for assay of fecal
glucocorticoids (fGC) (Goymann, et al., 1999), fecal estrogens (fE) (Van Meter et al.,
2008), and fecal androgens (fA) (Dloniak et al., 2003) have been previously validated.

For the analysis of fGC, extracts were assayed in duplicate with a corticosterone
radioimmunoassay kit (ImmuChem Double Antibody Corticosterone 125I RIA Kit, MP
Biomedicals (formerly ICN), 07-120102), which displays a high afﬁnity to the major
glucocorticoid metabolites present in the feces of spotted hyenas (Dloniak, 2004;

Goymann et al., 1999). Concentrations of fE were assessed with a modiﬁed enzyme-

irnmunoassay protocol using an estradiol antibody (R4972; Clinical Endocrinology

101

Laboratory, University of California, Davis) shown to cross-react with the major
immunoreactive estrogen metabolites in hyena feces (Van Meter et al., 2008). Spotted
hyena fA concentrations were analyzed with a modiﬁed enzyme-immunoassay using a
testosterone antibody (R156/7; Clinical Endocrinology Laboratory, University of
California at Davis), shown to display a high afﬁnity for the major immunoreactive
androgen metabolites in hyena feces (Dloniak et al., 2003). The precision of each assay
was monitored by assaying serial dilutions of pooled hyena fecal extracts. The intra-assay
coefﬁcients of variation for fGC, fE and fA were 4.79%, 3.48%, and 6.18% respectively,
and the interassay coefﬁcients of variation were 10.12% (n = 17 assays), 13.34% (11 = 25
assays), and 12.05% (n = 51 assays).
Statistical analysis

All statistical testing was performed using SPSS 16.0.0 (SPSS) and graphs were
made with Sigma Plot 8.0 (StatSoft). In our behavioral data, a value of zero was often a
meaningful datapoint, indicating that an individual failed to attack conspeciﬁcs when it
had opportunities to do so. Many of our datasets contained enough. zero values that the
distributions of the data were highly skewed, so did not meet basic parametric test
assumptions, even after transformations were applied. Therefore, we used nonparametric
statistical tests to assess the behavioral data. We used within-individual comparisons to
evaluate within-sex variation in rates or intensity of aggression directed toward animals
in various target groups (e. g., comparing behavior of the same females when they were in
different reproductive states and individuals among different contexts of aggression). We
used a Friedman AN OVA omnibus test to compare more than two dependent groups, and

when this was signiﬁcant, we used Wilcoxon signed-ranks tests for post-hoe

102

comparisons. To make comparisons between the sexes we used tests for independent
data. When making comparisons among multiple independent groups, as with our
assessment of sex differences in intensity of aggression directed toward various targets,
we used a Kruskal-Wallis test omnibus test, then used Mann-Whitney U tests for post-
hoc comparisons. All tests were two-tailed (01 = 0.05) and we corrected for multiple
testing using the sequential Bonferroni adjustment (Rice, 1989), and report and interpret
only adjusted p—values. We present means 1 standard errors throughout.

We used linear regression to determine whether fecal steroid hormones explained
variation in aggression rates. Here, we could log-transforrn the aggression rates for these
analyses to approximate normal distributions (assessed by looking at the qq-plots

generated by these data). We found moderate correlations in our data among the three

fecal hormones (R2 2 0.10 S 0.76), and to avoid issues of multicollinearity we entered

each hormone into the model one at a time (Keppel and Wickens, 2004).

RESULTS
Distribution of aggression among contexts for adult males and females
We ﬁrst evaluated the contexts in which adults of both sexes exhibited aggression

toward other adults. The percent of total attacks emitted varied signiﬁcantly among

contexts for both males (Friedman ANOVA X2450 = 64.42, p < 0.001) and females

(X2432 = 58.89, p < 0.001) (Figure 3.1). We found no signiﬁcant sex differences in

percent of attacks in feeding (Mann-Whitney U = 1732.00, p < 0.510), unprovoked (U =

1573.50, p < 0.458), “other” (U = 1622.00, p < 0.533), or unknown contexts (U =

103

1847.00, p < 0.941); however, females directed signiﬁcantly more aggression toward
pesky individuals than did males (Mann-Whitney U = 751.00, p = 0.005).
Sex differences in rates of aggression

To compare rates of aggression directed by adults of both sexes toward
conspeciﬁcs, we calculated a rate of aggression emitted by each adult, controlled for
opportunity, directed toward either juvenile or adult conspeciﬁcs (Figure 3.2). Adult
females (n = 73) directed aggressive behavior at signiﬁcantly higher rates than did
immigrant males (n = 58) toward both adults (Mann-Whitney U = 767.00, p < 0.001) and
juveniles (U = 24.00, p < 0.001). Both females (Wilcoxon Z = 2.69, p = 0.007) and males
(Z = 6.00, p < 0.001) were signiﬁcantly more aggressive toward adults than they were to
juveniles.

We next used within-female comparisons to assess variation in rates of aggression
directed by adult females (n = 71) toward their various adult targets: higher-ranking
females, lower-ranking females, and males (Figure 3.3). There was one female, the
highest-ranking female, who was never present with a higher ranking individual and one
female, the lowest-ranking female, who was never present with a lower ranking female,

and both females were excluded from this analysis. Rates of aggression emitted by adult

females varied signiﬁcantly among target types (Friedman AN OVA X2230 = 64.42, p <
0.001). Females directed signiﬁcantly higher rates of aggression toward males than
toward females lower ranking (Wilcoxon Z = 2.03, p = 0.042) or higher ranking than
themselves (Z = 7.18, p = 0.003). Females directed the lowest rates of aggression toward

females higher ranking than themselves (Z = 7.27, p = 0.003).

104

 

- Females
{:3 Males

40-

Percent of all aggressive acts

 

 

     

Unprovoked Pesky Other Unknown

Context

Figure 3.1. Mean (:t standard error) percentage of all attacks (11 = 10098) directed toward
all adult conspeciﬁcs by adult females (11 = 73; black bars) and males (11 = 51; gray bars)
observed in various contexts. The “other” context refers to sex-speciﬁc situations like
mating and maternal defense. The sexes only differed signiﬁcantly with respect to
aggression directed toward “pesky” conspeciﬁcs (p = 0.005).

105

A — Pd I

F1811

&

mail

bars,

SﬁXC

Were

 

— Females
Males

 

Total aggression rate (hourly)

 

 

 

 

Adults Juveniles

Recipient of aggression

Figure 3.2. Mean (i standard error) lifetime aggression rates, controlled for numbers of
available targets in each observation session, directed by adult females (n = 73; black
bars) and males (n = 58; gray bars) toward adult and juvenile conspeciﬁcs. Adults of both
sexes were signiﬁcantly more aggressive to adults than to juveniles, and adult females
were signiﬁcantly more aggressive to both adults and juveniles than were adult males.

106

Similarly, we used within-male comparisons to assess variation in rates of
aggression directed by adult males (11 = 57) toward their various adult targets: higher-
ranking males, lower-ranking males, and adult females (Figure 3.3). There was one male
(the highest-ranking male at the beginning of the study) who was never present with a

higher-ranking male, so he was omitted. As with females, rates of aggression emitted by
adult males varied signiﬁcantly among target types (Friedman ANOVA X2256 = 45.65, p

< 0.001). Males directed aggression at signiﬁcantly higher rates toward males lower
ranking than themselves than toward either higher-ranking males (W ilcoxon Z = 5.72, p =
0.003) or females (Z = 5.59, p = 0.003). Males directed signiﬁcantly more aggression
toward females than toward high-ranking males (Z = 2.48, p = 0.013).

Next, we directly compared rates of aggression emitted by females with those
emitted by males (Figure 3.3). Females were signiﬁcantly more aggressive toward
higher-ranking targets (Mann-Whitney U = 1584.00, p < 0.045) and toward members of
the opposite sex (U = 321.50, p < 0.001) than were males; however, males and females
were equally aggressive toward same-sex targets lower-ranking than themselves (U =
1844.00, p < 0.389). Females were signiﬁcantly more aggressive toward males than
males were to each other (U = 1605.00, p < 0.045).

As a qualitative measure of sex differences in aggression, we compared the sexes
with respect to the intensities of the aggressive acts they emitted. Figure 3.4 shows the
percent of aggressive acts of high intensity directed by adult males and females toward
different target types (Kruskal-Wallis H = 47.14, p < .001). Severe aggression occurred
more often among female-female dyads than among dyads of any other type (for all

comparisons Mann-Whitney U < 640.5, p < 0.006).

107

 

0.12 d - Females *
— Males 115

0.10 -
0.08 -
0.06 -
0.04 -

0.02 a

Total aggression rate (hourly)

 

 

 

up hierarchy down hierarchy between sexes
within sex within sex

Figure 3.3. Mean (i standard errors) rates of aggression directed toward adult
conspeciﬁcs by adult male (gray bars) and female (black bars) spotted hyenas. Rates of
aggression emitted by females (11 = 71) varied signiﬁcantly among target types (females
higher-ranking, females lower-ranking, and males). Rates of aggression emitted by males
(n = 57) also varied signiﬁcantly among target types (males higher-ranking, males lower-
ranking, and females). Asterisks indicate signiﬁcant sex differences in rates of aggression
directed toward target types.

108

( It s
1

0 FNV

 

5 O 5
3 3 2 2 11
meow 0>mmmvhwwm :6 HO HCOOHOAH

Figure 3_.

intensity

numbers
aim-"e Iht‘

 

35 ' b - Females
E: Males

Percent of all aggressive acts

 

 

 

 

 

down hierarchy between sexes
within sex

Figure 3.4. Mean percent (1 standard errors) of all aggressive acts that were of high-
intensity (n = 1232). Not all males were seen to emit high-intensity aggressions, and the
numbers under the bars indicate number of individuals used in this analysis. Letters
above the bars indicate signiﬁcant differences among groups.

109

We 11
using only '4
35A). Rate
contexts of
“other" (1" =
toward pest
differences
3.531. and
toward mal

Finl
males in 211T
0.001z01he

low-intensi

to direct hi
Activating

We
TCPFOductit
Walled “
emlllEd b\
COHESan,
miss it.
”OI W)“ 11.

byfemin,

 

We next compared the sexes with respect to their rates of intrasexual aggression
using only aggression directed down the hierarchy within each aggressive context (Figure
3.5 .A). Rates of intrasexual aggression did not differ here between the sexes within the
contexts of feeding (U = 1698.00, p = 0.216), unprovoked (U = 1891.00, p = 0.519) or
“other” (U = 1613.00, p = 0.129). However, females were signiﬁcantly more aggressive
toward pesky targets than were males (U = 1209.00, p < 0.001). We also evaluated sex
differences in rates of inter—sexual aggression within each aggressive context (Figure -
3.5 .B), and found that females consistently directed signiﬁcantly more aggressive acts

toward males than did males toward females (p _<_ 0.005 for all comparisons).

 

Finally, intrasexual aggression was of higher intensity among females than among
males in all contexts (feeding, U = 537.50, p = 0.004; unprovoked, U = 368.00, p =
0.004; other, U = 409.00, p = 0.004) (Figure 3.6). Although we saw a few instances of
low-intensity attacks directed by males toward other pesky males, males were never seen
to direct high intensity aggression toward pesky conspeciﬁcs.

Activating effects of hormones in adult females

We used the natural ﬂuctuation in steroid hormone concentrations among
reproductive states to investigate whether rates of aggression emitted by adult females
covaried with these hormone concentrations. First, we asked if rates of aggression
emitted by females were higher when they were pregnant than when they were lactating,
corresponding to the natural variation in steroid hormone concentrations. In a paired
analysis (n = 40 females) (Figure 3.7.A), rates of female aggression directed at males did
not vary with reproductive state (Wilcoxon Z = 1.57, p = 0.116), and rates of aggression

by females toward lower-ranking female targets were signiﬁcantly higher during lactation

110

 

 

- Females
E223 Males

0
#
O

0.04 -

.9
O
on

0.03 -

p
8

0.02 -

p
E

0.02 q 0.01 -

Intrasexual aggression rate (hourly) >

p
8

 

 

 

 

 

0.00 -

 

 

 

0.10 4 J — Females
0'04 Males

 

0.08 r
0.03 -

0.06 .
0.02 r
0.04 -

0.02 d 0.01 .

 

 

 

 

 

0.00 - 0.00 -

F U P O

Figure 3.5. Mean (:1: standard errors) rates of (A) intrasexual aggression toward lower-
ranking conspeciﬁcs, (B) and inter-sexual aggression. Because feeding aggression
could only occur when food was present, rates were calculated for this context on a
different scale than for all other contexts. Asterisks indicated signiﬁcant differences.
(Contexts are abbreviated such that: F = feeding, U = unprovoked, P = pesky, and O =
“other”).

Inter-sexual aggression rate (hourly) W

111

0
1
1 i 0 0
3
A.”

“Ur-Mum
O 9:0
>_LL0.~ww~w :3 h

$80“ 0 o- r

gure 3.6

F1
of hi

h-inl

0
C

intensity 1

Unprovok.

50

 

— Females
* E: Males
* =1:

401

Percent of all aggressive acts

 

 

 

F U P 0

Figure 3.6. Mean percent (i standard errors) of all intrasexual aggressive acts that were
of high-intensity (n = 925). Aggression among females was of signiﬁcantly higher
intensity in every context. (Contexts are abbreviated such that: F = feeding, U =
unprovoked, P = pesky, and O = “other”).

112

than d1
steroid
are sut
signifl
(Z = 3.
aggres
concel

found
rankir
AVO‘

IOWar

differ!

feces
COlel
Pregr.
femaT
rates
and f

P=O

than during pregnancy (Z = 4.12, p = 0.004), which is opposite to the pattern seen in
steroid hormone concentrations during these two states. That is, androgen concentrations
are substantially higher among pregnant than lactating females. Females were
signiﬁcantly more aggressive toward males than toward females during both pregnancy
(Z = 3.49, p = 0.004) and lactation (Z = 5.72, p = 0.004). Next, we compared rates of
aggression emitted by females among the trimesters of pregnancy, since steroid hormone
concentrations are highest during the last trimester of pregnancy (Figure 3.7.B). We

found no effect of trimester on rates of female aggression directed toward either lower-

ranking females (Friedman AN OVA X2237 = 0.09, p = .958) or males (Friedman

ANOVA X2237 = 2.85, p = 0.482). Females were more aggressive toward males than

toward females during all three trimesters; however, after Bonferroni adjustment, this
difference was signiﬁcant only during early pregnancy (W ilcoxon, Z = 3.19, p = 0.003).
Finally, we inquired whether or not steroid hormone concentrations measured in
feces sampled from individuals directly predicted their concurrent rates of aggression. To
control for reproductive state, we focused here only on females in the last trimester of
pregnancy (11 = 16). Fecal concentrations of three steroid hormones measured in adult

females during their third trimester of pregnancy failed to explain any variation in their

rates of aggression toward females (fA, R2 = 0.00, p = 0.978, fE, R2 = 0.03, p = 0.478,
and ice, R2 = 0.03, p = 0.556) or toward males (fA, R2 = 0.001, p = 0.908, fE, R2 = 0.07,

p = 0.305, and fGC, R2 = 0.06, p = 0.380).

113

fur
is 1*

do
(lit
10‘

di'

 

 

 

— against females
3“,. — against males n/s
E 0.20 -
:3
O
.5:
V
d) 0.15 '1
H
to
3—1
:1
.2 010 - --
(A
(I)
8’.)
on 0.05 -
<3

 

 

 

 

 

Pregnant Lactating

 

w

- against females
:23 against males

.O
N
O
4

9

ﬂ

LII
l

Aggression rate (hourly)

 

 

 

Pregnancy

Figure 3.7. Mean (:1: standard error) rates of aggression directed by females toward lower-
ranking female targets, and adult males (A) during pregnancy and lactation (n = 40
females), and (B) during successive trimesters of pregnancy (n = 28 females). Females
were signiﬁcantly more aggressive toward other adult females during lactation than
during pregnancy (p = 0.004). During pregnancy, rates of aggression emitted by females
did not vary among successive trimesters of pregnancy. Females were more aggressive
toward males than toward females during all trimesters of pregnancy, although this
difference was signiﬁcant only for the ﬁrst trimester (p = 0.003).

114

 

 

Orgt’

[011".
cell:

beer

pro
nit
ma
0.6

3“

Organizing effects of hormones in adult females

We evaluated the rates of aggression emitted by 14 female subjects as adults
toward other adults in relation to fA, fGC, and fE concentrations measured in samples
collected from their mothers during the pregnancies in which those same subjects had

been in the womb. Offspring aggression rates as adults were not correlated with either
maternal fGC (R2 = 0.14, p = 0.191) or fE (R2 = 0.003, p = 0.852); however, there was a
signiﬁcant positive correlation between offspring aggression rates as adults and maternal
fA concentrations (R2 = 0.84, p < 0.001) (Figure 3.8.A). There was one data point that

proved to be an outlier in both x-space (assessed with leverage) and y-space (assessed

with Cook’s distance), but the relationship between offspring aggression as adults and
maternal fA during pregnancy was still signiﬁcant even after removal of this point (R2 =

0.65, p = 0.001). We assessed day of gestation, maternal rank during gestation, and

average offspring rank as adults as possible covariates, but none of these factors

signiﬁcantly inﬂuenced aggression rate (day of gestation, R2 partial = 0.001, p = 0.930;

maternal rank, R2 partial = 0.01, p = 0.460; and offspring rank, R2 partial = 0.01, p =

0-467).
Next, we examined the relationship between maternal fA and rates of aggression
directed by adult offspring toward immigrant males and also that directed toward lower-

ranking females. Maternal fA remained positively correlated with rates of offspring

aggression toward males (R2 = 0.71, p < 0.001), and neither day of gestation, maternal

rank, nor average offspring rank explained any additional variation (Figure 38.8).

However, maternal fA was not correlated with offspring aggression toward lower-ranking

115

 

females (R2 = 0.005, p = 0.813) (Figure 3.8.C). Thus rates of inter-sexual aggression, but

not intrasexual aggression emitted by females, were correlated with maternal fA
concentrations. To make our results strictly comparable to those of Dloniak et al. (2006),
we restricted our analysis to females who survived into adulthood for which we had
hormone data from samples collected from their mothers only during the second half of
gestation (n = 9 females). As with the larger dataset, there was a signiﬁcant positive

correlation here between maternal fA and offspring rates of aggression directed at males ET

(R2 = 0.52, p = 0.028), but not with rates of aggression towards females (R2 = 0.02, p =

 

0.746).
Our data revealed a strong sex difference in the intensity of aggressive acts
emitted by adult hyenas; however, we found no relationship between maternal fA and the

proportion of high intensity aggression directed by adult offspring toward adult females

(R2 = 0.017, p = 0.957). Although we had hoped to investigate the relationship between

maternal fA and rates of aggression received by female offspring from adult males,
because dyadic aggression directed by adult males toward adult females was so rare, we
did not have sufﬁcient data for this analysis; only four of the 14 females, for which we
had hormone data, ever received any aggression from an adult male.

Our data indicated that rates of aggression emitted by adult females varied with
the reproductive state of the aggressor, so we thought it important to control for
reproductive state in our analysis. We calculated rates of aggression from females only

while they were lactating, and these rates were signiﬁcantly and positively correlated

with maternal fA concentrations (R2 = 0.86, p < 0.001).

116

ft

 

0.35 -
0.30 ~
0.25 -
0.20 -
0.15 i
0.10 -

0.05 -

 

 

 

Offspring aggression rate toward females

 

r l T T

0 200 400 600 800 100012001400
0.35‘

 

O
0.30 .

0.25 -
0.20 .
0.15 —

0.10 ‘

 

0.05 *

 

 

Offspring aggression rate toward males

O
O

O

T

 

I l T I I I

0 200 400 600 800 100012001400
0.35~

 

0.30 ‘
0.25 r
0.20 - O
0.15 r

0.10 2

0.05 5 ' 3 O

 

 

 

 

Offspring aggression rate toward females

. 3 t . . .
0 200 400 600 800 100012001400

 

I I

Figure 3.8. Relationship between maternal fA during gestation and rates of offspring (n =
14) aggression in adulthood toward (A) all other adults, (B) adult males, and (C) adult
females. All offspring included here were adult females.

117

DISCUSS]

Ag
form of an
in the pres
different c
advantage
frequent 8
females is
resources.
functions
and femul
tallies wi
constrain.
and the n
majority
difficult 1
[Ott'ard C,
Signiﬁca
Controni
IEtrgets a
the 811210

IOIC in I

 

DISCUSSION

Aggressive behavior serves different functions in different contexts. Although the
form of an aggressive act may look very similar when an aggressor attacks a conspeciﬁc
in the presence of food to when it attacks in the presence of a potential mate, these
different contexts suggest different functions. In species in which males gain reproductive
advantages through competition with each other, aggression among males is often
frequent and intense. However, there is a growing recognition that competition among 1.. -
females is critical in shaping dominance relationships that determine access to key

resources, such as food (Sterck, et al., 1997; Walters and Seyfarth, 1987), and the t‘ "

 

functions of aggressive behavior directed toward conspeciﬁcs often differ between males
and females. While winning conﬂicts may convey reproductive advantages, aggression
carries with it obvious costs, and the expression of aggressive behavior is often
constrained by social factors, such as the dominance relationship between the attacker
and the recipient, and the immediate social setting (Mason and Mendoza, 1993). In the
majority of mammalian societies, one sex tends to be dominant to the other, making it
difﬁcult to compare males and females with respect to aggressive behavior directed
toward conspeciﬁcs of the opposite sex. With this chapter, we demonstrate that
signiﬁcant sex differences in aggression exist among adult spotted hyenas even after
controlling for the sex and rank of the attacker in relation to the sex and rank of the
targets available in the immediate social group, as well as the aggressive context in which
the attack occurred. We then also provide evidence indicating that androgens may play a

role in regulating the expression of aggressive behavior among adults in this species.

118

1
Sex diffe rel

ln 2

et al., 2001
Glickman
and Hamil
toward co
males. Lil
sexual ag
aggressio
Dyadic 21
instead it
function
not subn
in the CL
Signific;

(3003) r

female 1

(1611mm

2008; S

SimUll'd

adult j,

Holekt:

i

 

differe

 

Sex diﬁerences in aggression among adult spotted hyenas

In agreement with anecdotal ﬁeld reports from many hyena researchers (Boydston
et al., 2001; East et al., 2003; East and Hofer, 2002; Frank, 1986; Frank et al., 1989;
Glickman et al., 1997; Hamilton et al., 1986; Hofer and East, 1993; Kruuk, 1972; Tilson
and Hamilton, 1984), we have shown that overall rates of aggression directed by females
toward conspeciﬁcs are almost three times higher than rates of aggression emitted by
males. Like the results reported by Szykman et al. (2003), we found that rates of inter- F74
sexual aggression emitted by females were signiﬁcantly higher than rates of inter-sexual

aggression emitted by males, and this was true in every aggressive context observed.

 

Dyadic aggression emitted by males toward females occurs very rarely in this species;
instead males seem to rely on coalitions with other males to attack female hyenas. The
function of this coalitionary aggression among males is poorly understood, as females do
not submit to, and frequently counterattack their male attackers (Szykman, et al., 2003).
In the current study we also found that females directed aggression toward males at
signiﬁcantly higher rates than toward other adult females. By contrast, Szykman et al.
(2003) reported no difference in rates of aggression emitted by females toward male and
female targets, but did show a trend in the opposite direction from what was
demonstrated here. Male spotted hyenas are less gregarious than females (Smith et al.,
2008; Smith et al., 2007), and the ratio of adult females to adult males present
simultaneously in this clan was close to 2 to 1, so nearly twice as many adult females as
adult immigrant males were present at any given time (Kolowski et al., 2007; Watts and
Holekamp, 2008). Szykman et al. (2003) did not control for these important sex

differences in sociality in their earlier analysis, but when the opportunities available to

119

M

of

24:

ﬂ‘.

each aggressor to attack conspeciﬁcs of each sex are controlled, as we have done here,
we see that females frequently attack males that are present with them while being
observed. These data suggest that there is very little risk to females when attacking adult
males, and that it may be more risky to attack other females, even those who are lower-
ranking than the aggressor.

We also directly compared rates of intrasexual aggression emitted by the two
sexes and showed that these rates are not signiﬁcantly different after controlling for the
number of opportunities available in which to attack. However, the intensity of
intrasexual attacks is consistently higher when emitted by female than male hyenas. This
sex difference is also reﬂected in patterns of wounding, which indicate that adult females
experience higher rates of injury than do males (Holekamp, unpublished data). Overall,
our results suggest that female spotted hyenas emit aggressive acts more frequently than
do immigrant males, and that they ﬁght with greater intensity than do male conspeciﬁcs.
These sex differences in aggressive behavior may be important for the maintenance of
female dominance over males in spotted hyena society.

Goymann et al. (2001a) suggested that a comparative study should investigate
whether female spotted hyenas are more or less aggressive than females of other species
with similar social systems. Few studies have investigated rates of aggression emitted by
females of species living in the wild to the extent we do here; however, we can make a
few comparisons between spotted hyenas and well-studied primate species that also live
in multi-male, multi-female groups. Ehart and Overdorff (2008) made a simple
comparison of rates of intrasexual aggression among females for several species of wild

lemurs, monkeys, and apes by using available data from the literature. For each species,

120

the aut
kmde
menu
aggres

nmge

super
{OWE
Snnn
hyen
5000
rank
010
Inah
and
the 5

0fh

hth

Daft

the authors divided the total number of observed attacks among females by the number of

females included in the study, further divided by the total number of observation hours in

the study. Using this exact protocol we calculated the hourly rate of intrasexual

aggression among female hyenas in our study, and found that it falls well within the

range of rates Ehart and Overdorff (2008) reported for female primate species (Figure

3.9). This suggests that female spotted hyenas are not “hyperaggressive” relative to

female primates. However, spotted hyenas differ from most primate species in that rates _ _4

of inter-sexual aggression emitted by female spotted hyenas are signiﬁcantly higher than

 

those emitted by males. Among adult primates, males generally enjoy physical
superiority and social dominance over females, and females rarely direct aggression
toward adult males (Campbell, 2003; Kuester and Paul, 1996; Smuts, 1987; Smuts and
Smuts, 1993). Therefore, while rates of intrasexual aggression among females of both
hyenas and primates are similar, female hyenas can frequently aggress against socially
subordinate males, but female primates cannot freely direct aggression against higher-
ranking males. Finally, female primates and hyenas also differ with respect to the quality
of the aggression emitted. There is an almost universal tendency for aggression among
male primates to be more intense than that among females (reviewed by Smuts, 1987),
and ﬁghting can even lead to death (e. g., Campbell, 2006; Watts, 2004), indicating that
the situation in hyenas is highly unusual insofar as aggression among females is usually
of higher intensity than inter-sexual aggression or aggression among males.

Some earlier hyena researchers (East and Hofer, 2002; Goymann et al., 2001a)
have suggested that the aggressive behavior of males in this species has been reduced by

natural or sexual selection, and that reduced aggressiveness among males merely makes

121

 

 

 

     

3‘.
g 0 08 - ll 2 8 this study
5
a)
E
a 0.06 -
.2
8
58 0 04-
m .
a)
‘3
8
9,- 0.02 -
o
'3
8
LL
Old World monkeys Apes Lemuroids Spotted hyena

Figure 3.9. Mean (i standard error) rates of intrasexual aggression emitted by adult
females. Rates are calculated as total aggressive acts among females, divided by the
number of study subjects, over the total observation time for the study period. Primate
data were adapted from Erhart and Overdorff (2008), and the numbers over each bar
indicates the number of species represented in each group.

122

 

 

females appear “hyperaggressive” by comparison. Others have suggested that natural
selection has favored the evolution of traits among females, including enhanced
aggressive behavior, that have promoted female dominance in this species (Dloniak et al.,
2006b; Engh et al., 2002; Frank, 1986; Frank, 1996; Glickman et al., 1993; Kruuk, 1972;
Muller and Wrangham, 2002; Tilson and Hamilton, 1984). Surprisingly, our data show
that male and female spotted hyenas direct similar rates of aggression toward same-sex
targets. From these data alone we cannot determine whether selection has favored 1
enhanced aggression among females, reduced aggression among males, or both (Frank,

1986; Frank et al., 1995; Glickman et al., 1993; Hamilton et al., 1986; Holekamp and f

 

Smale, 1999; Tilson and Hamilton, 1984). However, our results indicating that the
intensity of aggressive acts directed by males towards other males generally tends to be
quite low, suggest that selection may indeed have acted to reduce ﬁght intensity among
males. Among adult males in most other mammalian species, ﬁght intensity tends to be
very high (Bernstein, et al., 1983; Campbell, 2006; Clutton-Brock, et al., 1979; Creel et
al., 1997; Lincoln, et al., 1972; Scott, et al., 2005; Smuts, 1987). On the other hand,
aggression appears to have unusually important ﬁtness consequences for adult female
hyenas (Hofer and East, 2003; Holekamp and Smale, 1999; Holekamp et al., 1996), so
selection may have favored enhanced aggressiveness among females in addition to
reduced aggressiveness among males in this species.

Many observers have commented on the low frequency with which attacks are
seen among male spotted hyenas (East et al., 2003; East and Hofer, 2001; Engh et al.,
2002; Frank, 1986; Henschel and Skinner, 1987; Holekamp and Smale, 1998; Kruuk,

1972); however, we suggest that this perception might be related, in part, to the fact that

123

males are less gregarious than females (Kolowski et al., 2007; Smith et al., 2008; Smith
et al., 2007 ). East et al. (2003) suggest that, in this female-dominated society, males have
little to gain by competing among themselves for mates, and that selection should instead
favor submissive, non-aggressive males in this species. Our results here show clear
overall sex differences in aggression, suggesting that females are the more aggressive
sex; however, our data also show that females do not comer the market on aggressive
behavior in this species, as males do compete among themselves in a variety of contexts.
Some studies have demonstrated that male hyenas compete aggressively, albeit at low
intensity, for access to females (Dloniak et al., 2006a; Goymann et al., 2003; Szykman et
al., 2003). Also, males participate vigorously in attacking prospective immigrants and
other male intruders (Boydston et al., 2001). Finally, prior to dispersal, adult males ,
maintain positions in the dominance hierarchy immediately below those of their mothers,
as do their female siblings (Engh et al., 2000; Holekamp and Smale, 1993; Smale et al.,
1993), allowing adult natal males to dominate and direct aggression toward females
lower-ranking than their mothers. Taken together, these data indicate that adult males are
fully competent aggressors. Dispersal has been shown to have profound effects on both
the behavior and physiology of male spotted hyenas (Dloniak et al., 2006a; Holekamp
and Sisk, 2003; Holekamp and Smale, 1998). We speculate that dispersal may be
associated with induction of an inhibitory mechanism functioning to dampen the
aggressive behavior of males. One of us (K.E.H.) has observed that immigrant males
sometimes behave erratically when they have been administered an anesthetic, but before
the drug has taken full effect. Under these circumstances, males may attack

“inappropriate” individuals, including adult females and young cubs. This inappropriate

124

 

beha

of th

1116C

s‘il

0C1

inc

re]

in

behavior suggests the disinhibition of aggression while the hyena is under the inﬂuence
of the drug. Whether or not this is the case, further investigation of the proximate
mechanisms controlling aggressive behavior, in both males and females, should yield
greater insight into the mediation of sex differences in aggression in this species.
Steroid hormones and aggressive behavior by females

As we were unable to directly manipulate steroid hormone concentrations in our
wild spotted hyena study population, we used the natural ﬂuctuations that are known to
occur among reproductive states to investigate variation in aggressive behavior within
individuals. We found no evidence that rates of aggressive behavior were high during
reproductive states when steroid hormone concentrations were also high. Moreover,
individual variation in rates of aggression among pregnant females was not correlated
with fA, fE, or fGC concentrations. Thus our data failed to provide support for
activational effects on aggressive behavior emitted by adult female spotted hyenas by the
steroid hormones we are currently able to measure in hyena fecal samples.

On the other hand, our results add to a growing body of evidence in support of an
organizational role for androgens in the mediation of aggressive behavior among spotted
hyenas. We showed that rates of aggression emitted by adult females were positively
correlated with the prenatal androgen concentrations to which these females were
presumably exposed in utero. This builds on work done by Dloniak et al. (2006b), who

showed that maternal fA concentrations sampled during gestation were positively

correlated with offspring behavior, including aggression, in juveniles aged 2 to 6 months.

It thus appears that prenatal androgens may be organizing the neural substrates

controlling aggressive behavior and that this organization has long-lasting consequences

125

tre‘l

 

 

 

 

Hokkar

nednue

 

Species

the pos
dispers.
We do
Noen
miI‘rOr
aduh;

dint

andro-

 

“hen

9008

 

 

for offspring behavior among spotted hyenas. Dloniak et al. (2006b) also showed that
maternal fA concentrations during pregnancy were positively correlated with maternal
rank. These results echo those obtained in studies of maternal effects in many bird
species. Maternal effects occur when maternal phenotype inﬂuences offspring phenotype
independent of offspring genotype, and maternal effects in regard to androgen exposure
have been suggested to enhance offspring ﬁtness among several bird species (Groothuis,
et al., 2005; Groothuis and Schwabl, 2008). Spotted hyenas experience intense feeding
competition at kills, and because reproductive success among females is strongly tied to
their ability to secure food for themselves and dependent young (Holekamp et al., 1996;
Holekamp et al., 1999), it is reasonable to suggest that enhanced aggressive behavior,
mediated by prenatal androgen exposure, might be adaptive among females of this
species.

An adaptive function for prenatal androgenization of females does not preclude
the possibility that males might also beneﬁt from prenatal androgen exposure. As males
disperse out of their natal clans after puberty (Holekamp et al., 1996; Smale et al., 1997),
we do not have comparable behavioral data from male offspring when they are adults.
Nevertheless, we would not predict that the aggressive behavior of adult males would
mirror the relationship between maternal fA concentrations and aggression seen among
adult female offspring. Low social rank among adult male hyenas in their new clans after
dispersal inevitably constrains their ability to behave aggressively. However, prenatal
androgen exposure does appear to inﬂuence the sexual behavior of male hyenas, at least
when they are juveniles. Rates of mounting behavior in juveniles of both sexes were

positively correlated with maternal fA, and this relationship was much more robust

126

l
‘h

 

among
spotted
imascr
detertr

reprod

found
aggre:
possil
gestat
not 8:
MDOL
prenz
beha
are n
lack
Pfilr.
d0m
P085
Spot
mai:
fA r

30%

among males (Dloniak, et al., 2006b). Mating is uniquely challenging for the male
spotted hyena who must overcome both behavioral (female dominance) and physical
(masculinized external genitalia of female) obstacles, and it would be interesting to
determine whether variation in prenatal androgen exposure is related to male
reproductive success.

When we controlled for the sex of the target of aggression in the current study, we
found that maternal fA concentrations were much better predictors of inter-sexual
aggression than intrasexual aggression emitted by females. This result raises the
possibility that adult females exposed to high concentrations of androgens during
gestation are treated differently later in life by adult males than are other females. We did
not see any support for this idea in our aggression data, as males did not direct substantial
amounts of aggression toward females exposed to either high or low concentrations of
prenatal androgens; however, these females might still be responding to some other
behavior emitted by males that we did not measure in this study. Although female hyenas
are more aggressive than males overall, we have clearly shown that adult males do not
lack the ability to behave aggressively. We have also presented evidence from the
primate literature suggesting that female aggression in species with matrilineal
dominance hierarchies need not be associated with female dominance. Therefore, it is
possible that androgens function uniquely in the prenatal environment experienced by
spotted hyenas to enhance female aggressiveness toward males, and thereby achieve and
maintain social dominance over males. Alternatively, the relationship between maternal
fA and offspring aggression directed toward males may reﬂect aggressive behavior in the

absence of social constraints. As noted above, female hyenas have very little risk of

127

 

 

retrif

migl

‘17
"Q
”Q

11th

the
for
L0:
det
rec
me
3111

C0

hy

retribution when directing aggression toward adult males, whereas the social context
might impact their decision to attack another female, even a lower ranking one.

We emphasize that the occurrence of organizational androgen effects on adult
aggressive behavior by female spotted hyenas does not by any means rule out effects in
hyenas that might be induced by any of the myriad variables known to mediate
aggressive behavior in females of other species. For instance, we cannot currently
measure progesterone in hyena feces, yet progesterone has been linked to female
aggression in other species (Davis and Marler, 2003). There are also several steroid-
independent mechanisms that might be acting in spotted hyenas to mediate female
aggressive behavior. Maternal aggression has received little attention in this species, and
the rates of aggression we observed here during lactation may suggest an excitatory role
for prolactin in this species, as occurs in some other mammals (e. g., Gammie and
Lonstein, 2006; Lonstein and Gammie, 2002). There is also growing evidence
demonstrating that aggression in some species is inﬂuenced by serotonin and serotonin-
receptor distribution in the central nervous system. For instance, metabolites of serotonin
measured in the cerebrospinal ﬂuid are thought to reﬂect serotonin turnover in the brain,
and low cerebrospinal ﬂuid concentrations of serotonin metabolites are positively
correlated with high-intensity aggression among male (Higley, et al., 1996; Higley, et al.,
1992) and female macaques (W estergaard, et al., 2003). The possibility that one or more
of these additional factors inﬂuences expression of aggressive behavior among spotted

hyenas must await the attention of future investigators.

128

 

 

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CHAPTER FOUR
FUNCTIONS OF UNPROVOKED AGGRESSION
AMONG FEMALE SPOTTED HYENAS
INTRODUCTION
The beneﬁts associated with group living are well established (Cheney, et al.,
1986; Wrangham, 1987), and gregariousness itself may convey direct ﬁtness beneﬁts to
individuals (Silk, 2007; Silk, et al., 2003). However, group living is also associated with

costs, especially those related to competition for resources (Sterck, et al., 1997; van

 

Schaik, 1989), and this competition often involves aggressive interactions (Silk, 1993;
Silk, 2002; Walters and Seyfarth, 1987). Aggression frequently occurs among
conspeciﬁcs in the context of acquisition of resources, such as food, territory, or mates
(Archer, 1988; Silk, 1993; Walters and Seyfarth, 1987), in defense of young (Hrdy, 1979;
Silk, 1993; Walters and Seyfarth, 1987) or it may be a product of fear or frustration
(Archer, 1988; Lorenz, 1970; Wingﬁeld, et al., 2006). However, here we are particularly
interested in the occurrence of aggression in the apparent absence of causal factors in the
individual’s immediate environment, termed unprovoked aggression. Unprovoked
aggression has been investigated as a manifestation of disease or pathological anti-social
behavior, as among domestic dogs (Dodman, et al.,.1996; Velden, 1979; Velden, et al.,
1976) and humans (Reidy, et al., 2008). However, unprovoked attacks, particularly
among female conspeciﬁcs, have been described to occur frequently in a number of
primate species (Silk, 2002; Smuts, 1987; Walters and Seyfarth, 1987), as well as among
spotted hyenas (Crocuta crocuta) (Engh, et al., 2000; Smale, et al., 1993; Wahaj and

Holekamp, 2006). These ﬁndings suggest that unprovoked attacks are not merely the

139

result of pathology. Indeed unprovoked aggression might have important ﬁtness beneﬁts.
In this chapter we describe the unprovoked aggressive behavior that frequently occurs
among adult female spotted hyenas, and examine hypotheses suggesting possible
adaptive functions for this behavior.

Spotted hyenas live in large, multi-male, multi-female social groups, called
“clans.” Like those of many cercopithecine societies (Drea and Frank, 2003; Watts and
Holekamp, 2007), the society of the spotted hyena is structured by a rigid linear
dominance hierarchy based on transfer of rank among generations via maternal rank
“inheritance” in which daughters slot into the hierarchy just below their mothers (Engh et
al., 2000; Frank, 1986; Smale et al., 1993). Females are philopatric, but males generally
disperse between the ages of 2 and 5 years (Boydston, et al., 2005; Smale, et al., 1997),
and adult immigrant males are socially subordinate to all natal members of the group
(East and Hofer, 2001; Engh, et al., 2002; Frank, 1986; Kruuk, 1972; Smale et al., 1993;
Tilson and Hamilton, 1984).

In contrast to mammalian norms, female spotted hyenas are more aggressive to
conspeciﬁcs than are adult immigrant males (Szykman, et al., 2003), and we showed in
Chapter 3 that intrasexual aggression among female hyenas occurs just as frequently as
does intrasexual aggression among males in a number of different contexts, including
unprovoked aggression. Unprovoked attacks among spotted hyenas are characterized by
seemingly spontaneous interactions in which the aggressor and the recipient have had
little or no contact in the minutes preceding the attack. Both juveniles (Smale et al., 1993;

Wahaj and Holekamp, 2006) and adults (Engh et al., 2000) direct unprovoked aggression

140

 

towards conspeciﬁcs, and given its frequency in the natural habitat, we think it is unlikely
to reﬂect pathological behavior among adult females.

In cercopithecine primate societies, unprovoked aggression among females has
been proposed as one mechanism for teaching rank relationships. Adult females
preferentially direct unprovoked aggression toward the infants of lower-ranking females,
which facilitates rank acquisition among the infants (Berman, 1980; Horrocks and Hunte,
1983). However, there was no support for this hypothesis among spotted hyenas, as rates
of unprovoked aggression received by cubs do not vary with ranks of the adult female
aggressors (Engh et al., 2000). Therefore, the function of unprovoked aggression in
spotted hyenas, particularly among adult females, remains unexplained. Here we tested
predictions of twoghypotheses suggesting functional explanations for unprovoked
aggression among female spotted hyenas.

Hypothesis 1: Unprovoked aggression among female hyenas functions to suppress
reproduction in other females. Among female spotted hyenas, reproductive success varies
with social rank (Hofer and East, 2003; Holekamp, et al., 1996). While all females bear
young, hi gher-ranking females have longer reproductive lifespans, experience shorter
inter-birth intervals, and produce more surviving offspring than do lower-ranking females
(Holekamp et al., 1996). Much of this variation may be explained by access to food; for
instance, inter-birth intervals among low-ranking females are longer during times of low
prey availability than during periods of prey abundance (Holekamp et al., 1996;
Holekamp, et al., 1999). However, in addition to differential food access, it is possible
that high-ranking females may directly suppress reproductive function in subordinate

females via unprovoked aggression. The eusocial naked-mole rat is an example of a

141

 

species displaying this kind of socially-mediated reproductive suppression; despotic
reproductive skew is controlled in these rodents by aggression emitted by queens toward
subordinate females (Bennett, 2000; Faulkes and Abbott, 1997). Moreover, targeted
female aggression serving as a mediator of reproductive suppression has been shown to
occur in a number of callitrichid primate species (reviewed by Solomon and French,
1997), as well as in some social carnivores (Creel, et al., 1997; McLeod, et al., 1996).
Reproductive suppression mediated by coalitionary attacks has also been demonstrated in
the yellow baboon, a species exhibiting sociality and reproductive skew similar to those
of spotted hyenas (Wasser and Starling, 1988). If unprovoked aggression similarly
represents a mechanism for reproductive suppression among spotted hyenas, then females
should be the recipients of aggression most often when they are in reproductively
vulnerable states (Wasser and Starling, 1988). That is, females should be targets of
unprovoked aggression most frequently in early pregnancy, when the health of the
mother and fetus may be compromised, and/or around the time of ovulation, when the
probability of successful conception may be reduced.

Hypothesis 2: Females use unprovoked aggression to test existing rank
relationships. The simple rules of rank inheritance in both cercopithecine and hyena
societies provide great stability in dominance relationships over long periods of time.
Given the substantial ﬁtness advantage enjoyed by higher-ranking hyenas,'improving
one’s rank should be highly beneﬁcial for females. Rank reversals do occasionally occur
in both cercopithecine and hyena societies (Chapais, 1985; Holekamp, et al., 1993; Mills,
1990; Smuts, 1987; Walters and Seyfarth, 1987), supporting the notion that opportunities

for social climbing exist even in these largely stable societies. We propose that female

142

 

hyenas may be employing alternative strategies for testing the stability of social
relationships when directing unprovoked aggression toward either lower- or higher-
ranking females. Females may use unprovoked aggression directed toward lower-ranking
conspeciﬁcs as a low cost reminder of the attacker’s dominant status (Silk, 2002).
Alternatively, individuals might use unprovoked aggression directed toward higher-
ranking conspeciﬁcs to test the stability of their social relationships and thereby assess
opportunities for rank advancement (Ehardt and Bernstein, 1986). This type of —-.J
opportunistic testing has been proposed as a precursor to matrilineal overthrows among
captive rhesus macaques (Ehardt and Bernstein, 1986). If this type of testing also occurs
among hyenas, then we would expect females to direct unprovoked aggression toward
targets who are close in rank to themselves, as interaction between individuals most alike
in ﬁghting ability or social standing would most likely provide opportunities for
improvement in the attacker’s social status (Ehardt and Bernstein, 1986; Forkman and
Haskell, 2004).

Here we compare the frequency and intensity of unprovoked attacks directed
toward higher- and lower-ranking targets and examine factors, such as rank and
composition of the immediate subgroup (Silk, 2002), which might inﬂuence the
likelihood of attack. We predict that, when unprovoked attacks occur in the presence of
bystanders, females directing aggression toward lower-ranking females should do so in
groups containing other higher-ranking individuals, who might provide support to the
attacker (Engh, et al., 2005; Smith, et al., 2009). Alternatively, the likelihood of attacking

a higher-ranking female should be positively correlated with the number of females

143

 

lower-ranking than the attacker, as these females also have something to gain, and might

provide aid when challenging dominant individuals.

METHODS
Study site and subjects

A single large clan of spotted hyenas living in the Talek region of the Masai Mara
National Reserve was the subject of a longitudinal study of hyena behavior and ecology. m ‘_
Here we used data collected between July 1988 and December 2005; during this period
all clan members were individually recognized by their unique spot patterns, and were
sexed based on penile morphology (Frank, et al., 1990). Females over 2 years of age were
considered adult members of the dominance hierarchy, which was updated annually.
During the study period, the number of adult females in the clan ranged from 16 to 34
(mean = 23.2) individuals. Offspring joined the dominance hierarchy below their mothers
but above their older siblings (Engh et al., 2000; Holekamp and Smale, 1991). By
convention the highest-ranking female in the clan was assigned a rank of 1.

Reproductive histories were known here for all adult females, and only parous
females were used in this study. Birth dates (:1: 7 days) were assigned based on
appearance and behavior of cubs when they were ﬁrst seen (Holekamp et al., 1996).
Gestation in this species is 110 days (Kruuk, 1972), so counting backward from dates of
parturition, we could estimate dates of conception. To compare rates of behavior among
phases of gestation, pregnancy was divided into trimesters; early pregnancy (P1), middle

pregnancy (P2), and late pregnancy (P3). Duration of lactation is highly variable in this

species and can range from 7.5 to 24 months, and lower-ranking females take longer to

144

 

wean litters than do hi gher-ranking females (Holekamp et al., 1996). Lactation intervals
began at birth, and lasted until weaning of the litter or the conception of the next litter,
whichever came ﬁrst; incomplete lactation intervals caused by litter death were not
considered here. We assigned wean dates (i 10 days) based on observed nursing conﬂicts
between mothers and cubs, and the cessation of nursing, as has been previously described
(Holekamp et al., 1996; Holekamp et al., 1999). We partitioned each lactation cycle (L)
into thirds, and since many females conceive their next litter prior to weaning the last
(Holekamp et al., 1996), we omitted the last third of lactation, in this way we controlled
for the possibility that females had begun cycling during this time. Very little is known
about ovulation in this species, but it is estimated to occur in a 14 day cycle (Kruuk,
1972; Lindeque, 1981; Matthews, 1939). We focused on the 30 days prior to a known
conception as a reproductive period during which females were potentially cycling (C).
Behavioral observations

Spotted hyenas live in a ﬁssion-fusion social system, and clan members spend the
majority of their time in small subgroups, the size and composition of which changes
many times a day (Kolowski, et al., 2007; Smith, et al., 2008). Therefore, it is important
to control for the variation in availability of social partners available with which an
individual might interact. We collected daily behavioral data during morning (0530 —
0900 hours) and evening (1700 — 2000 hours) observation sessions on groups of two or
more adult female hyenas that were separated from other hyenas by at least 200 m.
Identities and social ranks of all hyenas in each group were known and recorded, and a
location was assigned to each session (communal den, kill scene, or “other” location).

Sessions lasted at least 15 minutes and ended when we moved away from each subgroup.

145

We observed groups containing two or more adult females for a total of 4823 hours
during 6222 observation sessions. All aggressive interactions among hyenas were
recorded as critical incidents (Altmann, 1974). For each aggressive interaction we
recorded the aggressor, behavior, recipient, response, and, if possible, the context in
which the aggression occurred. Here we considered only dyadic aggression, occurring
between one aggressor and one recipient, in an unprovoked context. Observers
categorized an attack as unprovoked when the aggression occurred in absence of any
apparent contested resource, and when there was little or no interaction between the
aggressor and recipient leading up to the attack that would indicate provocation by the
recipient. Our ethogram of aggressive behaviors included head-wave, displacement, stand
over, and point as low-intensity aggressive acts; lunge, push, rush, and chase, we
included as medium-intensity behaviors, and any biting attack was categorized as high-
intensity. Responses to aggression included appeasement behaviors (head bob, carpal
crawl, squeal, back-off) and counter-attacks.

To test the reproductive suppression hypothesis, we compared rates of
unprovoked aggression received by females among different reproductive states using a
repeated measures within-female design. Rates of aggression received were calculated for
each female during each trimester of pregnancy (P1, P2, and P3), early lactation (L), and
the period one-month prior to conception (C). Since conception dates are estimated from
birth dates, we also calculated rates of aggression received for the two weeks prior and
two weeks post conception. All females included in this analysis were observed in all

these reproductive states, and we included only those observation sessions in which

146

 

females were seen with at least one potential attacker. Hourly rates of aggression received

were calculated for every female in each session as:

 

(number of unprovoked attacks acts received + number of females present that were
higher-ranking than recipient)

 

number of hours in the observation session

 

 

 

This rate calculation thus controlled for variation in number of adult female hyenas
present in a given observation session that might have attacked the target female, as well
as for the amount of time each female was observed in that session. Aggression rates
were averaged over all sessions during which a female was in a particular reproductive
state, including those sessions when the female was seen with potential aggressors but did
not receive any attacks. In this way, the mean rates of aggression received by individuals
who were frequently seen with potential aggressors, but did. not receive aggression, were
weighted in comparison to individuals who were seen less often.

We refer to aggressive acts by a lower-ranking aggressor toward a higher-ranking
recipient as aggression directed “up the hierarchy,” and aggressive acts emitted by a
higher-ranking aggressor toward a lower-ranking recipient, as attacks “down the
hierarchy.” To examine the rank relationships among aggressors and recipients involved
in unprovoked aggression directed both up and down the hierarchy, we calculated the
rank distance for all interacting dyads in two ways. First, we used an absolute rank
distance, which was the difference between the aggressor and the recipient’s overall rank
position in the current female dominance hierarchy. Second, we calculated a relative rank

distance, which was the difference in rank between the aggressor and recipient relative to

147

 

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the other adult female hyenas present in the immediate subgroup at the time of the attack.
We used multivariate techniques to investigate whether rank and subgroup composition
inﬂuenced the likelihood of attack. To assess the inﬂuence of subgroup composition, we
analyzed all observation sessions in which both members of an interacting dyad were
present and asked whether the number of adult females higher- or lower-ranking than the
aggressor inﬂuenced the likelihood of an attack occurring within the dyad. We predicted
that higher-ranking bystanders would positively inﬂuence the likelihood of attacking
down the hierarchy, while lower-ranking bystanders would positively inﬂuence the
likelihood of attacking up the hierarchy.
Statistical analyses

We used a repeated measures design to assess changes in rates of aggression
received when females were in each reproductive state (P1, P2, P3, L, and C) and to
compare unprovoked aggression directed up and down the hierarchy. Since a value of
zero was often a meaningful datapoint, indicating that an individual was present with
potential aggressors but was not attacked, our dataset contained enough zero values that
data distributions were highly skewed, and therefore did not meet basic parametric test
assumptions. Therefore, we assessed these data with nonparametric statistical tests using
SPSS 15.0.0 (SPSS). We ﬁrst used a Friedman ANOVA omnibus test, then used
Wilcoxon signed-ranks tests for post-hoc comparisons. All tests were two-tailed (or =
0.05) and we corrected for multiple testing using the sequential Bonferroni adjustment
(Rice, 1989); we report only adjusted p-values. We investigated the inﬂuence of

aggressor rank, controlling for number of targets, on the rank distance within dyads with

148

 

an analysis of covariance (ANCOVA). We present means i standard errors throughout;
all graphs were made with Sigma Plot 8.0 (StatSoft).

To investigate whether the composition of the immediate subgroup inﬂuenced the
likelihood of an attack within interacting dyads we used generalized estimating equations
(GEE), a technique for ﬁtting generalized linear models to clustered data (Faraway, 2006;
Liang and Zeger, 1986). All observation sessions in which the interacting dyads were
seen represented the clustered units, and we categorized the occurrence of an attack as a
binomial response (attack occurred = 1, attack did not occur = 0). These models were ﬁt

using geepack in R 2.4.1 (R Development Core Team).

RESULTS

As has been previously described (Chapter 3) aggression among adult female
spotted hyenas occurs in a number of different “provoked” contexts, such as feeding,
defense of young, or in response to “pesky” behavior by conspeciﬁcs; contexts for
interactions were classiﬁed as “unknown” when it was unclear due to poor visibility.
Aggressive acts were only classiﬁed as unprovoked when they occurred under conditions
of good visibility, in the absence of contested resources, and with no interaction between
the aggressor and the recipient leading up to the attack. Frequently the recipient of
unprovoked aggression was sleeping, resting or nursing their cubs prior to the attack.
Hyenas engaging in unprovoked attacks were commonly new arrivals to the subgroup,
and they had. therefore had no opportunity for interaction with the recipient before the

attack.

149

We witnessed a total of 3762 aggressive acts among adult females, of which 614,
or 16.3%, were classiﬁed as unprovoked. We found that the large majority of unprovoked
attacks occurred at dens (Figure 4.1), which are communal locations where females raise
their young, and serve as social centers for clan members (Holekamp and Smale, 1998;
White, 2007). The communal den is a complex of burrows used by mothers to house
young cubs. The narrow diameter of the burrow opening allows entry to cubs, but not to
adult hyenas, and mothers frequently lie in the den opening to nurse. Therefore we
considered the possibility that the den itself might serve as a limited resource and thus
provoke conﬂict. As the only females who use den entrances for nursing have young cubs
(Kruuk, 1972; White, 2007), we compared rates of aggression emitted and received by
females at the den when they did or did not have a cub aged less than 3 months. Using a
within-female comparison (N = 41 females), we found no signiﬁcant difference in rates
of aggression emitted (W ilcoxon Z = -1.69, p = 0.091) or received (Z = -0.72, p = 0.471)
by females at the den when they did or did not have a young cub. We concluded that this
aggressive behavior occurred without provocation, and in the absence of obvious
competition for resources including den access.

Testing the reproductive suppression hypothesis

To address this hypothesis, we compared the rates of unprovoked aggression
received by 31 females observed in every reproductive state (P1, P2, P3, L, and C)

(Figure 4.2). There was an overall signiﬁcant effect of reproductive state on aggression
received by these females (Friedman ANOVA X2439 = 33.98, p < 0.001). This hypothesis

predicted that females should receive higher rates of aggression earlier rather than later in

gestation, when the fetus is most vulnerable; however, a Friedman ANOVA comparing

150

 

 

100
CD
ii 80
S
H
CU
To 60-
H
O
H
a...
0 40a
H
t:
O)
8
<1) 20-
Q...

 

 

 

 

 

Dens Kills Other

Figure 4.1. Percent of all unprovoked aggressive acts (n = 614) observed at each location.

151

 

 

 

0.035 -

.O
O
D.)
O
1

0.025 -

0.020 5

0.015 -

0.010 -

0.005 -

 

 

 

 

Aggressions received (hourly rate)

P1 P2 P3 L C

Figure 4.2. Mean (i standard error) rates of unprovoked aggression received by females
(11 = 31) when they were in each reproductive state, where P1, P2, and P3 refer to
successive trimesters of pregnancy, L refers to early lactation, and C indicates the month
prior to a known conception. Different letters above the bars represent signiﬁcant
differences among the states.

152

 

 

P1.PI
ngnﬁ
hzﬂsc
conce
duln
andf
oner
ofag
these

4108

unr
att

dn

 

an

 

hi

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11

 

P1, P2, and P3 was not signiﬁcant (X2229 = 2.33, p = 0.311), indicating that there was no

signiﬁcant variation in rates of aggression received among the three phases of pregnancy.
It also predicted that females should receive high rates of aggression while cycling when
conception might be disrupted; however, rates of aggression received prior to conception
did not differ from those received during any other reproductive state except lactation,
and here aggression received during lactation was signiﬁcantly higher than rates received
one month prior to conception (W ilcoxon Z = -3.34, p = 0.004). We also measured rates
of aggression received for the period i 2 weeks from our estimated conception dates;
these were also signiﬁcantly lower than rates received during lactation (W ilcoxon Z :- -
4.08, p < 0.001), but were not signiﬁcantly different from rates measured during the
month prior to conception (Wilcoxon Z = -0.357, p = 0.721). As these results were
inconsistent with predictions of the reproductive suppression hypothesis, it could be
rejected.

Females use unprovoked aggression to test existing rank relationships

The aggressor was higher-ranking than the recipient in 576, or 93.8% of 614
unprovoked attacks, and lower-ranking than the recipient in only 38, or 6.19% of these
attacks. We compared the intensity of unprovoked aggressive acts emitted by females
directed up the hierarchy with those directed down the hierarchy (Figure 4.3). In this
analysis we used aggressive acts emitted by 14 females who attacked females both
higher- and lower-ranking than themselves. The distribution of aggressive acts among our

different intensity levels varied signiﬁcantly regardless of whether they were directed up

the hierarchy (Friedman ANOVA X2113 = 9.67, p = 0.008) or down the hierarchy

(Friedman ANOVA X2 2,13 = 12.29, p = 0.002). The majority of attacks both up and down

153

 

the hierarchy were of low intensity; however, females directed signiﬁcantly more
aggressive attacks of high intensity towards lower-ranking females than toward higher-
ranking females (Wilcoxon Z = 2.81, p = 0.015). The rarity and relatively low intensity of
unprovoked attacks directed up the hierarchy suggests that this behavior may produce
more severe consequences for the attacker than when attacks are directed down the
hierarchy.

We ﬁrst examined the 38 attacks directed up the hierarchy, which occurred within
26 dyads. The absolute rank distance among 14, or 51.85%, of these dyads was within 5
rank places, so, the majority of the aggression directed up the hierarchy occurred among
closely ranked females (Figure 4.4.A). Likewise, the rank distance of the dyads
calculated using only females present in the immediate subgroup at the time of the attack
indicated that the majority of unprovoked aggression occurred among females that were
closely ranked within the observation session (members of 19 dyads, or 70.37% were
within 2 relative rank places) (Figure 4.4.B). We were interested to see if the composition

of the immediate social group inﬂuenced the likelihood of attack. Both the number of
females lower-ranking (R2 = 0.39, p = 0.042) and higher-ranking (R2 = 0.83, p = 0.001)

than the aggressor were positively correlated with the total number of females present at
the time of the attack. Surprisingly, the relationship between higher-ranking females and
total females present was much more robust, and indeed, there was always at least one
higher-ranking female other than the interacting dyad present at the time of the attack.
However, the likelihood of an attack toward a higher-ranking recipient was not

signiﬁcantly inﬂuenced by either the number of lower-ranking bystanders (Estimate:

154

 

 

0.02, Wald statistic: 0.03, p = 0.859) or the number of higher-ranking bystanders
(Estimate: 0.04, Wald statistic: 0.29, p = 0.593) present in the immediate social group.
We next examined the 576 attacks directed down the hierarchy, which occurred
within 444 different dyads. The absolute rank distance among 186, or 41.89%, of these
dyads was within 5 rank places, so, the majority of the aggression directed down the
hierarchy occurred among closely ranked females (Figure 4.5.A). Likewise, the rank
distance within the dyads calculated using only the females present in the immediate
subgroup indicated that the majority of the aggression occurred among females that were
closely ranked within the observation session (313 dyads, or 70.49% were within 2
relative rank places) (Figure 4.5.B). Again, we wanted to see if subgroup size or
composition inﬂuenced the likelihood of attacking a lower-ranking female. As with
attacks directed up the hierarchy, we very rarely saw an attack down the hierarchy when

the interacting dyad was alone; 14 out of 576 unprovoked attacks, or 2%, occurred when

the dyad was alone. Both the number of females lower- (R2 = 0.68, p < 0.001) and

higher-ranking than the aggressor (R2 = 0.63, p < 0.001) were positively correlated with

the total number of females present at the time of the attack. We found that the likelihood
of attacking a lower-ranking female signiﬁcantly increased with the number of higher-
ranking females present (Estimate: 0.07, Wald statistic: 8.94, p = 0.003), and signiﬁcantly
decreased with the number of lower-ranking females present (Estimate: -0.16, Wald
statistic: 34.45, p < 0.001). These results support the prediction that females directing
aggression toward lower-ranking females do so when the opportunity for support from

other higher-ranking females is high.

155

100

 

_ Aggressions up hierarchy
1:2] Aggressions down hierarchy

Percent of aggressive acts

 

 

 

Low Medium High

Intensity

Figure 4.3. Percent of total aggressive acts in each intensity category directed by females
(11 = 14) towards higher-ranking recipients (black bars) or towards lower-ranking
recipients (gray bars). Asterisk indicates a signiﬁcant difference (p = 0.015).

156

71>

N
O

 

Percent of dyads
S {I

LII
1

 

 

 

 

 

 

5 20 25 30 3

 

O _
5 10 1 5
B Absolute rank difference
60
50 -

A
O

Percent of dyads
8 8

H
O

 

 

 

1 2 3 4 5

Relative rank difference

Figure 4.4. Frequency distribution showing rank distances within 26 dyads in which the
aggressor was lower-ranking than the recipient. Rank distance was calculated for each
dyad as the difference between aggressor and recipient in a) absolute rank in the clan, and
b) relative rank among females present in the immediate subgroup.

157

3>

 

)—1
O

OO
1

Percent of dyads

 

 

5 10 15 20 25 30 35
B Absolute rank difference

 

Percent of dyads
N U) -P
O O O

p—r
O

 

 

 

 

1 2 3 4 5 >6
Relative rank difference

Figure 4.5. Frequency distribution showing rank distances within 444 dyads in which the
aggressor was higher-ranking than the recipient. Rank distance was calculated for each
dyad as the difference between aggressor and recipient in a) absolute rank in the clan, and
b) relative rank among females present in the immediate subgroup.

158

mm

{1100:

< 0.l
sigr.
ava
bet‘
0.1

wit

Dl

 

 

We used an AN COVA to inquire whether the social rank of the aggressor
inﬂuenced the absolute rank distance between the aggressor and the recipient (whole
model R2 = 0.08, F = 618.93, 22592.16, p < 0.001). Rank of the aggressor was a
signiﬁcant predictor in the model such that high-ranking females attacked females ranked
farther from them in the hierarchy than did low-ranking females (F = 17.90, 653.19.16, p
< 0.001). The number of targets available to the aggressor was included as a marginally
signiﬁcant covariate, so there was a trend for rank distance to increase as number of
available targets increased (F = 2.97, 108.41, p = 0.086). However, the interaction
between aggressor rank and number of targets was not signiﬁcant (F = 2.55, 93.23, p =
0.111); therefore, after controlling for targets available to the aggressor, the rank distance

within the dyads was not inﬂuenced by the rank of the aggressor.

DISCUSSION

A substantial proportion of aggressive acts among adult female spotted hyenas
cannot be immediately related to conﬂict over resources such as food or dens, defense of
young, or any other provocative behavior emitted by the recipient prior to the attack.
Nearly one ﬁfth of all aggressive acts emitted by females are unprovoked, which is
second only to ﬁghts over food. Here we tested predictions of two hypotheses suggesting
functional explanations for the occurrence of unprovoked aggression among adult
females. We did not ﬁnd any support for the hypothesis suggesting females were using
unprovoked aggression to suppress the reproduction of subordinates, but our data were
consistent with the hypothesis suggesting that this behavior functions to test existing rank

relationships.

159

Reproductive suppression hypothesis

An important cost associated with group living is the increase in competition for
resources necessary for reproduction (Sterck et al., 1997; van Schaik, 1989). Females
may be able to derive ﬁtness beneﬁts for themselves and their offspring by suppressing
the reproductive efforts of others (Creel and Wasser, 1997; Wasser and Barash, 1983;
Wasser and Starling, 1988). Social harassment is one behavioral mechanism known to
suppress reproduction among conspeciﬁcs in cooperatively breeding species (Creel et al.,
1997; Faulkes and Abbott, 1997; McLeod et al., 1996). In contrast to most other social
carnivores (Moehlman, 1997), spotted hyenas are not cooperative breeders, but they do
experience reproductive skew associated with social rank. As higher-ranking female
spotted hyena out-perforrn lower-ranking females on several measures of fertility (Hofer
and East, 2003; Holekamp et al., 1996), we looked for evidence suggesting that females
might use unprovoked aggression to hinder the reproductiVe efforts of lower-ranking
individuals. However, we found no evidence that females in reproductively vulnerable
states were preferentially receiving unprovoked aggression from dominants. Wasser and
Barash ( 1983) suggested that reproductive physiology is easiest to disrupt early in the
reproductive effort; therefore, the reproduction suppression hypothesis predicted that
females would receive higher rates of aggression early, rather than late in pregnancy, but
rates did not vary among trimesters. Among baboons, cycling females were shown to
receive the highest rates of aggression (W asser and Starling, 1988); however, among
female spotted hyenas rates were not elevated around the time of conception, when
females were presumed to be cycling. As reproductive success is strongly connected to

social rank in this species (Holekamp et al., 1996), and as rank determines priority of

160

access to food (Frank, 1986; Holekamp, et al., 1997b; Kruuk, 1972; Tilson and Hamilton,
1984), energy availability (Bronson, 1989) is likely a sufﬁcient explanation for the lower
reproductive output seen among lower-ranking female hyenas, and additional suppression
of reproduction via unprovoked aggression is unnecessary. Consistent with this idea is
the fact that the study clan experiences an annual peak in conceptions that coincides with
an inﬂux of prey species into the Masai Mara during the annual wildebeest migration
(Holekamp et al., 1999).

Challenging the status quo

As we have discussed, high social rank conveys a reproductive advantage
associated with access to resources in this species; therefore, females should be motivated
to elevate their position in the dominance hierarchy if presented with an opportunity to do
so (Smuts, 1987; Walters and Seyfarth, 1987). We proposed that unprovoked aggression
might serve as a tool for assessing such opportunities among adult females, and examined
the frequency and intensity with which adult females attacked higher-ranking females.
Female dominance relationships within this clan have been extremely stable over many
years (Engh et al., 2000; Frank, 1986; Holekamp et al., 1993), and by 2 years of age, a
female’s position in the dominance hierarchy is well established (Holekamp and Smale,
1993; Smale et al., 1993). Thus, it is perhaps not surprising that we recorded very few
cases of attacks directed up the hierarchy, and these attacks were of relatively low-
intensity. This result is consistent with prior work demonstrating that aggression directed
up the hierarchy occurs infrequently in this species (Chapter 3; Engh et al., 2005),
suggesting that there are severe risks involved in attacking higher-ranking individuals.

The large majority of unprovoked attacks directed up the hierarchy occurred among

161

females close together in rank, suggesting that, if rank reversals within the hierarchy did
occur, they would most likely occur between females ranked close together in the clan’s
hierarchy. Although we did not see an inﬂuence of subgroup composition on the
likelihood of attacking a higher-ranking conspeciﬁc, we believe it would be premature to
conclude that females ignore the subgroup composition when attacking higher-ranking
individuals. The non-signiﬁcant results obtained here may simply reﬂect inadequate

statistical power, and larger sample sizes may be required to conﬁrm or reject this idea.

3. ._ -~.- ‘ '

Rank reversals have been reported among adult female spotted hyenas living in
the wild (East and Hofer, 2002; Henschel and Skinner, 1987; Hofer and East, 2003; 4"
Holekamp etal., 1993; Mills, 1990; Tilson and Hamilton, 1984; White, 2007), but due to
the rarity of these reversals, the factors contributing to them remain unclear. Rank
reversals are also reported anecdotally in the cercopithecine primate literature. Reversals
among female baboons have been associated with the advanced age of the unseated
female (Hausfater, et al., 1982), suggesting that elderly females may not be able to
maintain their dominance over would-be usurpers. Several primate studies report that
rank reversals occur after the loss of a key ﬁgure in the deposed female’s social network
(Chapais, 1985; Ehardt and Bernstein, 1986; Hausfater et al., 1982; Raper, et al., 2006).
These scenarios suggest that even in these societies in which social rank is “inherited,”
and dominance hierarchies are relatively stable, stochastic events may promote
weaknesses in the dominance hierarchy, and a tool for probing for weakness would be

valuable. Unprovoked aggression might function as such a tool.

162

Maintenance of the dominancy hierarchy

Our results suggest that most unprovoked aggression serves the same function
among adult spotted hyena females as it serves in many cercopithecine primate societies
(Silk, 1993; Silk, 2002; Smuts, 1987; Walters and Seyfarth, 1987), which is to maintain
existing dominance relationships. Among adult female spotted hyenas, most unprovoked
aggression occurs downward in the existing dominance hierarchy, and among individuals
close together in rank, suggesting that females are using unprovoked aggression to
reinforce the status quo. Despite the fact that higher-ranking females have a wider range
of targets available to them, when we controlled for opportunity we saw that both high-
and low-ranking females attack conspeciﬁcs that are close to themselves in rank. This
suggests that closely ranked females may pose the biggest threat with respect to rank-
reversals, and that these relationships therefore need the most maintenance and attention
(Forkman and Haskell, 2004).

In the ﬁssion-fusion society of spotted hyenas, lower-ranking females are less
gregarious and spend more time alone than do higher-ranking females (Boydston, et al.,
2003; Holekamp, et al., 1997a; Smith et al., 2008; Smith, et al., 2007), and it is difﬁcult
to assess the frequency with which particularly solitary clan members interact. Holekamp
et al. (1993) describe several events in which females returned to the clan’s home range
after small absences of one to three months with little or no penalty; however, clan
members that returned after absences of six months or more experienced a drop in rank
and elevated rates of aggression. Therefore, mechanisms for maintaining the dominance

hierarchy might be particularly critical in this dynamic social system, and hierarchy

163

maintenance appears to be a life-long necessity (Silk, 1993; Silk, 2002; Smuts, 1987;
Walters and Seyfarth, 1987).

In addition to understanding how the relationship between the aggressor and the
recipient inﬂuences unprovoked aggression, we were also interested in how the
immediate social environment might affect the likelihood of attack. Only 2% of
unprovoked attacks down the hierarchy occurred when the interacting dyad was alone,

which suggests that the audience of an unprovoked attack may be nearly as important as

the recipient of the attack (Zajonc, 1965; Zuberbuhler, 2008). An example of this type of

I-ALnx

audience effect was demonstrated in a group of wild chimpanzees (Pan troglodytes).
Victims of aggression enhanced their recruitment screams when in the presence of an
individual higher-ranking than their attacker, as intervention by a third party generally
occurs in support of the victim among chimpanzees (Slocombe and Zuberbuhler, 2007).
However, among spotted hyenas, females tend to intervene on behalf of the higher-
ranking female (Engh et al., 2005; Smith, et al., 2007) indicating that higher-ranking
bystanders usually represent allies for the attacker rather than the recipient. We also
found that females are more likely to attack down the hierarchy when the presence of
lower-ranking females, other than the recipient, is low, suggesting that lower-ranking
bystanders might be potential allies for the attacked female. The positive relationship
between the probability of attack and the number of higher-ranking bystanders seen here
in dyadic aggression is opposite to that seen during coalitionary aggression, in which two
or more hyenas attack a single target animal. In coalitionary aggression the likelihood of
a female joining an on-going dyadic ﬁght decreases as the number of higher-ranking

bystanders increases (Smith et al., 2009). It may be that higher-ranking bystanders

164

perceive dyadic aggression toward lower-ranking females as situations in which
intervention is unnecessary, whereas coalitionary aggression might represent escalated
aggression in which beneﬁts accrue to higher-ranking females that intervene to “police”
or terminate the aggressive interaction (Slocombe and Zuberbuhler, 2007).

Silk (2002) suggests that recipients of unprovoked aggression cannot predict
when attacks will occur, how long they will last, or when the threat has ended. This
creates continuous uncertainty among subordinate individuals, and this uncertainty may
represent a form of social stress. Social stressors may be potent stimulators of the
hypothalamic-pituitary-adrenal (HPA) axis, often measured as an increase in circulating
glucocorticoids (GC) (Sapolsky, 1982; Sapolsky, 2002; Sapolsky, 2005). Acute
activation of the HPA axis, and short term release of GC concentrations from the adrenals
is an adaptive part of the “ﬁght or ﬂight” system; however, chronic elevation of GC
concentrations can lead to pathology, including compromise of cardiovascular, immune,
neural, and reproductive function (Sapolsky, 2002). If the hypothesis that unprovoked
aggression negatively inﬂuences the stress physiology of recipients is true (Silk, 2002),
we might predict that subordinates would have higher GC concentrations than would
dominant individuals. While there does not appear to be a strong relationship between
social rank and individual variation in GC concentrations among female hyenas, a study
has demonstrated that fecal GC concentrations were elevated among females after a
serious ﬁght (Goymann, et al., 2001). Although these authors did not indicate who won
these conﬂicts, it seems reasonable to assume that recipients of unprovoked aggression

might experience acute activation of the HPA access. To conclude, we ﬁnd that

165

unprovoked aggression is a useful means for testing the stability of social relationships,

with minimal risk for the attacker, and a potentially large cost to the recipient.

166

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