Investigating the Functions of Rough-and-Tumble Play in American Mink, Neovison vison
by
Jamie Ahloy Dallaire
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Doctor of Science
in
Animal and Poultry Science
Guelph, Ontario, Canada
© Jamie Ahloy Dallaire, November, 2015
ABSTRACT
INVESTIGATING THE FUNCTIONS OF ROUGH-AND-TUMBLE PLAY IN AMERICAN MINK,
NEOVISON VISON
Jamie Ahloy Dallaire
Advisor:
University of Guelph, 2015
Professor Georgia Mason
Play is very widespread among young mammals, yet its functions remain
mysterious. Ethologists have largely relied on social deprivation experiments or
correlational studies to determine how play might promote survival or reproduction.
Using American mink (Neovison vison) as a model, I aimed to develop an alternative
experimental approach, not relying on deprivation, to test two hypotheses about the
functions or long-term benefits of rough-and-tumble play: that it prepares animals to
cope emotionally with unexpected, frightening situations, and that it prepares them for
adult sexual behaviour. I aimed to do this using longitudinal studies of mink raised in
multiple different housing treatments that independently modulate the frequency of
rough-and-tumble play. If treatments have convergent effects on juvenile play and adult
outcomes, then we may confidently ascribe these outcomes to differential juvenile play,
rather than to other confounding effects of treatments on juveniles.
In Chapter 2, in preliminary research into the basics of mink play, I showed that
rough-and-tumble play is subject to modest litter effects, only somewhat stable over the
juvenile period, and motivationally distinct from object play in mink. In Chapter 3, in
research conducted over three summers, I tested a wide variety of experimental housing
treatments but found none that had consistent effects on rough-and-tumble play
frequency. In subsequent chapters, I was therefore limited to correlational, and not
experimental, hypothesis tests. In Chapter 4, I found no evidence for a relationship
between juvenile rough-and-tumble play and adult fearfulness. The most playful juveniles
were no less likely to react fearfully to an experimenter’s approach or to scream when
handled, and did not have smaller increases in corticosteroid hormone production when
restricted in a carrying cage. In Chapter 5, I found that the most playful juvenile males
copulated for longer durations in adulthood, but that the most playful females were
slower or less likely to begin copulating with males. To my knowledge, these are the first
demonstrations that juvenile rough-and-tumble play predicts adult sexual behaviour in
any species. Further refinement of experimental methods will be required to understand
whether and how rough-and-tumble play affects the sexual development of juvenile mink.
For James Leo
iv
Acknowledgments
First and foremost, I would like to thank my family for supporting me not only during
these doctoral studies, but in everything that has led up to this. My parents Danielle and Jean
and my brother Jason have been nothing but encouraging throughout. My love Liana has been
my number one friend, psychologist, cheerleader, sounding board, and an inexhaustible source
of comfort and energy. Thanks to you all.
Major thanks to my advisor, Georgia Mason, for giving me her trust when I first asked, in
the vaguest of terms, if I could stay on in the lab after my Master’s “to do a Ph.D. on play”.
Taking on an ambitious self-directed project has been a truly valuable learning experience, and
Georgia’s keen and critical eye was constantly there to help refine my methods and keep me
focused and sensible. After just over seven years there, I will really miss being a part of her lab,
but look forward to collaborating for years to come.
Many colleagues have come and gone during my tenure in the Mason lab, and all
contributed to the development of the ideas in this thesis. In particular, thanks to the mink team
with whom I directly collaborated on this and related projects: Maria Díez-León, Rebecca
Meagher, Misha Buob, Dana Campbell, and Lauren Dawson. A large number of other
colleagues and students provided assistance with data collection and/or processing: Michael
Walker, Kaitlin Bahlmann, Sarah Bowyer, Amanda Saldivia-Woo, Cammie Punniamoorthy,
Carla Wsiaki, James Templeman, Noah Nimelman, Siobhan Speiran, Kathryn Reynolds,
Spencer Woodley, and Liana Ahloy Dallaire.
Thanks are also due to Lee Niel and Derek Haley for sitting on my advisory committee
and helping guide me at all stages of the project; to Lee and Derek along with Alexandra
Harlander and Pat Barclay for their involvement in my qualifying exams; and to Lee, Georgia,
Andrew McAdam, and Sergio Pellis for reading this and for serving on my examining committee.
v
Thanks also to Andy Robinson and Trevor DeVries, respectively, for chairing my qualifying
exams and dissertation defense, and to Wendy McGrattan for making these events happen.
All of the work detailed in this thesis would, of course, have been impossible without the
mink farmers themselves, who were constantly gracious and welcoming. By their combined
decades of experience and knowledge, and by their curious and helpful nature, they made the
project much better than it would otherwise have been. At Millbank Fur Farm, many thanks to
Ross, Ted, Elaine, Cassie, and Aaron Parkinson, to Dean and Jenny Broadfoot, and to Joey
Molto and the rest of the gang. At RBR Fur Farm, thanks to Jim, Kirk, Curtis, Jamie, Steve, and
Daniel Rankin and the entire team. Thanks also to Angelo Napolitano and Andrew CohenBarnhouse at Michigan State University’s Experimental Fur Farm for helping with what turned
out to be a short-lived pilot study that did not quite make it into the thesis.
Many thanks also to Laura Arrindell for hosting me in St. Marys whenever I went out
there to work at RBR.
Thanks are also due to the subjects themselves, to the thousands of mink I watched,
handled, measured, carried, or scared deliberately or accidentally.
Thanks also to all the dogs! To Tilly and Gus and Jake for being awesome; to Brody for
never getting around to eating me after all; and to my poor little Basil for enduring those long
days on the farm last summer. I know he hated every second.
Finally, I would like to thank those who funded both my experiments and my continued
existence as a human being during my studies: the Natural Sciences and Engineering Research
Council, the government of Ontario, the Universities Federation for Animal Welfare, the Keyes
Family (via their Ontario Agricultural College scholarship), and the Canada Mink Breeders
Association.
vi
Table of Contents
Acknowledgments __________________________________________________________ iv
Table of Contents___________________________________________________________ vii
List of Tables ______________________________________________________________ xii
List of Figures ____________________________________________________________ xiii
Chapter 1 – Investigating the Nature of Animal Play _______________________________ 1
Defining animal play _________________________________________________________ 1
Evidence for functions of animal play ____________________________________________ 6
Hypotheses about the functions of play _________________________________________ 11
Project overview ___________________________________________________________ 16
Chapter 2 – Characterizing Play in Juvenile Mink ________________________________ 18
Abstract _________________________________________________________________ 18
Introduction_______________________________________________________________ 19
Ontogeny and husbandry of young mink ______________________________________ 19
Play in mink and related species ____________________________________________ 20
Definitions and classifications _____________________________________________ 20
Development of play ____________________________________________________ 23
Causes of variation in play _______________________________________________ 25
Consequences of play deprivation _________________________________________ 29
Unknowns in mink play ____________________________________________________ 31
Project overview _________________________________________________________ 35
Methods _________________________________________________________________ 36
Subjects and housing _____________________________________________________ 36
Behavioural observations __________________________________________________ 38
Data analysis ___________________________________________________________ 39
Results __________________________________________________________________ 41
Descriptive analyses – total play ____________________________________________ 41
Descriptive analyses – subtypes of play _______________________________________ 46
Hypothesis tests _________________________________________________________ 47
Discussion _______________________________________________________________ 53
vii
Chapter 3 – Experimentally Manipulating Play in Juvenile Mink ____________________ 59
Abstract _________________________________________________________________ 59
Introduction_______________________________________________________________ 60
Manipulating the subject itself _______________________________________________ 60
Manipulating the subject’s social environment __________________________________ 65
Manipulating the subject’s physical environment ________________________________ 71
Project overview _________________________________________________________ 74
General Methods __________________________________________________________ 75
Subjects and housing _____________________________________________________ 75
Behavioural observations __________________________________________________ 76
Data analysis ___________________________________________________________ 77
Experiment 1a ____________________________________________________________ 79
Methods _______________________________________________________________ 79
Subjects and housing ___________________________________________________ 79
Behavioural observations ________________________________________________ 82
Data analysis __________________________________________________________ 83
Results ________________________________________________________________ 83
Experiment 1b ____________________________________________________________ 84
Methods _______________________________________________________________ 85
Subjects and housing ___________________________________________________ 85
Behavioural observations ________________________________________________ 88
Data analysis __________________________________________________________ 88
Results ________________________________________________________________ 89
Experiment 1c ____________________________________________________________ 91
Methods _______________________________________________________________ 92
Subjects and Housing ___________________________________________________ 92
Behavioural Observations ________________________________________________ 93
Data Analysis _________________________________________________________ 93
Results ________________________________________________________________ 93
Discussion – Experiment 1 ___________________________________________________ 94
Experiment 2a ___________________________________________________________ 101
Methods ______________________________________________________________ 101
Subjects and housing __________________________________________________ 101
Behavioural observations _______________________________________________ 101
viii
Data analysis _________________________________________________________ 102
Results _______________________________________________________________ 102
Experiment 2b ___________________________________________________________ 103
Methods ______________________________________________________________ 104
Subjects and housing __________________________________________________ 104
Behavioural observations _______________________________________________ 105
Data analysis _________________________________________________________ 106
Results _______________________________________________________________ 106
Experiment 2c ___________________________________________________________ 107
Methods ______________________________________________________________ 108
Subjects and housing __________________________________________________ 108
Behavioural observations _______________________________________________ 109
Data analysis _________________________________________________________ 109
Results _______________________________________________________________ 110
Discussion – Experiment 2 __________________________________________________ 111
Exploratory Analyses ______________________________________________________ 113
Experiment 3a ___________________________________________________________ 122
Methods ______________________________________________________________ 122
Subjects and housing __________________________________________________ 122
Behavioural observations _______________________________________________ 122
Data analysis _________________________________________________________ 123
Results _______________________________________________________________ 124
Experiment 3b ___________________________________________________________ 126
Methods ______________________________________________________________ 126
Subjects and housing __________________________________________________ 126
Behavioural observations _______________________________________________ 128
Data analysis _________________________________________________________ 129
Results _______________________________________________________________ 129
Experiment 3c ___________________________________________________________ 131
Methods ______________________________________________________________ 131
Subjects and housing __________________________________________________ 131
Behavioural observations _______________________________________________ 131
Data analysis _________________________________________________________ 132
Results _______________________________________________________________ 133
Discussion – Experiment 3 __________________________________________________ 134
ix
General Discussion _______________________________________________________ 137
Chapter 4 – Decreasing Fearfulness through Play _______________________________ 140
Abstract ________________________________________________________________ 140
Introduction______________________________________________________________ 140
Project overview ________________________________________________________ 146
Methods ________________________________________________________________ 147
Subjects and housing ____________________________________________________ 147
Stick and glove tests _____________________________________________________ 148
Handling test ___________________________________________________________ 150
Carrying cage test _______________________________________________________ 151
Data analysis __________________________________________________________ 154
Results _________________________________________________________________ 156
Test consistency and construct validity ______________________________________ 156
Convergent validity ______________________________________________________ 159
Rough-and-tumble play and fearfulness ______________________________________ 160
Play, fearfulness, and alert behaviour _______________________________________ 161
Discussion ______________________________________________________________ 162
Chapter 5 – Increasing Sexual Prowess through Play ____________________________ 165
Abstract ________________________________________________________________ 165
Introduction______________________________________________________________ 165
Male sexual behaviour, social isolation, and rough-and-tumble play ________________ 166
Female sexual behaviour, social isolation, and rough-and-tumble play ______________ 171
Causality in social isolation experiments _____________________________________ 174
Project overview ________________________________________________________ 175
Methods ________________________________________________________________ 177
Subjects and housing ____________________________________________________ 177
Farm mating regimen ____________________________________________________ 180
Behavioural observations _________________________________________________ 181
Data analysis __________________________________________________________ 184
Results _________________________________________________________________ 188
Male sexual behaviour ___________________________________________________ 188
Female sexual behaviour _________________________________________________ 193
Discussion ______________________________________________________________ 196
x
Chapter 6 – Conclusion _____________________________________________________ 203
General project summary ___________________________________________________ 203
The state of play in ethology and psychology ___________________________________ 205
Bibliography ______________________________________________________________ 209
Appendix _________________________________________________________________ 238
xi
List of Tables
Table 2–1: Ethogram of recorded behaviours. ______________________________________ 37
Table 3–1: Treatment effects on rough-and-tumble play in Millbank litters (Experiment 1a). __ 83
Table 3–2: Treatment effects on rough-and-tumble play in Millbank and RBR pairs (Experiments
1b and 1c). _________________________________________________________________ 89
Table 3–3: Treatment effects on rough-and-tumble play in Millbank litters and in Millbank and
RBR pairs (Experiments 2a, 2b, and 2c). ________________________________________ 103
Table 4–1: Contingency table of responses of individual mink on the first and second days of
glove or stick testing, summed across experiments and sexes. _______________________ 157
Table 5–1: Number of breeding males observed from each juvenile housing treatment described
in Chapters 2 and 3._________________________________________________________ 179
Table 5–2: Effects of juvenile rough-and-tumble play on adult male sexual outcome measures in
farm-wide survival analysis models. ____________________________________________ 189
Table 5–3: Effects of including male attributes as covariates on the relationships between
rough-and-tumble play and copulation duration in RBR (both experiments) and Millbank (2012)
males. ___________________________________________________________________ 192
Table 5–4: Effects of single-housing during the pair phase on adult male sexual outcome
measures, tested across both farms. ____________________________________________ 193
Table 5–5: Effects of juvenile rough-and-tumble play and of single-housing during the pair
phase on adult female sexual outcome measures at RBR Fur Farm. ___________________ 194
xii
List of Figures
Figure 2–1: Juvenile mink time budgets by housing condition and time period. ____________ 40
Figure 2–2: Juvenile mink time budgets by time of day. ______________________________ 43
Figure 2–3: Correlation between frequency of mutual rough-and-tumble play in enriched and
non-enriched pairs from the same litter. __________________________________________ 48
Figure 2–4: Correlation between frequencies of play in periods one (July) and two
(August/September). _________________________________________________________ 49
Figure 2–5: Correlation between frequency of mutual R&T and solitary object play in enriched
pairs. _____________________________________________________________________ 52
Figure 3–1: Satellite image of Millbank Fur Farm. __________________________________ 114
Figure 3–2: Diagram of a typical mink shed at Millbank Fur Farm. _____________________ 115
Figure 3–3: The inside of a Millbank Fur Farm shed late on a sunny afternoon. ___________ 116
Figure 3–4: Mutual rough-and-tumble in relation to peak daily temperature.______________ 121
Figure 4–1: Fecal corticosteroid metabolites by collection period over two days at RBR Fur
Farm. ____________________________________________________________________ 159
Figure 5–1: Survivorship curves comparing copulation duration in males according to their levels
of juvenile rough-and-tumble play. ______________________________________________ 190
Figure 5–2: Survivorship curves comparing latency to copulate in females according to their
levels of juvenile rough-and-tumble play. _________________________________________ 195
xiii
Chapter 1 – Investigating the Nature of Animal Play
Play has long puzzled students of behaviour. More or less universal among human
children, it is also very widespread in young mammals, and occurs in many vertebrates and
even some invertebrates (Burghardt, 2014; Fagen, 1981). Members of these various species
can undeniably look like they are having fun (Bekoff, 1972). However, defining play in a way that
capture’s the phenomenon’s breadth – children turning somersaults, young rats wrestling, adult
dogs chasing each other, and monkeys gleefully destroying household objects – has proven to
be difficult.
Equally mysterious is the question of why play arose and has been maintained by
evolution. Despite its extensive phylogenetic distribution, empirical evidence for functional
explanations of the behaviour is scarce. Indeed, many definitions of play explicitly focus on its
apparent lack of purpose (Fagen, 1981; Smith, 1982). The absence of any obvious function for
play, combined with the peculiar difficulties involved in testing hypotheses about its effects, has
curbed progress in the field.
This literature review concerns the challenges of defining play and explaining its
occurrence from a functional perspective. In the first section, I will survey existing definitions.
Then, I will discuss the types of evidence previously used to support or falsify functional
hypotheses about play. Next, I will summarize and evaluate existing hypotheses in the literature.
Finally, I will give an overview of my approach to testing such hypotheses in my doctoral
research project.
Defining animal play
A common sense definition is that play is any behaviour that is fun for its performer.
Several authors define play in such phenomenological terms (Blanche, 2002; Scarlett et al.,
1
2005; Suits, 1978). While I believe that this is the gold standard distinguishing play from nonplay, it is not easily operationalized. Tellingly, these are definitions by psychologists and
philosophers reflecting on play in humans, not animals. Perhaps more tractable for animal
research is a formulation that does not refer to private experience: play is engaged in because
“the activity, rather than the outcome, is reinforcing” (Bernstein, 1982). Play has been shown to
be rewarding, at least when it comes to rough-and-tumble play (play fighting) in laboratory
Norway rats (Rattus norvegicus) (reviewed in Trezza et al., 2011; Vanderschuren, 2010). This
has been demonstrated using conditioned place preference (Burgdorf et al., 2008; Calcagnetti
and Schechter, 1992) and operant maze-learning (Humphreys and Einon, 1981; Normansell
and Panksepp, 1990). Further, playing rats emit ultrasonic vocalizations like those produced
across a range of positive affective contexts (Burgdorf et al., 2008; B. T. Himmler et al., 2014).
Young common chimpanzees (Pan troglodytes) given the choice to pull a lever for rough play or
for other social interactions, like petting or grooming, all with a human experimenter, also most
often chose play (Mason et al., 1962). This exception aside, few experiments have been
performed on the rewarding nature of play outside of rat play fighting, and none distinguish
between reinforcement by the activity and by its outcome. Furthermore, showing that an activity
is rewarding does not show that it is specifically fun (North, 2015).
Speculating on the nature of the mental states associated with play, then, Bateson and
Martin (2013) have suggested a distinction between play – defined largely according to
morphological and situational criteria – and ‘playful play’, further accompanied by a mental state
that predisposes individuals to behave spontaneously and flexibly. Similarly, Špinka and
colleagues (2001) proposed that fun is a combination of pleasure, excitement, and relaxation:
though one may play hard or compete intensely, the outcome (e.g. winning or losing) is not of
vital importance. The subjective experience of play may also be akin to ‘flow’, a positive mental
state in which full attention is devoted to an optimally-challenging task, a “merging of action and
2
awareness”, reported by humans engaged in enjoyable, skilled activities (Nakamura and
Csikszentmihalyi, 2002; Sutton-Smith, 1997). These suggestions are intriguing, but still not
easily verified or operationalized, especially in animals. Ethological definitions therefore for the
most part substitute or add morphological criteria, identifying play by its form in addition to or
instead of its consequences and associated mental states (reviewed in Burghardt, 2005).
Fagen (1981) surveyed the literature and identified the attributes widely accepted as
characteristic of play. These are that play is similar in form to certain “functional” (non-play)
juvenile or adult behaviour patterns but lacks their consummatory consequences; that it may
combine actions from different functional categories; and that these actions are exaggerated
and repeated more often within a bout than in functional equivalents. Most authors also agreed
that the sequence of actions in play is more variable or unpredictable than in functional
behaviour. A definition constructed from these attributes, however, would disqualify many types
of behaviour otherwise regarded as play. For example, play fighting and genuine fighting look
very similar in domestic piglets (Sus scrofa domesticus), with little agreement on what
differences in form might exist (Hohenshell et al., 2000; Šilerová et al., 2010). Play outcomes
may also matter to animals: young monkeys preferentially initiated play fights with smaller
individuals whom they were likely to defeat (Thompson, 1998). No single criterion is satisfied by
all instances across categories of play and species.
Recognizing this, some authors have instead proposed defining play in terms of “family
resemblance” (Burghardt, 2005; Caillois, 1967; Scarlett et al., 2005). In this conception, all
instances of play are united by an overlapping set of features, but no single feature is necessary
on its own. Perhaps it is no accident that philosopher Ludwig Wittgenstein (1967) used the
example of defining games when he popularized the concept of ‘family resemblance’. While
many features are shared by many or most games, no feature applies to all games: for
example, chess involves little physical activity; solitaire is played alone; professional sports can
3
be more serious than amusing; snakes and ladders requires no skill; “Ring around the Rosey”
cannot be won or lost; and throwing a ball against a wall may have no rules to speak of. With
this in mind, Burghardt’s (2005) definition lists five criteria that must all apply for an action to
qualify as play, but each of these criteria can be satisfied in more than one way. Thus, particular
instances of play may not share any specific features. In condensed form, the criteria are that
play 1) includes elements that do not contribute to current survival or reproduction, 2) is
voluntary, reinforcing, or done for its own sake, 3) differs from “serious” behaviour in form or
timing, 4) is performed repeatedly but not stereotypically, distinguishing it from exploration, and
5) takes place in a low-stress, low-threat environment. Criterion 3, for example, may be satisfied
by exaggerated movements, by self-handicapping movements (e.g. voluntarily adopting a
disadvantageous position in a play fight), by performance at the “wrong” life stage (e.g. sexual
behaviour in juveniles), by the inclusion of specific signals that do not occur in “serious”
behaviour (e.g. open-mouth play face or play bow, not seen in aggressive encounters), or by
appetitive behaviours without the normally associated consummatory actions. Thus, each of
these attributes is sufficient, but not individually necessary, to satisfy Criterion 3.
Another definitional issue concerns the classification of play types. Traditionally, animal
play has been divided into three broad categories: locomotor (or “locomotor-rotational”) play,
play with objects, and social play, including play fighting and chasing (Fagen, 1981; Smith,
1982). Specific examples of play often blur the lines between these categories: especially in
animals, much play with objects or with social partners has a locomotor aspect (e.g. pouncing
on toys, chasing conspecifics), and social play can incorporate objects (e.g. tug-of-war) (Barrett
and Bateson, 1978; Thompson, 1998). All three types can be combined, for example in
Japanese macaques (Macaca fuscata) chasing each other while competing for possession of
an object (Shimada, 2006). Classifications of play in humans (Homo sapiens sapiens) typically
include more categories, such as construction play, language play, imitation play, games of
4
chance, and pretend play, which are either difficult to detect or may not exist in animals
(Caillois, 1967; Gómez and Martín-Andrade, 2005; Sutton-Smith, 1997). Other classifications of
animal play have proposed a hierarchy of complexity in play types, generally progressing from
locomotor to social play, and in some cases to social play involving pretense (reviewed in
Burghardt, 2005). Most general definitions, like Burghardt’s, aim to encompass all categories of
play.
Burghardt’s broad definition is the most satisfactory to date, but requires information that
can be difficult to verify, and is likely still too narrow to include all of play (Bateson and Martin,
2013). The first and third criteria exclude any behaviour with an obvious consummatory
outcome, but psychologists and anthropologists often consider that play and work are not
mutually exclusive (Caillois, 1967; Cohen, 1993; Huizinga, 1950; Suits, 1978; Sutton-Smith,
1997). Ethologists have also argued for this, though it is rarely reflected in definitions (Bekoff,
1982; Berman, 1982; Burghardt, 1982). Behaviour like hunting or courtship could be fun and
clearly functional (Emery and Clayton, 2015). The fourth criterion excludes stereotypic
behaviour, yet certain stereotypies could conceivably be an abnormal form of play for animals in
barren cages. Finally, the fifth criterion pre-supposes that play only occurs when basic needs
are met, in the absence of threats or intense competing motivations (e.g. hunger). While this
appears to be largely true, there are many counter-examples (reviewed in Held and Špinka,
2011). For example, early weaning unexpectedly increases object play, and possibly some
forms of social play, in domestic kittens (Felis silvestris catus), as does food rationing of the
mother and litter (Barrett and Bateson, 1978; Bateson and Young, 1981; Bateson et al., 1990). If
some types of play serve a preparatory function or are otherwise particularly beneficial in harsh
environments, they may violate this criterion. Burghardt likely uses such a conservative
definition because his aim is to identify the phylogenetic limits of play. Only a definition that can
regroup very disparate cases, yet exclude obviously questionable examples, can convincingly
5
be used to recognize play in the European paper wasp (Polistes dominulus) (Dapporto et al.,
2006), the African softshell turtle (Trionyx triunguis) (Burghardt et al., 1996), a spider
(Anelosimus studiosus) (Pruitt et al., 2012), and the common octopus (Octopus vulgaris) (Kuba
et al., 2006). Burghardt’s definition, then, is excellent for identifying clear instances of play
across species. I will be using a slightly modified version of Burghardt’s criteria to establish my
operational definition of play in mink, as detailed in Chapter 2.
Evidence for functions of animal play
The bulk of the ethological literature on play has sought to identify its ultimate,
evolutionary causes (reviewed in Graham and Burghardt, 2010; Martin and Caro, 1985; Pellis
and Pellis, 2009; Sutton-Smith, 1997). The typical assumption is that play must have positive
effects on fitness, as these must outweigh its costs (Fagen, 1977). Play is sensitive to energy
intake, and consumes energy at the expense of growth (Miller and Byers, 1991; MullerSchwarze et al., 1982; Sharpe et al., 2002). It may also increase the risk of predation: juvenile
South American fur seals (Arctocephalus australis), for example, are less vigilant and more
likely to be taken by sea lions while playing (Harcourt, 1991). Others, however, have found
measured fitness costs to be relatively minimal (Caro, 1995; Martin and Caro, 1985) or
suggested that play is an ‘opportunity behaviour’, performed only when costs are low (Barber,
1991; Fraser and Duncan, 1998). While some hold that play could simply be a non-adaptive byproduct of selection for other traits (Alford, 1982), the vast majority of hypotheses do hold that
play promotes fitness. Because play is typical of young animals, and because its benefits are
not immediately obvious, these benefits are largely thought to be developmental in nature: a
trade-off between short-term costs, reducing survival in juveniles, and long-term benefits,
increasing later survival and/or reproduction in adulthood. However, increasing attention has
been paid to more easily measurable short-term benefits, with direct positive impacts on juvenile
6
survival. Before surveying existing hypotheses about the functions of play, in the next section, I
will first discuss the strengths and weaknesses of the types of evidence presented for them.
Evidence for functions of play can be divided into three categories (Martin and Caro,
1985; Smith, 1982). Correlational evidence is obtained by showing that an attribute of play (e.g.
its frequency) is predictive of a later outcome measure, across individuals. Experimental
evidence is obtained by showing that manipulating some aspect of play results in subsequent
differences between treated animals and controls. Finally, evidence from design is obtained by
showing that previously unknown features of play are consistent with predictions made on the
basis of hypothesized functions. I will now consider the difficulties involved in each approach.
Because functional hypotheses about play are about consequences – about whether
play has a certain effect – correlational evidence is necessarily weaker than experimental
evidence. Outcomes could easily be confounded with other, non-measured variables. For
example, Blumstein et al. (2013) interpreted a correlation – that young yellow-bellied marmots
(Marmota flaviventris) who won the most play fights became dominant adults, as ranked by the
outcome of agonistic interactions – as evidence for the hypothesis that play fights help establish
adult dominance ranks. An alternative explanation, however, is that fighting ability is stable over
the marmot’s lifetime, and determines success both in play fights and in adult agonistic
interactions. While the authors did control for body weight, some other unmeasured determinant
(e.g. strength or reflex rapidity) could explain their results. Correlational evidence may be more
appropriate for falsifying functional hypotheses rather than for supporting them. For example,
Vincent and Bekoff (1978) interpreted the lack of a relationship between juvenile play frequency
and later mouse-killing ability as evidence against the hypothesis that play is practice for
predation in coyotes (Canis latrans). This type of falsifying evidence is more convincing, but not
wholly so. First, confounding explanations are still possible, though usually more far-fetched
than for positive correlations. Second, especially if play is costly, it could be that animals only
7
play as much as they need to reach a certain threshold of ability (see “self-assessment” below:
Thompson, 1998). It might then be wrong to expect a correlation between the amount of play
and trained adult behaviour. Where possible, it is therefore better to test for correlation between
play and improvement in an outcome measure, rather than between play and a final measure
(e.g. Barnett, 1990; Lillard et al., 2013; Nunes et al., 2004). Though still inherently limited, the
best correlational studies take all plausible confounds into account – Fagen and Fagen (2004),
for example, found that juvenile brown bear (Ursus arctos) play frequency predicts later survival,
even controlling for body condition, maternal behaviour, and local salmon availability.
Experimental studies of play have the potential to provide convincing evidence for or
against hypotheses about benefits of play, but are extremely difficult in practice. This is
especially true when evaluating hypotheses about long-term benefits, because the experimental
treatments used to manipulate play (reviewed in depth in Chapter 3) must impose their effect
over a substantial portion of the juvenile period, enough to result in lifelong differences in play
experience. These treatments therefore almost inevitably introduce confounds of their own. The
most frequently-used paradigm – social play deprivation – has often been implemented via
complete social isolation (Chivers and Einon, 1982; Einon et al., 1975; Pellis and Pellis, 2009;
van den Berg et al., 1999). Isolates are deprived of all forms of social contact, not just play, so
play deprivation does not necessarily explain any deficits they develop. This paradigm,
however, can convincingly show that play is not crucial to the development of a certain faculty, if
it appears to be unimpaired in isolates. The best play deprivation research addresses the issue
of confounds by using treatments intermediate between pure isolation and social housing with
playmates. For example, subjects are deprived of play by being housed with non-playful adults
(Bell et al., 2010; Himmler et al., 2015), or across a barrier from other juveniles which they can
see, smell, hear, and touch but not play with (Hole, 1991; Holloway and Suter, 2004). Isolates
with a running wheel can be physically active without social play (Holloway and Suter, 2004).
8
Partially isolated rats given one hour of daily contact with another juvenile spend the majority of
that hour in play rather than in other social behaviour, playing nearly as much as non-isolates do
in an entire day (Einon et al., 1978). Partial isolates can thus, with relatively minimal confounds,
be compared to full isolates, or to partial isolates whose partners are rendered non-playful using
drugs (Einon et al., 1978). While these are significant improvements on full social isolation
paradigms, they are still rather blunt manipulations that do not affect play alone.
Given the aforementioned problems with correlational and experimental studies – and
because simply demonstrating that playing provides a certain benefit to an animal in the present
does not necessarily imply that this is the evolutionary function for which the behaviour was
initially selected (Buss et al., 1998; Gould and Vrba, 1982) – evidence from design has proven
to be particularly popular among play researchers. This type of evidence focuses on the form or
timing of play, rather than on its correlates or consequences. The “design features” approach
generates predictions that follow the form: if play evolved to have effect X, then it should
possess feature Y (Buss et al., 1998; Symons, 1978). For example, Byers and Walker (1995)
predict that if play in cats, rats, and laboratory mice (Mus musculus) promotes particular types of
long-term physical and cerebral development, then age-related peaks in play should fall within
the critical periods for these. Hypotheses about the functions of play have been tested using
such design-based predictions about the occurrence of particular movements in play (Pellis and
Pellis, 2009; Špinka et al., 2001), about social contexts that elicit play (Palagi et al., 2004), and
about preferred play objects (Negro et al., 1996), partners (Byers, 1980), and surfaces (Byers,
1977), among others. Evidence from design can be used to demonstrate that the form of play is
incompatible with a certain functional hypothesis, but can only provide much weaker evidence
for a hypothesis (Barber, 1991). Design-based predictions, in some cases, can seem circular,
with already-known or strongly suspected features of the behaviour actually having inspired the
hypothesis they are meant to provide support for (Thornhill, 1997). Thus, evidence from design
9
can be weak except where it uses a truly novel feature of play to falsify a functional hypothesis.
Otherwise, it may be more appropriate for hypothesis generation than for hypothesis testing.
Overall, the type of study that can most convincingly demonstrate that play does or does
not confer certain benefits on animals is the experimental one. Especially by combining it with
corroborating evidence from design-based and even comparative approaches, one could build a
very strong case for or against hypotheses about the evolutionary functions of play. To achieve
this, ideal experimental treatments would either stimulate or depress levels of play, thus
introducing much less severe confounds than play deprivation, which eliminates the behaviour
altogether. Enough is known about the social and environmental factors affecting play in various
species – reviewed in Chapter 3 – to attempt this approach (Barrett and Bateson, 1978;
Donaldson et al., 2002; Pellis and McKenna, 1992; Vinke et al., 2005), but this has generally not
been done in experiments that manipulate play over the length of the juvenile period, as
required for tests of long-term outcomes. In addition to using such less heavily confounded
experimental treatments, one should seek to use multiple treatments that influence play, but
have different effects on non-play behaviour. For example, in testing whether rats would learn
an operant rewarded by social play, Humphreys and Einon (1981) decreased the playfulness of
‘non-play’ stimulus rats using either physical restraint or one of two different drugs, selected for
their opposite effects on non-play social behaviour. If an experimental outcome is common to all
of these treatments, this provides convergent evidence that it can be attributed to play, rather
than to confounding factors. One danger is that treatments that increase or decrease play,
rather than eliminating it, might not generate large, detectable differences in outcome measures.
For example, ceiling effects are possible if only a minimal amount of play is required for
developmental change in outcome variables (Martin and Caro, 1985). Knowing this risk of false
negatives, however, convergent results from multiple non-heavily-confounded experimental
10
treatments – including some that increase or decrease play rather than eliminate it – would
provide the most convincing evidence for functional hypotheses about play.
Hypotheses about the functions of play
I now turn to discussing the breadth of hypotheses about the functions of play in the
literature, with a brief evaluation of the rationale and existing evidence for them, where
available. These hypotheses fall into three categories: that play is non-adaptive, that it has
short-term benefits, or that it has long-term benefits.
The classic non-adaptive explanation for the evolution of play is known as “surplus
energy theory” (Schiller, 1967 (original 1795); Spencer, 1872, cited in Burghardt, 2005; Špinka
et al., 2001). Higher animals such as mammals, as a result of their superior nutrition, were
thought to have more energy and time than necessary for survival. Built-up energy, when there
were no appropriate outlets for it (e.g. no current need to hunt), was then released through
frivolous lively activity, or play. While this theory has been abandoned, its assumptions do
accord with common observations that play is most frequent when animals need not devote
themselves to activities necessary for survival: in young and/or captive animals, who do not
forage for themselves, and in those not under threat from predation. Surplus energy theory may
thus have identified some of the conditions favourable for the initial emergence and expression
of play. Once play entered a species’ behavioural repertoire, natural selection could of course
co-opt it, modifying its function and form through a process of exaptation (Buss et al., 1998;
Gould and Vrba, 1982). This process is at the heart of Burghardt’s (2005) “surplus resource
theory”, which posits that play emerged at multiple points in evolutionary history, and tends to
persist at high rates, in species that meet physiological and ecological prerequisites. Recent
findings have bolstered this theory: primates have been found to expend remarkably low
amounts of energy on a daily basis compared to other mammals (Pontzer et al., 2014); their
high potential for surplus may explain why they are among the most playful of mammals
11
(Burghardt, 2014). Having emerged in this way, play could then be co-opted to serve potentially
quite different short-term or long-term functions in different taxonomic groups.
The idea that play can have short-term benefits – instead of or in addition to long-term
ones – is suggested, in part, by the observation that adults sometimes play (Smith, 1982;
Špinka et al., 2001). Long-term trade-offs are less likely explanations in these cases. Short-term
benefit hypotheses fall into three loose camps: learning, social, and self-regulatory. Learning
hypotheses propose that some types of play are ways of exploring actions to generate insights
about problems at hand (Sutton-Smith, 1997). For example, in Köhler's (1925) early
experiments on cognition in apes, a captive chimpanzee attempting to retrieve a banana outside
his cage only succeeds after playing with two sticks and apparently realizing that he could
combine them into one long perch to drag the food in (see also Birch, 1945). A bit of designbased and experimental evidence also exists, though some of the former is confounded.
Preschoolers presented with a jack-in-the-box-like toy operated by multiple levers played with
the toy for longer if the relationships between levers and different outcomes were confounded,
and thus hard to learn (Schulz and Bonawitz, 2007). Hybrids of the polecat (Mustela putorius)
and its domesticated form, the domestic ferret (Mustela putorius furo), given tubes to play in and
with then more readily learned mazes than those without, and this was domain-specific, as they
were no better at visual discrimination (Weiss-Bürger, 1981). Common ravens (Corvus corax)
given the opportunity to play-cache objects then monitored human experimenters and learned
which of them would raid the cache: they later avoided hiding food within sight of the pilferer
(Bugnyar et al., 2007). This last finding, of course, concerns both learning and social behaviour.
Hypotheses about the social functions of play in group-living species propose either that
play has group-wide benefits, or that it helps individuals navigate a complex social world. There
is equivocal design-based and correlational evidence for the hypothesis that social play
promotes group cohesion and individual bonds, and decreases social stress, in various group-
12
living primates (Pellis and Pellis, 2009). Play was most frequent in common chimpanzees and
common marmosets (Callithrix jacchus) at a time of peak stress, shortly before feeding, when
diffusing tensions is most necessary (Norscia and Palagi, 2011; Palagi et al., 2004). However,
at least in pigs, experiments show that anticipation of rewarding stimuli elicits play, providing an
alternative explanation (Dudink et al., 2006; Reimert et al., 2013). Chimpanzee dyads who
preferred to play together also later showed other types of affiliative behaviour with each other
more than with others (Palagi et al., 2004); scratching (taken as an index of stress) decreased
following spontaneous play bouts in marmosets (Norscia and Palagi, 2011); and so did
aggression between resident and intruder male sifakas (Propithecus verreauxi) (Antonacci et
al., 2010). None of these relationships convincingly demonstrate causality. Similarly, the finding
that young chimpanzees who played most often gained the most central (most connected)
positions in the troop’s social network (Shimada and Sueur, 2014) may mean that they develop
affiliative relationships through play, or that another characteristic (e.g. sociability) predicts play
and social position. Relatedly, young chacma baboons (Papio ursinus) preferred to play with
offspring of high-ranking mothers, perhaps developing relationships that would eventually give
them access to valued resources, though other explanations are possible (Cheney, 1978).
Fagen (1981) proposed that adult social play could serve a proceptive function in mammals, as
an energetic display of mate quality (Byers et al., 2010). Finally, play fighting could in some
cases be a way of subtly asserting dominance over others, either because of the implied threat
of escalation, or when aggressive gestures are given an overtly playful appearance to avoid
external interference (Jones, 1983; Mitchell, 1991; Pellegrini, 2002).
Besides learning and social hypotheses, play has been proposed to be a mechanism by
which individuals regulate energy and nutrient levels, and as a way of coping with stress and
promoting healing. One intriguing and oft-cited hypothesis is that play is a mechanism for
adaptive energy loss: animals with excess dietary energy play to trigger brown adipose tissue
13
thermogenesis, thus concentrating protein for growth and defending against obesity, parasitism,
and cold (Barber, 1991). Barber’s novel predictions have not been tested extensively, though to
my knowledge the evidence is negative: protein-restriction in rats did not lead to extra energy
expenditure (Even et al., 2003), and group-housing did not protect overfed lab rats from obesity,
compared to play-depriving isolation (Sahakian et al., 1982). Applied behavioural researchers
have taken particular interest in hypotheses about stress and healing. Psychologists have
successfully used play therapy to treat children with both physical and mental illnesses, though
it is difficult to disentangle the specific contribution of play from other features of the treatment,
such as relationship-building between patient and therapist (Ray et al., 2001; Scarlett et al.,
2005; Schaefer, 2003; Wishon and Brown, 1991). A number of researchers have also found
evidence of stress reduction – reduced self-scratching (a displacement activity) or glucocorticoid
levels – immediately following play in wild and captive animals (Arelis, 2006; Horváth et al.,
2008; Norscia and Palagi, 2011). In one of these experiments, the effect of rough-and-tumble
play on glucocorticoid levels could not be replicated by non-playful social contact or by physical
activity (wheel-running) alone (Arelis, 2006). The dichotomy between short- and long-term
benefits is not absolute, of course: short-term learning, strengthened social bonds, low stress
and good health may all accumulate over time.
Hypotheses about long-term, developmental functions of play are all to some extent
descended from Groos’ (1898) practice theory, which proposed that in play, juveniles rehearse
the actions needed for adult behaviours such as hunting, fighting, or mating (Smith, 1982).
Modern offshoots of this theory continue to be applied to specific types of play, with
investigations generating design-based, correlational, and experimental evidence. Consistent
with the hypothesis that play is training for hunting, young American kestrels (Falco sparverius)
preferentially play with prey-like objects over non-prey-like ones (Negro et al., 1996). In contrast,
play fighting lion cubs (Panthera leo) wrestle and do not practice stalking, while adults wrestle
14
very little and stalk prey extensively (Schaller, 1976). Juvenile coyote play frequency was
uncorrelated to later prey-killing ability, but this was in a very small sample (Vincent and Bekoff,
1978). Depriving domestic kittens of object play did not result in predatory skill deficits, though
the author suggests that they may instead have learned by playing with siblings (Caro, 1980).
Play fighting juvenile rhesus (Macaca mulatta) do not practice threat and submission, the
behavioural elements most common in adult fights, suggesting that play does not prepare them
for agonistic encounters (Symons, 1974). Meanwhile, this hypothesis predicts a lack of sexual
dimorphism in meerkats (Suricata suricatta) play fighting (adults of both sexes compete
aggressively for dominance), and observation bears this out; however, play fighting frequency
showed no correlation with aggressive adult outcomes (Sharpe, 2005). Thus, evidence for these
hypotheses is generally mixed, perhaps in part because functions of play differ between the
species investigated. The hypothesis that play prepares animals for sexual behaviour will be
reviewed in greater detail in Chapter 5, of which it is the focus. Note that for this variant, play
need not constitute direct rehearsal of relevant actions, but may instead act indirectly through
physiological mechanisms.
Play may instead cultivate more general capacities, rather than individual types of
behaviour. The hypothesis that play helps develop long-term motor capabilities – either directly
through physical training, or by promoting the physiological development of the motor system –
has some degree of support, though none that I know of is experimental. Design-based
predictions have been supported: Siberian ibex kids (Capra ibex sibirica) chose to play on (more
challenging) sloped rather than flat surfaces (Byers, 1977); another study correctly predicted
that age-related peaks in play in three species should occur during the sensitive periods for two
types of physiological maturation – muscle-fibre differentiation and cerebellar synapse
distribution – that could have long-term impacts on motor competence (Byers and Walker,
1995). Juvenile Belding’s ground squirrels (Urocitellus beldingi) who spent the most time playing
15
also showed the largest improvements in a task that involved balancing on a rod (Nunes et al.,
2004). Another possible function is the development of a general capacity for behavioural
plasticity (Bateson and Martin, 2013; Pellis and Pellis, 2009; Špinka et al., 2001; Sutton-Smith,
2002). This hypothesis is the focus of Chapter 4, and will be described in detail there. Finally,
Thompson (1998) hypothesized that in addition to having developmental effects such as those
above, play is also a self-assessment mechanism used to manage the pace of development, in
which success or failure guides each individual animal towards a level of challenge optimal for
their current stage of development.
Project overview
The aim of my doctoral research is to test two functional, long-term hypotheses about
social play in American mink (Neovison vison). The first is that a function of social play is to
prepare mink for a specific challenge/opportunity faced in adulthood: sexual behaviour,
particularly in the case of males. The second is that social play functions to develop a general
capacity to cope with unexpected events. Tests of this latter hypothesis will focus on outcome
measures related to fearfulness or stress reactivity, as these are of applied interest in captive
animals. It is important to note that the two hypotheses are not mutually exclusive, as play may
have more than one function, even within the same species.
Mink are a suitable research species for this investigation for several reasons: they are
highly playful as juveniles (Vinke et al., 2005); their sexual performance can be evaluated in
captivity, as they are not artificially inseminated (breeding relies on natural mating); social
deprivation of juvenile males is already known to impair adult sexual behaviour in mink and
closely related species (Bassett et al., 1959; Eibl-Eibesfeldt, 1963, cited in Eibl-Eibesfeldt, 1982;
Hansen et al., 1997; Gilbert and Bailey, 1969); farmed mink are diurnal or crepuscular in
captivity (Hansen and Møller, 2008) and can readily be observed by humans with minimal
changes to their behaviour (Svendsen et al., 2004); and, finally, they are available in very large
16
numbers on local farms. Thus, they are both useful research subjects and important subjects for
applied research.
The research will take place in three general phases, during which I will look for both
correlational and experimental evidence for my hypotheses. The first phase will be used to
conduct some basic investigations into the nature of play in mink, to test correlational
predictions, and to develop and refine my methods. In the second and third phases, I will test
experimental predictions, in addition to looking for further correlational evidence. My goal is to
acquire experimental evidence based on multiple housing treatments that persistently affect
social play, without the use of deprivation. The second phase will be used to identify successful
treatments from among a large number of candidates. Recognizing the high likelihood of false
positives when running multiple tests, I will replicate treatments that show initial promise in the
third phase. Ideally, this project will produce multiple lines of convergent evidence about the
functions of mink social play, based on correlation and various experimental treatments.
17
Chapter 2 – Characterizing Play in Juvenile Mink1
Abstract
This chapter details my first year of doctoral research, the aim of which was to refine
observation methods, to generate descriptive statistics, and to test three hypotheses about the
basic nature of play in juvenile farmed mink by analyzing data from 186 pairs of juveniles from
93 litters, where one pair per litter was provided with objects to play with (two balls and a plastic
chain). The chapter opens by extensively reviewing the current state of knowledge about play
and its development in mink and related species. The first hypothesis, that rough-and-tumble
play (R&T) is subject to litter effects due to genetic and/or maternal influences, was supported
by a positive correlation between R&T frequency in enriched and non-enriched pairs from the
same litter. This relationship, however, was likely driven by much stronger litter effects on
overall activity. The second hypothesis, that individual differences in play are stable over the
juvenile period, was supported for total play and R&T, and to some degree for object play,
though object play was not stable when controlling for activity. The third hypothesis, that object
play and other types, such as R&T, share a common motivational basis, was not supported:
object play did not act as a motivational substitute for non-object play in enriched mink, and nor
were the frequencies of these types of play correlated. Other differences between play types
further supported this conclusion, as object play declined less with age than did R&T, and only
the former showed consistent diurnal patterning independently of overall activity levels. These
data will inform the design of experimental manipulations of play in Chapter 3.
1
A modified version of this chapter is currently under revision at Developmental Psychobiology.
18
Introduction
Existing research on play in American mink (Neovison vison) is limited, relative to that in
some other species (especially laboratory Norway rats (Rattus norvegicus), reviewed in Pellis
and Pellis, 2009). The aim of this chapter is to investigate untested hypotheses concerning the
basic characteristics of mink play. The research was conducted in the first year of my Ph.D. as
part of a larger project on environmental enrichment of commercially-farmed mink (Meagher et
al., 2014). In addition to testing the aforementioned hypotheses, I had two further practical aims:
first, to refine my methods of observing juvenile mink play; and second, to obtain information
useful for planning experimental manipulations of the frequency of mink play (the subject of
Chapter 3).
The remainder of this introduction is an overview of our current state of knowledge of
play in mink. I begin with a brief overview of mink ontogeny and husbandry. I then review
existing research on play in mink and in the related polecat (Mustela putorius) and its
domesticated form, the ferret (Mustela putorius furo). Some authors have studied play in both
species, and research on the latter may complement our incomplete knowledge of play in the
former. I then detail some remaining areas of uncertainty in mink play, and conclude the
introduction with a general overview of my first-year project.
Ontogeny and husbandry of young mink
American mink are altricial, with their infant ‘kits’ born in the spring in litters of two or
three in the wild (Dunstone, 1993), but averaging up to 5 or 6 in captivity (Korhonen et al.,
2002). Several extra kits may be produced but die shortly after birth, before remaining kits are
counted (Malmkvist et al., 2007a). In the wild, females are promiscuous, and their litters may
include kits from several sires (Yamaguchi et al., 2004). Adult mink are solitary, and kits receive
care exclusively from their mothers (dams) (Dunstone, 1993). Kits begin transitioning from
19
nursing to ingesting solid food between about 3 and 5 weeks of age (Dunstone, 1993; Mason,
1994), at which time they are only just beginning to walk (Jonasen, 1987), and may not disperse
from their mothers until 11-16 weeks (Dunstone, 1993; Gerell, 1970; Jonasen, 1987). In typical
farm practice, kits are separated from dams after nutritional weaning, but before dispersal age:
e.g. current codes of practice call for separation between 6 and 10 weeks in Canada and after 8
weeks in Europe (European Fur Breeders’ Association, 1999; National Farm Animal Care
Council, 2013). Littermates are usually kept together for a few weeks beyond removal of the
dam, before being moved into pair-housing. For example, a typical North American farm may
remove the dam around 6-7 weeks, then pair off kits around 8-10 weeks. The pair-housing
arrangement may persist until September, when the sub-adult animals are about 5 months old,
or until mink are pelted (killed for their fur) at around 7 months, with remaining breeding stock
then housed individually. In the last twenty years, researchers and farmers, particularly in
Europe, have been experimenting with group-housing of more than two kits (even entire litters),
or family-housing of litters with dams, far beyond conventional separation ages (Hänninen et al.,
2008b) (Hänninen et al., 2008b; Hansen and Møller, 2012; Hansen et al., 1997).
Play in mink and related species
Juvenile mink, polecats, ferrets and other mustelids engage in social, object, and
locomotor play (e.g. Poole, 1978; Vinke et al., 2005). In this section, I review the distinctions
between play and other behaviour in these species, the developmental time course of play,
effects of the environment on play, and the effects of play deprivation on later behaviour.
Definitions and classifications
The boundaries between play and related non-play behaviours have not been clearly
defined in mink and polecats/ferrets. The most thorough investigation of this distinction – with a
large influence on subsequent research in both species – was performed by Poole (1978, 1973,
1967, 1966), using slow-motion analysis of staged, videotaped dyadic encounters in a 4 m x 4
20
m arena. Poole declined to provide an explicit definition of play, instead differentiating
polecat/ferret rough-and-tumble play and aggression primarily based on age: juveniles play,
while adults fight, with fuzzy demarcations between these and intermediate forms along a
developmental gradient. Poole described several features distinguishing rough-and-tumble play
from aggression: rough-and-tumble play involved inhibited biting attacks and clumsy, bouncy, or
extravagant movements (Poole, 1978, 1973, 1966); aggressive attacks tended to be sustained
despite a partner’s screams, defensive threats, or attempts to flee, sometimes involved rapid
sideways approaches absent in play, and appeared to be subjectively more “serious” or
“intense” to the observing researcher than rough-and-tumble play (Poole, 1973, 1966). The
most predominant actions in play were chasing and fleeing, neck and chin biting, and rolling
onto backs or sides (Poole, 1978). Playing polecats often opened their mouths wide, with lips
not retracted (an ‘open mouth play face’ similar to that described in canids and primates: Fox,
1970; Pellis and Pellis, 1997). At 7 to 9 weeks, they did this only while trying to bite opponents,
but by 15 weeks it was a standalone action during rough-and-tumble play (Poole, 1978). Thus,
Poole suggested that the open mouth play face, along with the bounciness of movements, is a
signal of playful intent in polecats. Other mustelids may not be as expressive as polecats: mink
rarely produced the open mouth play face in Poole’s study, and in another study, it was difficult
to separate open mouth displays of oriental small-clawed otters (Amblonyx cinerea) from
attempts to bite (Pellis, 1984). Mink rough-and-tumble play otherwise largely resembled polecat
play, and in fact the two species readily played together, but mink moved “much more rapidly”
and, unlike polecats, played both socially and with objects in water if given the opportunity to
swim (Poole, 1978).
The high speed at which mink play may explain why researchers other than Poole have
not described specific play signals in this species. Consider, for example, the contrast between
the same author’s definitions of locomotor play in adult ferrets (Vinke et al., 2008) and juvenile
21
mink (Vinke et al., 2005): the former gallop (“bouncing jerky gait”) while the latter simply run
“with occasional jumping”. The lack of clear signals complicates the task of distinguishing play
from non-play in mink, especially in the case of rough-and-tumble play and aggression. One
potential social play signal in mink and polecats is the staccato “chuckle” vocalization (Gilbert,
1969; Held and Špinka, 2011; Poole, 1966), frequently produced during rough-and-tumble play.
However, the same vocalization (or a superficially similar one) is also produced in aggressive or
sexual contexts by mink (Enders, 1952; MacLennan and Bailey, 1969) and other mustelids
(Belan et al., 1978; Poole, 1967). One experienced mink rancher was of the opinion that the
chuckle is primarily related to aggression, even in juveniles (T. Parkinson, personal
communication, June 2012). No formal test of whether chuckling is a play signal has been
conducted.
Authors vary in their definitions and classifications of play in mink and polecats/ferrets.
One author sub-divided ferret object play into predatory play (chasing and attacking moving
objects), object manipulation, and “care-giving play” (grooming, feeding, and moving objects
between nests) (Shimbo, 2006). Vinke et al. (2008) classified galloping and jumps onto/over
opponents as locomotor play, while others have used intermediate categories, such as
“transitory play” to encompass such behaviour, as it links locomotor and rough-and-tumble play
(Vargas and Anderson, 1998). Vinke et al. (2008) classified what they called “inhibited chin
biting”, with associated actions aimed at achieving or avoiding chin bites, as rough-and-tumble
play. Unlike Poole, these authors considered all other bites, especially those directed to the
neck, as aggressive or sexual. In juvenile mink, Vinke (2005) differentiated social play from
aggression based on inhibited vs. “fierce” or prey-shaking-like biting, and noted that the latter
sometimes involved loud screams or hissing. Working on farmed mink in Denmark, Jonasen
(1987) did not explicitly define play, but did state that between 8 and 13.5 weeks of age, male
mink play frequently, and also mount and drag their siblings and mothers (though it was not
22
clear whether this last was considered play). In some mink and ferret studies, neck-biting and
mounting behaviour have been considered social play (Biben, 1982; Jeppesen and Falkenberg,
1990; Poole, 1966; Stockman et al., 1986). Pedersen et al. (2004), uniquely compared to other
authors, claimed that social play movements in 4- to 6-month-old mink are usually soft and slow.
Brink and Jeppesen (2005) also reported gentle social biting play between kits lying on their
backs, but it was characteristic of very young mink (e.g. 5-6 weeks), becoming more vigorous
over time.
Development of play
The time course of play in young mink and polecats/ferrets is fairly similar. Jonasen
(1987) found that captive mink engaged in social play during the “socialization” (5.5 to 8 weeks)
and “exploration” phases (8 to 13.5 weeks), with object play starting around 7 weeks. Gilbert
and Bailey (1969) also noted that social behaviour in farmed mink began around 5.5 weeks, in
the form of biting and fighting between juveniles, and that time spent in (presumably social) play
increased after 6 weeks, but they did not present these data. Brink and Jeppesen (2005)
reported play in litters as early as 27-32 days, though it was very rare until 5.5 weeks. Solitary
play (manipulating straw or own tail) was more frequent than social play, and both rose in
frequency over time through weaning at 7 weeks, until the end of their study at 8 weeks.
Another study of 7- to 11-week-old mink litters found that overall play (social, object, and
locomotor) peaked between 8 and 10 weeks (Vinke et al., 2005). “Social biting play”, by far the
most frequent form, peaked at 9 weeks. Hansen et al. (1997) noted that in pair- and grouphoused mink born in (presumably early) May, social play peaked in July (thus at some time
between 8 and 12 weeks of age). Social and locomotor play in domestic ferret litters was more
frequent at 10-12 than at 5-7 weeks (Biben, 1982). In black-footed ferrets (Mustela nigripes),
play was the most common social behaviour of wild juveniles (who were observed only while
outside their burrows), peaking in late July and early August (at 2 months of age or more) (Clark
23
et al., 1986); and captive juveniles showed the most play at 12-16 weeks (Vargas and
Anderson, 1998).
Poole (1978, 1973, 1967, 1966), meanwhile, studied play and aggression primarily in
polecats and ferrets, with a secondary focus on mink. Polecat/ferret hybrids had “almost
exclusively playful” social interactions with familiar individuals between 6 and 20 weeks of age,
beginning at low frequencies as early as 4 weeks and peaking between 6 and 14 weeks (Poole,
1978, 1966). Rough-and-tumble play was more frequent at 7-9 weeks than at 15 weeks, while
the opposite was true of locomotor play (Poole, 1978). Note, however, that many elements
classified as locomotor by Poole (e.g. chasing, pouncing, and fleeing) would be considered
social play by others (including in the present thesis). In both polecat/ferret hybrids and in mink,
the most complex forms of play (of maximal roughness and including all behavioural elements
found in play) emerged around 14 weeks (Poole, 1978, 1966). The typical polecat/ferret
response to an introduced stranger was social play at 13 weeks, but aggressive fighting at 1820 weeks (Poole, 1967). In a different study, farmed mink in staged encounters between
unfamiliar same-sex pairs began displaying adult-like aggression at the age of 14 weeks and
threats and dominance signs at 17 weeks, and all individuals behaved aggressively by 20
weeks, though most also continued to play socially beyond this age (MacLennan and Bailey,
1969). Indeed, social play has also been observed in older animals: between 4 and 7 months in
sub-adult mink (Jeppesen and Falkenberg, 1990; Jeppesen, 2004; MacLennan and Bailey,
1969; Malmkvist et al., 2007b; Pedersen et al., 2004) (even in unfamiliar pairs) and in adult
ferrets (Vinke et al., 2008) and river otters (Lutra canadensis) (Beckel, 1991). However, it has
not been reported in adult mink – perhaps because they are usually housed singly after sexual
maturity. Adult mink housed with small objects, however, manipulate them in a way consistent
with object play (Axelsson et al., 2009; Dallaire et al., 2012).
24
Though the above suggests a lack of aggression until at least 3 months of age, and
reports of fighting in the wild pertain only to adults in breeding condition (Dunstone, 1993),
farmed kits are known to cannibalize their littermates (even while they are alive) during the
weeks before and after weaning. Kits may severely injure each other, biting off skin, ears, and
muscle tissue, and this may lead to death (Agriculture Canada, 1976). One study on several
American farms reported that 5% of kits aged 4-6 weeks had been thus injured by littermates
(Gorham, 1966). Agonistic behaviour between kits, consisting mostly in “biting and jerking of the
opponent while growling”, was found to peak between the start of solid food ingestion and the
later onset of water drinking – it began at 27-32 days of age, peaked between 33 and 44 days,
and was not seen after weaning at 49 days (Brink and Jeppesen, 2005; Brink et al., 2004). Diets
deficient in fat, along with dehydration, may promote live cannibalism (Agriculture Canada,
1976; Brink et al., 2004; Gorham, 1966). Thus even from a young age, kits have the potential to
harm each other, making distinguishing between play and aggression important for them as well
as for play researchers.
Causes of variation in play
As well as the age of the player, the frequency or style of play can be influenced by
several features of the housing or social environment. In one extreme example, Erlebach
(1993,1994) found that 3- to 8-month-old mink living in mixed-sex groups in ~20 m2 semi-natural
enclosures with swimming water, vegetation, and climbing branches played more (all types
combined) than controls alone in standard cages. Thus, potentially important environmental
features include space and complexity. Other mink families in semi-natural enclosures (~9 m2)
with grass, swimming water, and multiple nest boxes showed more running and more “hide and
seek” play (presumably a form of chasing) than families in standard cages (~0.3 m 2), which the
author attributed to extra space (Jonasen, 1987). Vargas and Anderson (1998) studied blackfooted ferrets housed either in 18.2 m2 semi-natural enclosures or in 1.44 m2 cages with two
25
external .09 m2 nest boxes. They did not compare play frequency between housing conditions
(perhaps because enclosure subjects could not be seen when in burrows), but the balance of
observed play types differed: the ratio of locomotor and chasing/pouncing play to contact play
fighting was higher in enclosure than in cage ferrets, and in the latter was higher while subjects
were in the cage than in the nest box. In another case (Vinke et al., 2005), mink families in large
cages (0.765 m2) with multiple nest boxes, cylinders, and raised platforms performed more play,
both social and solitary locomotor, if they also had access to an additional compartment of
similar size containing a 45 cm deep water bath. However, mink spent very little time in the bath
compartment itself, so it does not seem that extra play took place in water or in the additional
space. Besides the sheer amount of space, access to novel areas may temporarily stimulate
play: sub-adult and adult river otters wrestled at higher rates when allowed into areas of their
outdoor enclosures that were usually blocked off (Beckel, 1991), while being placed in novel
areas or introducing novel objects can either stimulate or inhibit rough-and-tumble play in
juvenile polecats (Poole, 1978, 1966).
Indeed, the presence of enrichment objects may affect the frequency and types of play.
In polecat/ferret hybrids, young animals given access to loose tubes to play with/in very
frequently transitioned from tube play to rough-and-tumble play, though whether the latter
stimulated the former is unknown (Weiss-Bürger, 1981). In another study of polecat/ferret
hybrids, Poole (1978) placed various small objects, as well as large ones that subjects could
walk into (tubes and boxes), in the arena in which encounters took place. Here, enrichments led
to a decrease in the frequency of social play, as subjects spent time manipulating or chasing
objects. Perhaps similarly, Hansen et al. (2007) found that pair-housed juvenile male mink in
home cages furnished with large tubes, chewing ropes, and play balls initiated fewer social
interactions with female cagemates. Unfortunately, they did not differentiate social play from
aggression, and suggested that males directed play at objects rather than at females, and/or
26
that females used tubes as a refuge from aggression. In another study of 6- to 7-month old
male-female pairs of mink, however, those given plastic balls played with them, but still engaged
in as much social play as those without balls (Jeppesen and Falkenberg, 1990). Relevant to
these examples of enriched housing is that subjects may ultimately, however, after a period of
habituation and/or with increasing age, come to spend hardly any more time playing with objects
than do their non-enriched counterparts (Jeppesen and Falkenberg, 1990; Meagher et al.,
2014). The presence of enrichment objects, then, has in some cases increased play, and
decreased it in other cases.
The identity and number of social partners is also likely to affect play, as suggested by
the existence of spontaneous and experimentally-induced individual differences. Poole (1978)
found that in some ferret/polecat hybrid dyads, partners consistently adopted attacker and
defender roles. Mink from a genetic line selected for confident temperament played more,
socially and with objects, than mink from a fearful line both before weaning (~5.5 to 7.5 weeks)
and after (9 to 22 weeks) (Malmkvist et al., 2007b). The sex of social partners may be important.
Young male ferrets performed more social play than females, though this may have been limited
to “standing over” behaviour, which includes neck-biting and mounting (Biben, 1982; Stockman
et al., 1986). At least for neck-biting by males, play was preferentially directed at same-sex
partners (Biben, 1982). Standing over and biting play were reduced in gonadectomized females
unless they were given androgen implants, as was the case for the former behaviour in males
(Stockman et al., 1986). In mink housed in male-female pairs until they were killed and their
pelts examined at ~7 months, males made relatively more bite marks on females’ necks, and
less elsewhere on their bodies, than females did to males (Jeppesen and Falkenberg, 1990),
consistent with increased frequency of aggression and/or rough social play. Surgically- and
chemically-castrated singly-housed adult male ferrets, on the other hand, were found to play
more than intact males during staged encounters with adult males or females, coincident with a
27
reduction in aggression and in sexual behaviour (Vinke et al., 2008). In addition, there was no
sex difference in frequency of play wrestling in sub-adult/adult river otters in mixed-sex groups,
and mixed-sex wrestling bouts were most often initiated by females (Beckel, 1991). Juvenile
males of promiscuous species commonly play more often and/or more roughly than females,
particularly with respect to rough-and-tumble play (Chau et al., 2008; Meaney et al., 1985), but
sex differences have not to my knowledge been investigated in mink. The familiarity of social
partners may also play a role, though it is uncertain in what direction. Pet ferrets as well as
young polecats/ferrets/hybrids are stimulated to play specifically by the arrival of strangers
(Poole, 1966; Shimbo, 2006). Adult male polecats/ferrets/hybrids, in contrast, attacked
unfamiliar males (and females) introduced into an arena they occupied, but instead reacted to
the introduction of familiar male cagemates with a form of inhibited fighting that Poole (1973)
classified as intermediate between play and aggression. Cagemates were treated as unfamiliar
after only two days of separation. The previously reviewed age-based differences in play
suggest that the age of social partners may affect play frequency. This has not been formally
tested in mink or related species, but juvenile polecats/ferrets/hybrids of different ages play
together readily unless the age difference is considerable and same-age partners are available
(Poole, 1966). Some older adult pet ferrets are reported to rarely engage in social play unless
solicited by younger animals (Shimbo, 2006). The number of play partners may also matter.
Solitary juvenile mink played more with their own tails, extremities, or straw than did mink in
pairs or families (Hansen et al., 1997), while juvenile polecats/ferrets/hybrids played with objects
when alone, but largely ignored them in the presence of playmates (Poole, 1966). In other
experiments on family- or group- housing (respectively with or without the mother present), pelts
of large-caged group-housed sub-adults had more bite marks, on average, than those of pairhoused controls (without the mother) (Hänninen et al., 2008a, 2008b; Hansen and Møller, 2012;
Hansen et al., 2014). However, when directly observed, 4- to 6-month-old mink given extra
space and housed in litter groups of 4 to 10 did not play more than brother-sister pairs in smaller
28
cages (Pedersen et al., 2004). Overall, then, existing results are not entirely conclusive as to the
ways in which the play of mink might be influenced by the number and identity of social
partners.
Consequences of play deprivation
Play is obviously constrained in nature and maybe in overall frequency in impoverished
housing conditions where animals are deprived of social partners or play objects. Studies have
revealed various impairments in mink, ferrets, and polecats previously raised in social isolation
or without environmental enrichments.
Early social isolation has profound effects on sexual behaviour. Male ranch mink isolated
immediately after weaning (at 6 weeks) were much less likely to mate successfully as adults
than males raised in groups of 3 or 4 until 11 weeks (Bassett et al., 1959), or than males
weaned at 8 weeks and subsequently pair-housed (Hansen et al., 1997). Male mink weaned
and isolated earlier than 8 weeks of age almost never achieved intromission, and sired no kits,
while females weaned and isolated before 8 weeks were less aggressive toward males and
mated with them more readily than later-weaned females (Gilbert and Bailey, 1969). The
authors of the latter study specifically hypothesized that their findings reflected the lack of
aggressive and play interactions with peers in early-weaned isolates, suggesting that
experience fighting and chasing are necessary for development of hunting and sexual
techniques. Male polecats raised in social isolation from the age of eye opening later failed to
properly orient neck bites on females to initiate copulation, and were less successful at biting
the necks of and killing rats than non-isolates (Eibl-Eibesfeldt, 1982, 1963). The author again
proposed that these deficits stemmed from the lack of experience normally gained in play,
further noting, however, that copulatory neck bite deficits were partially negated following initial
adult sexual experiences with females. Rough-and-tumble play in mink and polecats could thus
conceivably provide early training for any or all of hunting, mating, and fighting. Indeed, while
29
Poole (1973) noted that polecat rough-and-tumble play resembled (and apparently developed
into) genuine aggression, another author (Kuby, 1982, cited in Vinke et al., 2005) thought that
rough-and-tumble play in mink over 10 weeks old more closely resembled adult mating or
predatory behaviour. For males, all three types of behaviour involve attempts to bite other
individuals in the back of the neck.
Early social deprivation can also affect non-social outcomes. Isolation-rearing increases
the frequency of later stereotypic behaviour in mink in adulthood (Hansen et al., 1997; Jeppesen
et al., 1990, cited in Pedersen et al., 2004). Weaning and isolation at 6 weeks also led to later
increased fear of other mink, objects, and humans (Hansen et al., 1997). In another study,
ferrets were raised from age 10 weeks to 5 months either socially, in partial isolation (2 hours
daily contact with conspecifics), or in total isolation, then tested repeatedly in an open field with
novel objects (Chivers and Einon, 1982). Their methodology was adapted from research on
rats, in which partial isolates engage in abundant social play during brief reunions, while limiting
other types of social contact (Einon et al., 1978). Though the authors did not test whether
partially isolated ferrets likewise play nearly as much as non-isolates do, they concluded that it
was specifically deprivation of social play that induced hyperactivity (an effect seen in full
isolates only), while general lack of social interaction led to slow habituation to novel objects
(observed in both partially and totally isolated subjects). These studies suggest that effects of
social or play deprivation are not necessarily specific to one domain – for example, the welldocumented inhibition of sexual behaviour caused by isolation-rearing could conceivably be a
consequent of generalized fearfulness.
The effects of providing objects for juveniles to play with (among other environmental
enrichments) are fairly well-studied in mink, having been investigated both in extra-large double
cages, joined by a raised tunnel and containing swimming water and objects (Campbell et al.,
2013; Dallaire et al., 2011; Díez-León et al., 2013; Meagher and Mason, 2012; Meagher et al.,
30
2013), and in standard farm cages containing more limited enrichments such as objects and
raised platforms (Hansen et al., 2007; Malmkvist et al., 2013; Meagher et al., 2014). Although
whether play is a mediator is unknown, these types of enrichments had various effects including
reduced tail-biting or fur-chewing (Hansen et al., 2007; Malmkvist et al., 2013; Meagher et al.,
2014), reduced stereotypies (Campbell et al., 2013; Díez-León et al., 2013; Hansen et al., 2007;
Meagher et al., 2013), increased activity (Meagher et al., 2013), reduced fecal corticosteroid
metabolite levels (Díez-León et al., 2013; Hansen et al., 2007; Meagher et al., 2014, 2013),
higher androgen levels, more highly developed bacula, larger spleens and lower fluctuating
asymmetry in males (Díez-León et al., 2013), reduced fearfulness (Meagher et al., 2014), better
male sexual performance (Díez-León et al., 2013; Meagher et al., 2014), higher female fertility
(Meagher et al., 2014), reduced perseveration and increased spontaneous alternation
(Campbell et al., 2013; c.f. Dallaire et al., 2011). Polecat/ferret hybrids raised with loose tubes to
play in/with later outperformed non-enriched controls in maze acquisition and reversal learning,
but not in visual discrimination learning (Weiss-Bürger, 1981).
Thus, there is extensive evidence that depriving young mink, polecats, and ferrets of
social interactions or of environmental enrichments produces various impairments. However, it
is not clear which if any of these effects are due specifically to lack of social or object play, and
which to other features of social or enriched housing.
Unknowns in mink play
Some characteristics of play have been studied in other species, but very little in mink
and related species. Some of these are investigated in the present chapter because they are
interesting in their own right and can inform my efforts to experimentally manipulate mink play in
Chapter 3.
31
Some existing research suggests that there are some familial effects on play and
playfulness, attributable to both genetic and early environmental (maternal) factors. In two
strains of laboratory mouse (Mus musculus) that show large differences in rates of locomotor
play, hybrids played at intermediate rates, but genetic contributions and maternal effects could
not be distinguished (Walker and Byers, 1991). Mink from a strain selected for confidence also
played more than those from a fearful strain (Malmkvist et al., 2007b). Rats exhibit consistent
strain differences in the frequency of rough-and-tumble play (e.g. Reinhart et al., 2004) or in the
manoeuvres most commonly used (S. M. Himmler et al., 2014), some of which persist even
when pups are cross-fostered across strains, showing that genetic effects can trump maternal
ones (Siviy et al., 2011, 2003). The early maternal environment, however, is also known to
affect subsequent rough-and-tumble play in rats: high levels of licking and grooming of pups
decrease the later frequency of rough-and-tumble play in offspring of both sexes (Birke and
Sadler, 1987; Moore and Power, 1992; Parent and Meaney, 2008; Parent et al., 2013); and both
prenatal treatment with an androgen receptor blocker and prenatal stress lead to female-like
(i.e. low) levels of rough-and-tumble play in male pups (Casto et al., 2003; Ward and Stehm,
1991). In a large sample of German shepherd and Rottweiler dogs (Canis lupus familiaris)
subjected to a standardized behavioural test battery, general playfulness was moderately
heritable within breed, with both genetic and early environmental (litter) effects (Saetre et al.,
2006; Strandberg et al., 2005). Dog breeds also differ consistently in playfulness, a trait found to
be positively associated with confidence or boldness (Starling et al., 2013; Svartberg, 2006).
Research on the stability of individual differences in play over time is limited in nonhuman animals, including mink. More has been done in humans (Homo sapiens sapiens), much
of it showing stability over time (e.g. Yager et al., 1997), though individual differences may be
not or only weakly stable across distinct developmental periods (e.g. childhood vs. young
adulthood: Casas et al., 2003). While most such research relies on questionnaire-based
32
assessments of playfulness, direct observation of spontaneous play has shown that individual
differences in frequency of young children’s solitary and social play were consistent both across
contexts (classroom vs. playground) and across school terms, several months apart (Roper and
Hinde, 1978). Similarly, rats tested at 40-50 days and 80-90 days of age showed consistent
individual differences (Pellis and McKenna, 1992). In mink aged 7 to 11 weeks (Vinke et al.,
2005), however, large differences in frequency and preferred type of play existed between
litters, but there was little evidence that these were stable over time (C. Vinke, personal
communication, January 2013). In dogs, individual differences in playfulness as measured in a
series of brief staged interactions with humans and dogs were consistent when re-tested after
one month, and were predictive of owner-rated interest in playing with humans and other dogs
one to two years later (Svartberg, 2005; Svartberg et al., 2005).
Finally, existing evidence is somewhat mixed as to whether or not play is a general,
unitary category. Though we generally think that social, object, and locomotor play have a
common foundation that makes them all play – they are all fun (see Chapter 1) – it could be that
they are otherwise distinct phenomena, with different motivational bases. In support of play
being unitary, an empirically-identified personality dimension ('playfulness') predicts multiple
types of play in children (Trevlas et al., 2003). The same is true in dogs, where those most likely
to chase a rag are also most likely to play with a human stranger (Svartberg and Forkman,
2002). If play is unitary, these different types of play should act as motivational substitutes. For
example, polar bears (Ursus maritimus) (Pluta and Beck, 1979, cited in Fagen, 1981) played
with objects more in the absence of a playmate; and as we have already seen,
polecats/ferrets/hybrids paired in an arena engaged in less social play if a manipulable object
was present, and in less object play if a social partner was present (Poole, 1978, 1966). Isolated
juvenile mink played with straw and their own extremities more than pair- or group-housed mink
(Hansen et al., 1997). Social play signals produced during solitary object play, e.g. a common
33
chimpanzee (Pan troglodytes) making an open mouth play face while playing with leaves and
water (McGrew, 1977, cited in Smith, 1982), may be further evidence for their common nature.
Juvenile dwarf mongooses (Helogale undulata rufula) also seem to produce the same
vocalization during social play, object play, and play with their own extremities (Rasa, 1984).
However, other data suggest that different types of play are motivationally distinct, for
instance not acting as motivational substitutes, being independently influenced by experimental
manipulations or external stimuli, or following different developmental trajectories. For example,
the opportunity to play with other dogs did not reduce the amount of time dogs spent playing
with humans (Rooney et al., 2000). Injecting bonnet macaques (Macaca radiata) with gonadal
hormones did not affect object play, but social play was replaced by huddling, an alternative
affiliative social behaviour more typical of adults (Rosenblum and Bromley, 1978, cited in
Rosenblum, 1990). In captive oriental small-clawed otters, in which play with objects often
resembles food handling, object play peaked before feeding, but social play after (Pellis, 1991).
Cuvier’s gazelle (Gazella cuvieri) locomotor and object play was highest early in ontogeny, with
play fighting and sexual play predominating in older juveniles (Gomendio, 1988). Similarly, as
already mentioned, fifteen-week-old polecats performed more locomotor play than 7-9-weekolds, but less play fighting (Poole, 1978). Besides the aforementioned dwarf mongoose,
examples of play signals given outside of social play are rare outside of primates (Fagen, 1981;
Smith, 1982). Poole (1978) reported the open mouth play face during social but not object play
in polecats. Finally, comparative studies have also found dissociations between types of play:
rodent species with the most complex play fighting show the least complex locomotor play
(Pellis and Iwaniuk, 2004), while across primate species, the size of the striatum correlates with
the frequency of social, but not non-social, play (Graham, 2011).
34
Project overview
In my first year of doctoral research, I observed pairs of juvenile farmed mink living in
enriched cages containing play objects and, for each, a non-enriched control pair from the same
litter. I assessed the frequencies of total play, of rough-and-tumble (R&T) and object play to test
three hypotheses, refine my observation methods and generate descriptive statistics useful for
my next experiment.
First, I hypothesized that overall levels of play are subject to litter effects, as are levels
specifically of rough-and-tumble play (the one sub-type suitable for testing this hypothesis in the
dataset). This predicts a positive correlation between R&T frequency in enriched and nonenriched pairs from the same litter. Note that such litter effects do not distinguish between the
relative contributions of genetic and maternal or other early environmental influences.
Littermates were housed in neighbouring cages, so family was confounded with location.
Therefore, the correlation between littermate/neighbour pairs should additionally be stronger
than that between non-littermate neighbours.
Second, I hypothesized that individual differences in total play, rough-and-tumble play
and object play are stable over the juvenile period. This predicts that the frequency of each
should be positively correlated across pairs between mid-summer and late summer observation
periods.
Finally, I hypothesized that object play shares a common motivational basis with other
play types, such as rough-and-tumble play. This predicts that enriched pairs should perform less
R&T or other types of non-object play than non-enriched pairs, because playing with objects
reduces their motivation for other types of play. Further, among enriched pairs, there should be
a positive correlation between the frequencies of object play and R&T and/or other types of
play.
35
Another aim of my first-year research was to test for correlations between juvenile play
and sub-adult/adult behaviour, but these data will not be presented until Chapters 4 and 5,
which focus on the relevant longitudinal hypotheses.
Methods
Subjects and housing
Subjects were black American mink at Millbank Fur Farm in Rockwood, Ontario, a
subset of those used in a larger experiment on environmental enrichment (see Experiment 2,
"Farm A" in Meagher et al., 2014). Subjects were pair-housed starting in late June, at ~8 weeks
of age, having lived with littermates in their whelping cage since being weaned at 6-7 weeks.
Two female-male pairs, one enriched and one non-enriched, were randomly selected from each
litter. Litters known to contain fosterlings were avoided. Records of fostering of very young kits
(newborn to ~1 week) were unavailable, however, so a small proportion of subjects may in fact
be fosterlings. Cross-fostering to reduce large litters, or away from inadequate mothers, is
common in farmed mink (National Farm Animal Care Council, 2013; Tauson et al., 2004). Pairs
were managed according to normal farm routine, fed their primary meal of meat-based paste in
the afternoon, and a smaller amount in the morning. Pairs were kept together until pelting
(slaughter) in late November and early December, after which remaining breeding stock were
housed individually until the early March breeding season.
All pairs were housed in a single row of 61 cm L x 30 cm W x 46 cm H wire mesh cages.
Towards the end of the first observation period (see below), all were vaccinated and their cages
furnished with an elevated wooden nest box. Enriched and non-enriched pairs were housed in
alternating order, with pairs from the same litter in adjacent cages. Enriched animals were
provided with a golf ball, a perforated plastic ‘whiffle’ ball, and a length of garden hose. These
had previously been rated as durable and attractive to mink by farmers (Meagher et al., 2014).
36
Hoses were hung from a horizontal wire running along the row of cages, so that pulling by one
mink would cause hoses in nearby cages to jiggle, hopefully preventing habituation. Missing or
destroyed enrichment items were replaced when noticed, up to once a month.
Table 2–1: Ethogram of recorded behaviours. Categories are mutually exclusive except
where otherwise indicated.
Inactivity
Head is down and mink is immobile except for minor positional adjustments and
limb twitches (as in REM sleep: Blumberg and Lucas, 1994).
Rough-and-tumble
play (R&T)
Mink bites, pushes (with nose), spars (with forepaws), chases, pounces on, or
wrestles with another mink, without signs of clear aggression (see below).
Unidirectional
One mink engages in R&T, but the other does not do so within two seconds of
initial observation (see note in text).
Mutual
Both mink engage in R&T simultaneously, or the second mink does so within two
seconds of initial observation. For enriched mink, also includes social object play
(see below).
Low intensity
Unidirectional or Mutual R&T with actions that are noticeably slower or weaker
than is normal, usually performed while lying down.
Clear aggression
As for R&T, but with audible hissing, screaming and/or persistent escape
attempts by one mink.
Object play
Mink bites, pushes (with forepaws or nose), lifts, carries, manipulates with
forepaws, pounces on, or chases an object (ball, hose, or suspended nest box)
Social
Mink compete for control of objects (stealing, chasing, tug-of-war), usually
simultaneous with other elements of R&T.
Solitary
Only one mink engages with the object.
Locomotor play
Mink performs forward somersault, jump, spins or chases own tail, or writhes on
back on cage floor, without interacting with other mink or objects.
Of the 400 pairs in the larger enrichment project, I selected 208 (from 104 litters) in one
continuous block of cages as my subjects. This number was chosen based on what preliminary
attempts showed I could feasibly observe. When any mink went missing (died or escaped), both
pairs from its litter were dropped from my study. Several pairs were later discovered to
accidentally contain two mink of the same sex; these families were also dropped. The final
sample consisted of 93 enriched pairs and their 93 non-enriched littermate pairs.
37
Behavioural observations
I observed mink over 6 days in early to mid-July (age ~10-11 weeks) and over 11 days in
late August and early September (age ~16-20 weeks). Further observations were conducted in
November (age ~6.5 months) to quantify stereotypic behaviour, rather than play, as part of the
enrichment project. I used instantaneous scan sampling (Martin and Bateson, 2007), at 25
minute intervals, starting one hour after sunrise and ending one hour before sunset, with breaks
at 11 am and 4 pm. This ensured adequate visibility, and captured as much as possible of the
mink’s diurnal behaviour (Hansen and Møller, 2008). On some days, observations could not be
conducted without interruption, so I collected extra data at the missing times on other days to
balance observations over time as much as possible (the first and last scans of the day had to
be phased out as the days became shorter, however). I obtained a total of 112 and 268 scans in
the first and second periods, respectively. To avoid disturbing mink, I observed them quietly
from behind another row of cages, ~3 m away. The order in which cages were observed was
reversed from day to day, such that no cages were observed systematically earlier than others.
Clear instances of aggression were therefore distinguished from R&T on the basis of
those marks that were observed: aversive or threatening vocalizations, or sustained interaction
with a fleeing partner (Poole, 1973, 1966; Vinke et al., 2005). In ambiguous cases, I observed
apparent instances of R&T or aggression as long as needed to e.g. determine whether one
mink was fleeing. Unidirectional R&T was always observed for an extra period of 2 seconds to
determine whether the second mink would reciprocate play (see ethogram in Table 2–1).
This operational definition of mink rough-and-tumble play is, except on two counts, in
agreement with Burghardt's (2005) definition of play, reviewed in Chapter I: it provides no
obvious fitness benefit, is performed repeatedly, and informal observations of young mink not in
close confinement suggest that it is voluntarily sought out (see also Poole, 1966). However, my
definition does not exclude the possibility that mink might play even when sick, hungry, or
38
scared. In addition, while mink R&T does sometimes include exaggerated movements or selfhandicapping manoeuvres, these are usually either absent or hard to perceive in high-speed
play. One potential play marker that is readily detectable is what I have called “low intensity”
rough-and-tumble play, in which one or both mink, often lying down, play much less vigorously
than is usual. Low intensity R&T was recorded separately, as it may or may not be play: it could
either represent non-playful warding off of partners, or be a clear form of self-handicapping in
play.
Data analysis
Individual mink could not be reliably identified – sexual dimorphism in body length, the
primary characteristic used to visually sex mink from a distance, is not very pronounced in
juveniles (Hansen, 1997) – so observations were not recorded separately by sex: behavioural
data were averaged for both mink within a cage. Overall play frequencies for each pair were
calculated by combining data from both summer observation periods. Solitary object play is
reported on its own, and in combination with social object play as ‘total object play’. Social
object play is also included in mutual rough-and-tumble play. Behavioural frequencies were
arcsine-square-root transformed where necessary to meet assumptions of parametric testing,
which were verified by visual inspection and using the Shapiro-Wilk test of normality. Where
these could not be met, the non-parametric Spearman’s correlation test with blocking factors
was used (Taylor, 1987). Sample and subsample means are presented below +/- standard
error.
Data were otherwise analyzed using paired t-tests, Pearson’s correlations, and general
linear models (GLM) and general linear mixed-effects models (GLMEM) run with R statistical
software and the “nlme” package (Pinheiro et al., 2015; R Core Team, 2015). GLMs were used
for analyses of total play or rough-and-tumble play where it was necessary to control for housing
(specified as a fixed effect). GLMEMs comparing behavioural means in early and late
39
observation periods included pair, nested in litter, both as random effects. Split-half analyses for
reliability were conducted by testing for correlation between separate averages calculated for
behavioural data collected on odd and even days. Where differential activity levels could
plausibly provide confounding explanations for observed play frequencies, tests were also run
using play frequency as a proportion of active time, in addition to as a proportion of overall time.
All tests are two-tailed and conducted at alpha = 0.05.
Figure 2–1: Juvenile mink time budgets by housing condition and time period. Frequency
of behaviour (mean +/- SE) is shown as a proportion of all observations. Dark grey bars
represent enriched housing, light grey bars non-enriched. Solid bars represent the July
period (10-11 weeks of age), hatched bars the August/September period (16-20 weeks).
A) Frequency of overall activity and total play.
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
Activity
Total Play
40
B) Frequency of different subtypes of play. Non-enriched mink object play
frequencies are slightly higher than zero, because they sometimes played with
their suspended nest boxes.
12%
10%
8%
6%
4%
2%
0%
Mutual R&T
Mutual R&T
Total Object Play
Without Objects
Solitary Object
Play
Locomotor Play
Results
Descriptive analyses – total play
Before reporting on tests of my three hypotheses, I first present descriptive analyses that
will be of use in planning future experiments on mink play. Time budgets for overall activity and
play are presented in Figure 2–1a. Mink spent the bulk of their time resting (65.9 +/- 0.22%).
They spent 6.59 +/- 0.12% of their overall time, and 19.3 +/- 0.3% of their active time, playing.
Total play as a proportion of overall time was strongly positively correlated with overall activity
level (F1,183 = 57.92, r = .500, p < .0001), and total play as a proportion of active time also
tended to be (F1,91 = 3.29, r = .140, p = .073). Clear aggression was extremely rare (0.04 +/0.01%).
41
Split-half analyses indicated a positive correlation between odd and even day averages
for total play (F1,91 = 11.90, r = .247, p = .0009). However, overall levels of activity were also
correlated (F1,91 = 76.97, r = .569, p < .0001); effects on play were therefore reanalyzed
controlling for this. Odd-even day consistency remained a trend for total play even when
expressed as a proportion of activity (F1,91 = 3.22, r = .132, p = .076). The odd/even day
correlation could not be tested for aggression because no pair registered more than one
instance.
Activity and play were most frequent in the early morning and late evening, as well as
around feeding and feed-related events (Figure 2–2a-b).
Subjects were more active overall and performed significantly more play at 10-11 weeks
than at 16-20 weeks (activity: 41.85 +/- 0.28% vs. 30.89 +/- 0.27%, F1,185 = 981.29, p < .0001;
play as proportion of overall time: 12.55 +/- 0.23% vs. 4.13 +/- 0.11%, F1,185 = 1809.84, p <
.0001; play as proportion of active time: 29.96 +/- 0.50% vs. 13.27 +/- 0.33%, F1,185 = 1079.31, p
< .0001). Play was extremely rare in November observations, accounting for less than 0.5% of
observations.
42
Figure 2–2: Juvenile mink time budgets by time of day. Frequency of behaviour is shown
as a proportion of all observations (solid line) and of active behaviour (dashed line).
Error bars represent SE of the mean of daily behaviour frequencies for a particular scan.
For object play, means are for enriched pairs only. The first scan began at 6:45, and the
last and 28th ended at 20:05. The observer took breaks at 11:00 and 16:00. Vertical dotted
lines show average times of management events: small morning feeding (feed 1),
redistribution of feed from cages with leftovers to cages without (spread), removal of
leftovers from previous day and morning (scrape), and large afternoon feeding (feed 2).
Note that spreading sometimes preceded and sometimes followed feed 1.
A) Total activity level over time.
Frequency of activity (% all scans)
80%
70%
feed 1
spread
scrape
60%
50%
40%
30%
20%
10%
0%
Time at start of scan
43
feed 2
B) Total play over time.
30%
Frequency of total play
feed 1
scrape
spread
feed 2
25%
20%
15%
10%
5%
0%
Time at start of scan
C) Mutual rough-and-tumble play over time.
30%
Frequency of mutual R&T
feed 1
scrape
spread
25%
20%
15%
10%
5%
0%
Time at start of scan
44
feed 2
D) Total object play over time.
Frequency of total object play
10%
9%
feed 1
scrape
spread
feed 2
8%
7%
6%
5%
4%
3%
2%
1%
0%
Time at start of scan
E) Solitary object play over time.
Frequency of solitary object play
7%
feed 1
spread
scrape
6%
5%
4%
3%
2%
1%
0%
Time at start of scan
45
feed 2
Descriptive analyses – subtypes of play
Time budgets for different types of play are presented in Figure 2–1b. Overall, the most
frequent type of play was mutual rough-and-tumble (5.05 +/- 0.10%), followed by object play
(enriched pairs only: total: 1.90 +/- 0.07%; solitary: 1.40 +/- 0.05%), unidirectional R&T (0.55%
+/- 0.02%), and finally locomotor play (0.28 +/- 0.01%). Only a small fraction of R&T was low
intensity (mutual: 5.7 +/- 0.4%; unidirectional: 5.3 +/- 0.9%). Analyses below are presented only
for total (high and low intensity) R&T, except in one case where excluding low intensity R&T
qualitatively changed results.
Split-half analyses indicated positive correlations between odd and even day averages
for mutual rough-and-tumble and object play as a proportion of total observations (mutual R&T:
F1,91 = 8.63, r = .232, p = .004; total object play: r = .287, p = .005; solitary object play: r = .221,
p = .033). Thus, the amount of data collected was sufficient to achieve some internal
consistency on these different forms of play. The correlations were unsurprisingly weaker, and
some not significant, when repeating this analysis for play as a proportion of active observations
(mutual R&T: F1,91 = 2 .38, r = .125, p = .126; total object play: r = .256, p = .013; solitary object
play: r = .153, p = .144). Unidirectional R&T was not consistent (as proportion of overall time:
rho = 0.080, p = .276; as proportion of active time: rho = 0.072, p = .327), while the odd/even
day correlation was in fact slightly negative for locomotor play. Analyses are therefore typically
presented below for those variables that were consistently measured.
A temporal pattern of early morning and evening peaks, and of peaks triggered by
feeding-related events, was apparent for rough-and-tumble play both as a fraction of all
observations, and as a fraction of observations where mink were active (general activity too
showed similar circadian variation). For total and solitary object play, however, no such pattern
was discernible when correcting for activity levels (Figure 2–2d-e).
46
Subjects performed significantly more of each sub-type of play, as a proportion of all
observations, at 10-11 weeks than at 16-20 weeks, the effect being most marked for R&T
(mutual R&T: 9.96 +/- 0.21% vs. 3.01 +/- 0.09%, F1,185 = 1334.66, p < .0001; total object play:
2.67 +/- 0.13% vs. 1.58 +/- 0.07%, t(92) = 7.93, p < .0001; solitary object play: 1.75 +/- 0.10 vs.
1.25 +/- 0.06, t(92) = 4.22, p < .0001). As a proportion of active behaviour only, there was no
decrease with age in solitary object play, only in R&T (overlapping, as social object play, with
total object play) (mutual R&T: 23.77 +/- 0.48% vs. 9.69 +/- 0.27%, F1,185 = 772.69, p < .0001;
total object play: 6.37 +/- 0.31% vs. 5.06 +/- 0.21%, t(92) = 3.53, p = .0005; solitary object play:
4.17 +/- 0.23 vs. 4.02 +/- 0.17, t(92) = 0.50, p = .618).
Hypothesis tests
Total levels of play covaried between enriched and non-enriched littermate pairs (r =
.245, p = .018), but not between non-littermate neighbours (r = .105, p = .325). The latter was
tested for using 89 rather than 93 data points, because some non-littermate neighbours were
lost to mortality, escape, or same-sex pairing. In corresponding tests, overall activity levels were
also highly similar between littermates (littermate pairs: r = .506, p < .0001; non-littermate
neighbours: r = -0.017, p = .879), so this needed to be controlled for. Overall levels of play did
not covary between littermates when counted as a proportion of active behaviour (r = .137, p =
.192). Mutual R&T, the one sub-type of play that could reliably be compared across littermates,
showed a similar pattern. Mutual rough-and-tumble play covaried between enriched and nonenriched littermate pairs (Figure 2–3), but not between enriched pairs and their other nonlittermate neighbours (r = .098, p = .359), and nor did it correlate between littermate pairs when
expressed as a proportion of active scans (r = .120, p = .253).
47
Figure 2–3: Correlation between frequency of mutual rough-and-tumble play in enriched
and non-enriched pairs from the same litter. Total mutual R&T (high and low intensity) is
shown here as a proportion of all observations (r = .200, p = .054). The relationship is
significant when excluding low intensity R&T (r = .222, p = .033).
Mutual R&T in non-enriched pair
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.00
0.02
0.04
0.06
0.08
0.10
Mutual R&T in enriched pair
There was a positive correlation between total play in the early and late observation
periods (Figure 2–4a). This was also true of rough-and-tumble play (Figure 2–4b), but only a
trend for total object play (Figure 2–4c) and non-significant for solitary object play (Figure 2–4d).
Overall activity was also stable between observation periods (F1,183 = 5.94, r = .177, p = .016),
so this was then controlled for. When play was re-tested as a proportion of activity, the
correlation held for total play (F1,91 = 10.43, r = .210, p = .002) and mutual R&T (F1,91 = 7.32, r =
.191, p = .008), but not for total object play (r = .160, p = .126), and solitary object play was still
uncorrelated (rho = -0.899, p = .392).
48
Figure 2–4: Correlation between frequencies of play in periods one (July) and two
(August/September). Behavioural frequencies are a proportion of all observations.
A) Total play in enriched (circles, solid line) and non-enriched (triangles, dashed line)
pairs (F1,91 = 15.05, r = .257, p = .0002).
0.09
Total play in period 2
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.00
0.05
0.10
0.15
0.20
0.25
Total play in period 1
B) Mutual rough-and-tumble play (total) in enriched (circles, solid line) and nonenriched (triangles, dashed line) pairs (F1,91 = 11.57, r = .232, p = .001).
0.08
Mutual R&T in period 2
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.00
0.05
0.10
0.15
Mutual R&T in period 1
49
0.20
C) Total object play in enriched pairs (r = .180, p = .084).
Total object play in period 2
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0.00
0.01
0.02
0.03
0.04
0.05
0.06
Total object play in period 1
D) Solitary object play in enriched pairs (rho = -0.079, p = .452).
Solitary object play in period 2
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0.00
0.01
0.02
0.03
0.04
Solitary object play in period 1
50
0.05
Next, I investigated whether provision of enrichment objects reduced the performance of
non-object types of play. Housing did not have any significant effect on general activity levels
(34.34 +/- 0.28% vs. 33.86 +/- 0.34%, t(92) = 1.54, p = .127), so I did not control for this here.
Despite enriched mink spending 1.4% of their overall time in solitary object play, and 1.9% in
total object play (0.5% social object play), they performed no less mutual rough-and-tumble play
(a category which includes social object play) than their non-enriched siblings (5.11 +/- 0.14%
vs. 4.99 +/- 0.14%, t(92) = 0.67, p = .506). Thus, solitary object play did not substitute for, nor
reduce the frequency of, mutual rough-and-tumble play among enriched mink. However, the
result would have been different had I used a more restrictive definition of rough-and-tumble
play that excludes social object play: mutual R&T without objects was less frequent in enriched
pairs (4.61 +/- 0.13% vs. 4.99 +/- 0.14%, t(92) = 2.15, p = .034). Similarly, enriched mink
performed less overall non-object play (unidirectional R&T, mutual R&T without objects, and
locomotor play) than non-enriched mink (as proportion of overall time: 5.39 +/- 0.14% vs. 5.87
+/- 0.15%, t(92) = 2.39, p = .018). Thus, mutual rough-and-tumble play with and without objects
did appear to substitute for each other among enriched mink. Taken together, these results
suggest that mutual rough-and-tumble play without objects shares a common motivational basis
with social object play, but not with solitary object play. Mink given enrichment objects did not
engage in less mutual R&T, but instead incorporated the objects into this play. An alternative
explanation for the lack of a significant effect of housing on mutual R&T is that solitary object
play simply took up too little time to have an impact on the frequency of R&T. This is extremely
unlikely to be the case, however: if mutual R&T in enriched mink had actually been partially
replaced by solitary object play, and accordingly been reduced by the latter behaviour’s
frequency (1.4% of all observations), the housing effect on mutual R&T would have been very
strong (3.71 +/- 0.14% vs. 4.99 +/- 0.14%, t(92) = 6.96, p < .0001).
51
Finally, there was no correlation between time spent in solitary object play and in mutual
R&T (Figure 2–5). While I focus here on solitary object play, because social object play appears
to have merged with mutual rough-and-tumble play among enriched pairs, further tests showed
that no subset of object play was correlated with any subset of non-object play. Mutual R&T
without objects covaried with neither solitary object play (r = -0.071, p = .495) nor total object
play (r = .114, p = .278). There was also no correlation between overall non-object play and
solitary object play (r = .021, p = .840) or total object play (r = .137, p = .192).
Figure 2–5: Correlation between frequency of mutual R&T and solitary object play in
enriched pairs. This correlation was not significant as a proportion of all observations
(illustrated; r = .064, p = .540), nor as a proportion of active time (r = -0.012, p = .907).
0.030
Solitary object play
0.025
0.020
0.015
0.010
0.005
0.000
0.00
0.02
0.04
0.06
0.08
Mutual R&T
52
0.10
Discussion
Play, across all types, dominated the young minks’ active time budgets, and each pair
showed levels that were relatively consistent day to day. Total play also showed clear diurnal
and longitudinal patterns, consistent with existing research on mink. Play was thus most
frequent near dawn and dusk, and around feeding or related events, according with other
observations of diurnal activity patterns in farmed mink (e.g. as measured by wheel-running:
Hansen and Jensen, 2006) and with a previous finding that play was higher in the morning than
at noon (Hansen et al., 1997). Play, in total, was less frequent at 16-20 weeks than at 10-11
weeks, again consistent with previous research (Hansen et al., 1997; Poole, 1978; Vinke et al.,
2005). Though quite rare, I also did observe play in ~6.5-month-old sub-adults (Jeppesen and
Falkenberg, 1990; Jeppesen, 2004; MacLennan and Bailey, 1969; Malmkvist et al., 2007b;
Pedersen et al., 2004). Play and overall activity levels covaried, but the patterns of playfulness
described here were typically not just by-products of general activity: pairs were consistent in
levels of play from day-to-day, and overall play declined with age, even after controlling for
overall activity levels. Finally, clear instances of aggression were surprisingly rare, which could
be because subjects were too young for adult-like aggression (MacLennan and Bailey, 1969;
Poole, 1978), but past the typical age for live cannibalism (Brink et al., 2004; Gorham, 1966).
Turning to the various sub-types of play, mutual rough-and-tumble was by far the most
common sub-type. Low intensity (slow, weak) rough-and-tumble play represented a small
enough proportion of R&T that the decision to include or exclude it was mostly inconsequential.
The fact that the proportion of low intensity play was as high in unidirectional as in mutual R&T
suggests that low intensity play is a voluntary, relaxed, possibly self-handicapping form of
genuine play. If it were merely the way in which mink who are trying to rest non-playfully defend
themselves from a partner’s harassment, we would not expect to see it performed
unidirectionally toward non-playing, non-aggressive partners. I also observed some fairly rare
53
instances of R&T (both mutual and unidirectional) that included dorsal-ventral pins or mounts
combined with nape bites, much like the pre-sexual behaviour observed by Jonasen (1987). For
object play, mink played much less frequently with balls than with the hose, the one enrichment
that had purposefully been set up to minimize habituation, by allowing mink in nearby cages to
jiggle the wire from which it hung. In terms of locomotor play, the fact that individual differences
were not consistent is, in retrospect, not surprising. Throughout the study, writhing increasingly
struck me as a possible form of social play solicitation, while jumping mink may just have been
trying to get a bite of food (mink are fed on top of the cage, and typically climb to eat). My
ethogram may not have adequately classified reality in this respect. In any event, solitary
locomotor play appears to be quite rare in mink. While this has been found even in juvenile mink
provided with larger cages in which to run (Vinke et al., 2005), it may be that, as in black-footed
ferrets, locomotor play is only expressed at high frequencies in very spacious housing, such as
outdoor enclosures (Vargas and Anderson, 1998).
The hypothesis that play is subject to genetic and/or maternal effects was supported by
significant litter effects. Likewise, the same hypothesis with respect to mutual rough-and-tumble,
the most common sub-type, was somewhat supported by a weak-to-moderate positive
correlation (trend) between littermate pairs. Both these correlations were detected despite the
fact that an unknown, minor proportion of subjects were fosterlings, who did not share the same
prenatal and perinatal environment as their littermates. Genetic effects may be further
underestimated due to multiple paternity in mink litters: some subjects were undoubtedly half
rather than full siblings (Shackelford, 1952; Yamaguchi et al., 2004). Furthermore, these
relationships between littermate pairs could not have been due to the confounding influence of
location, as there were no such correlations between unrelated neighbouring pairs. Thus, these
relationships are due to genetic and/or maternal effects, consistent with the previous finding that
selective breeding for temperament affects social and object play in mink (Malmkvist et al.,
54
2007b). However, accounting for levels of overall activity, themselves strongly influenced by
litter, eliminated the effects of litter on overall play and on mutual R&T. The litter effects on play
and R&T thus appear to be largely due to this stronger relationship. Similar investigations could
not be made of object play because only one pair per litter was enriched, and individuals within
a pair were not differentiated in observations; nor of locomotor play because the data collected
appeared too unreliable. Future research is thus needed to determine whether these are subject
to litter effects. More research is also needed to disentangle the relative contributions of genetic,
maternal, and other early environmental effects on total play and rough-and-tumble play.
Overall, the results for overall play and for R&T join findings from other species in indicating
maternal and/or genetic effects on mink play, but they emphasize the importance of considering
overall levels of activity/inactivity as potential confounds in certain studies (Bekoff and Byers,
1992), such as those investigating playfulness as a trait.
The hypothesis that individual differences in play are stable over time was partially
supported, depending on the sub-types of play considered. There were weak-to-moderate
positive correlations between early and late observation periods for total play and for mutual
rough-and-tumble play, revealing stability in these behaviours over the 10 weeks of the study.
These were also stable when considered as a proportion only of active behaviour: thus not mere
by-products of pairs’ stable activity levels. In adult mink, similarly stable individual differences
over time have been found in enrichment use (though not restricted to play) (Dallaire et al.,
2012). However, despite this, there was only a positive trend for stability over time for total
object play, but no correlation for solitary object play. Further, the trend for stability in object play
was no longer present when controlling for activity levels. Thus, the little stability in object play
observed seemed merely to have been a side effect of the stability observed in activity levels. It
might be tempting to ascribe the weak relationships for object play to its low frequency – more
observation is required to accurately quantify rare behaviours – but split-half analyses showed
55
that the object play data were no less reliable than R&T. The weak consistency of object play
over time could possibly instead be explained by different pairs of mink habituating at different
rates, especially because destroyed or lost objects were replaced during the study (which cages
was not recorded). Future research could usefully experiment with repeatedly adding new
objects for all subjects, to see if this reveals or engenders more consistent object play over time.
In addition to these differences in stability over time between sub-types of play, two other
dissociations suggested that different sub-types of play in mink have different motivational
bases. First, object play declined much less than did rough-and-tumble play between the first
and second observation periods. Subjects in enriched cages spent only just over a third as
much time playing at 16-20 as at 10-11 weeks, and this decrease was almost entirely due to
reduced rough-and-tumble play: mutual R&T dropped to 29% of its original level, compared to
just 71% for solitary object and 59% for total object play. Put differently, solitary object play
accounted for 13% and mutual R&T for 76% of all play at 10-11 weeks, but by 16-20 weeks
these figures were respectively 26% and 63%. Furthermore, when accounting for the
decreasing activity levels in older mink, the age-related decrease in solitary object play became
insignificant (unlike the decline in R&T, which was robust to this correction). The second line of
evidence that different sub-types of play are fundamentally distinct came from examining
temporal patterns. Contrary to mutual R&T, object play showed very little diurnal patterning
when controlled for overall activity. This is particularly surprising given that object play is
stimulated by hunger or the imminence of scheduled feeding in cats and otters, though it is
unknown whether increases in play in these species were accompanied by increased general
activation (Hall and Bradshaw, 1998; Pellis, 1991). In the present study, however, it does
appear that the frequency of object play across individuals, times of day, and ages is to a large
extent driven by overall activity levels. Rough-and-tumble play, in contrast, showed stable
56
individual differences, diurnal patterns, and age-based differences that were all independent of
overall activity.
The formal tests of the hypothesis that rough-and-tumble and object play share a
common motivational basis did not support it either. There was no relationship between the
frequencies of object play and R&T, nor between object play and all other types of play
combined: thus they did not appear to share a common basis in ‘playfulness’. In addition,
playing with objects did not appear to be a motivational substitute for rough-and-tumble or nonobject play: while being able to play with objects did cause a decrease in overall non-object
play, enriched subjects made up for this by spending time engaging in R&T with objects (social
object play) – thus, rather than play with objects instead of R&T, they integrated objects into
R&T, performing as much R&T overall as non-enriched mink. Thus, this study provides no
evidence for a unitary playfulness trait in mink. This accords with a previous study that found no
difference in social play between mink given plastic balls and those without (Jeppesen and
Falkenberg, 1990). The lack of substitution between types of play does conflict with some other
previous studies, but there are potentially relevant differences between experiments. Mink
deprived of social partners played more with straw and their own body, but social play occupies
a much larger portion of young mink’s time budget, and would leave a larger void than object
play (Hansen et al., 1997). Objects did reduce R&T in polecat/ferret hybrids, but habituation
would have been minimal in these short-term arena tests (unlike my own experiment which
lasted 10 weeks), and so object exploration could have simply depleted time budgets.
This research also generated some potential methodological refinements for future mink
play observation. First, split-half analyses showed that the amount of data collected was
sufficient to reveal individual differences in total, object, and mutual rough-and-tumble play, but
investigations focusing on rarer forms (e.g. unidirectional rough-and-tumble play) would require
additional observation. Second, time budget estimates might remain nearly as good even if the
57
observer took a longer mid-day break, when mink are largely inactive and non-playful, and
avoided taking a late afternoon, post-feeding break. Finally, one-zero sampling might lead to
better reliability, because the start and end of a play bout may be ill-defined (Martin and
Bateson, 2007). I sometimes found myself wondering e.g. whether a subject had been in
locomotion at the time of sampling and then began playing with its cagemate, or whether the
locomotion itself was in fact the start of a play chase.
To conclude, the results of this experiment can help determine which experimental
manipulations of housing or social groups are likely to have an effect on play frequencies in
Chapter 3. One potentially promising way to manipulate rough-and-tumble play is to pair
subjects with unrelated mink from litters with very high or very low levels of play, quantified in
litters before they are moved to pair-housing. My data suggest that this could work, because
individual differences in R&T are stable over time. Additionally, littermates housed in different
cages show similar levels of R&T, important because kits within a litter are largely
indistinguishable, and individual behaviour difficult to measure. The relatively weak correlations
involved in both findings, however, also suggest that this manipulation might not have very large
effects. This and other experimental manipulations will be tested in the next chapter.
58
Chapter 3 – Experimentally Manipulating Play in
Juvenile Mink
Abstract
This chapter details three experiments, conducted in consecutive summers, the purpose
of which was to identify experimental housing treatments that chronically elicit or suppress
rough-and-tumble play in juvenile mink. These experiments were carried out on young mink
housed in litters, and on older pair-housed juveniles. In Experiment 1, a large number of
potential treatments were evaluated, and I identified three in which play was significantly higher
than in controls: in subjects housed in extra-large cages; in males pair-housed with another
male, rather than with a female; and in subjects pair-housed with animals of a different strain
and coat colour. In Experiment 2, I replicated these three treatments, and mostly obtained
contradictory results: treatments that had formerly had positive effects now largely had negative
effects. The likely reason for this is that, due to management-based constraints associated with
working on a commercial farm, treatment was inevitably confounded with location in all three of
these cases, and the location of each treatment changed between experiments. At least some
of these results must be type I errors brought on by pseudo-replication. In Experiment 3, I
attempted to identify environmental factors that vary between locations and have an effect on
rough-and-tumble play, in order to reconcile past results. None of the hypotheses tested at this
stage were supported. Thus, it remains unclear whether any of the experimental housing
treatments had a genuine effect on rough-and-tumble play frequency, and if so, in which
direction. Therefore, the hypotheses about the function of rough-and-tumble play that are the
subjects of subsequent chapters will have to be tested using only correlational evidence.
59
Introduction
As discussed in the previous chapter, experimental evidence can provide the strongest
support for or against functional hypotheses about play. Hypotheses pertaining to long-term
benefits require experimental manipulations with chronic rather than acute influences, affecting
the frequency of play over all or most of the juvenile period. Depriving captive animals of
playmates or toys is one straightforward, very reliable method. This can introduce various
confounds, however, so more subtle alternatives should be used where possible.
Play can be elicited or inhibited in different species by manipulating either the subject
itself, its social environment, or its physical environment. In this introduction, I review the
literature on each type of intervention in turn, paying particular attention to factors with chronic
effects.
Manipulating the subject itself
Neuropharmacological interventions can acutely suppress or elicit play. This has been
studied fairly extensively, especially for play fighting in juvenile Norway rats (Rattus norvegicus),
revealing effects of many types of agonists or antagonists – e.g. opioid, cannabinoid,
dopaminergic, and noradrenergic (reviewed in Siviy and Panksepp, 2011; Trezza et al., 2011;
Vanderschuren, 2010). Here, I give only a few examples to illustrate the approach and its
potential usefulness. Some types of drugs generally increase social play (e.g. opioids: in rats
Normansell and Panksepp, 1990; and in common marmosets (Callithrix jacchus): Guard et al.,
2002), but drugs that reduce play seem to be more common, likely because there are many
ways to disrupt the process. For example, the antipsychotic haloperidol virtually eliminates play
fighting by rendering rats cataplectic and unresponsive (Pellis and McKenna, 1995). Other
drugs have more selective effects on social play, preserving or even elevating other forms of
activity (e.g. the muscarinic antagonist scopolamine: Deak and Panksepp, 2006; Thor and
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Holloway, 1983). Such drugs act on rats by reducing the frequency of nape attacks (play
initiation) and/or by reducing responsiveness to these attacks; similar reductions in dyadic social
play have been achieved by selectively anaesthetizing the nape of one partner (Lawrence et al.,
2008; Siviy and Panksepp, 1987). Some drug effects may be sexually dimorphic and differ
according to the site of injection (e.g. vasopressin antagonist: Veenema et al., 2013). The
effects of drugs on social play are typically tested in short trials, usually on a pair of rats who are
kept isolated beforehand so that social play will be maximally motivated during the trial. Thus,
effects may not be sustained with chronic drug administration, as required for testing long-term
functional hypotheses about play. In one study, for example, scopolamine no longer suppressed
play after five days of daily administration (Thor and Holloway, 1983; but see also Deak and
Panksepp, 2006). With this in mind, the ideal candidate drug for chronic play suppression would
possess the characteristics shown in one study by the stimulant and dopamine/norepinephrine
reuptake inhibitor methylphenidate, also known as Ritalin: it sharply reduced both play initiation
and receptiveness, but affected neither general activity nor other social behaviour, and subjects
did not develop tolerance or sensitization over the six days of testing (Vanderschuren et al.,
2008). As an alternative to neuropharmacological treatment, cerebral lesions can also have
long-term impacts on play. Prefrontal cortex and basal ganglia lesions typically do not affect the
overall frequency of social play in rats, but instead affect its modulation according to factors like
age and dominance, or the defensive manoeuvres used (Bell et al., 2009; Pellis and Pellis,
2009; Pellis et al., 1992b). Mild traumatic brain injury similarly altered the types of defense used,
and reduced overall rough-and-tumble play in rats (Mychasiuk et al., 2014).
Activational effects of hormones can also bring about acute increases or decreases in
play. Adrenocorticotropic hormone, at doses meant to mimic moderate to severe stress, for
example, stimulated social play in rats (Arelis, 2006). Meanwhile, injecting juvenile bonnet
macaques (Macaca radiata) with gonadal hormones (estrogen for females, androgen for males)
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mostly eliminated social play, with adult-like social huddling behaviour taking its place
(Rosenblum and Bromley, 1978, cited in Rosenblum, 1990). At least some of these activational
effects may be sustainable over long periods. The effect of gonadal hormones on macaques
persisted for three weeks, and subjects reverted to juvenile-typical play once hormone
administration ceased (Rosenblum and Bromley, 1978, cited in Rosenblum, 1990). Castration of
adult male ferrets (Mustela putorius furo) also reduced social play in dyadic encounters seven
weeks later, likely because of decreased aggression and sexual behaviour (Vinke et al., 2008).
The organizational effects of hormones on play, particularly gonadal ones, are more
consistent and better understood, and are by their very nature sustained over a long period.
Across a wide range of species, males play more often and sometimes more roughly than
females, especially when it comes to rough-and-tumble play, and sexes may also differ in the
manoeuvres they use (Fagen, 1981; Pellis and Pellis, 1990; Pellis, 2002a). Interesting
exceptions to this rule include that: among spotted hyenas (Crocuta crocuta), in which sexual
dimorphism and androgenization are reversed from the typical pattern, females play more than
males (Pedersen et al., 1990); sexual differences in play appear to be rare in monogamous
species, which typically show limited sexual dimorphism in adulthood (Chau et al., 2008); and
likewise, the males of many canids and felids do not play more than females (Fagen, 1981).
Where sex differences in play do exist, they are mediated by sexually dimorphic exposure to
androgen (and in some cases ovarian) hormones in the prenatal or perinatal periods: they can
be reduced or eliminated by interventions that expose females to male-typical hormone levels,
or vice versa (Beatty, 1984; Casto et al., 2003; Cooke and Shukla, 2011; Hotchkiss et al., 2002;
Pellis, 2002; but see also Colbert et al., 2005). For example, female and male rats injected with
testosterone propionate on the day of birth and the following day later initiated more rough-andtumble play than did controls (Pellis and McKenna, 1995; Pellis et al., 1992a). In ferrets, roughand-tumble play appears to be under both organizational and activational control by gonadal
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hormones: gonadectomy between 5 and 35 days of age reduced later juvenile rough-andtumble play in both males and females, and androgen implants partially reversed this effect
even if given as late as 70 days of age (Stockman et al., 1986). Early exposure to adrenal
hormones can also have a lasting effect: rats born to prenatally stressed mothers showed less
rough-and-tumble play than controls (Morley-Fletcher et al., 2003; Ward and Stehm, 1991), and
offspring of common marmosets treated with the synthetic glucocorticoid dexamethasone during
gestation initiated less social play than controls (Hauser et al., 2008). Human girls (Homo
sapiens sapiens) born to mothers who reported stressful events (e.g. job loss, death in the
family) during pregnancy later showed more boy-typical types of play than girls of unstressed
mothers (Barrett et al., 2014). At least some of the effects of prenatal stress on play may be due
to gonadal hormone disruption (Barrett et al., 2014; Ward and Stehm, 1991)
The long-term frequency of social play can be influenced by maternal care and other
early experiences. In infant rhesus macaques (Macaca mulatta), for example, transient
separation from the mother temporarily depresses social play (Seay et al., 1962), and chronic
early maternal deprivation can have lasting effects: rhesus who spent their first 6-12 months
alone almost never played when later paired with conspecifics (Harlow et al., 1965). High levels
of licking and grooming of infants by rat mothers are associated with a low frequency of roughand-tumble play in later juveniles (Parent and Meaney, 2008; Parent et al., 2013; but see also
for opposite result: van Hasselt et al., 2012). Experimental manipulations that decreased licking
and grooming frequency correspondingly led to increased play (Birke and Sadler, 1987; Moore
and Power, 1992). Though such effects have been observed in offspring of both sexes, some
sex differences observed within studies led to the suggestion that they may somehow be
mediated by androgen hormones (Birke and Sadler, 1987; van Hasselt et al., 2012). In another
study, rat pups who were handled and separated from their mothers for 15 minutes daily for the
first two weeks of life later showed a higher frequency of rough-and-tumble play than non-
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handled controls (Siviy and Harrison, 2008); this is somewhat surprising, given that mothers of
handled pups are known to respond with increased licking and grooming (Francis et al., 1999).
Finally, the amount of food available may also affect the frequency of play. For example,
play is responsive to artificial weaning, which causes a transient decrease in calves (Bos taurus)
(Krachun et al., 2010; Miguel-Pacheco et al., 2015; Rushen and de Passillé, 2014), but an
increase in domestic kittens (Felis silvestris domesticus) (Barrett and Bateson, 1978; Bateson
and Young, 1981). In the longer term, experimentally-imposed low milk rations decreased play
in white-tailed deer fawns (Odocoileus virginianus) and calves (Krachun et al., 2010; MullerSchwarze et al., 1982; but see also an increase: Rushen and de Passillé, 2014), as did forcing
squirrel monkeys (Saimiri sciureus and/or S. oerstedii) to forage for food (Baldwin and Baldwin,
1976), while experimental food provisioning increased play in meerkats (Suricata suricatta) and
Belding’s ground squirrels (Urocitellus beldingi) (Nunes et al., 1999; Sharpe et al., 2002).
Domestic kittens, again, played more with objects when they and their mothers received a
limited food ration (Bateson et al., 1990).
Thus, it is quite possible by a variety of methods to increase or decrease the frequency
of play by directly intervening upon subject animals. The acute nature of some interventions,
however, may render them unsuitable for tests of long-term functional hypotheses, which
require chronically elevated or suppressed play. Experimental manipulations with long-term
effects on play – such as cerebral lesions, early hormone exposure, or imposing differences in
maternal care – also tend to lack specificity, in that besides modulating play, they are likely to
have other, possibly confounding long-term effects on subjects. Where possible, these problems
may be circumvented by imposing such manipulations not on subjects themselves, but on their
social partners.
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Manipulating the subject’s social environment
The amount of time subjects spend playing can be manipulated by providing them with
more- or less-playful social partners, here termed stimulus animals. Differentially playful
stimulus animals can be created using the experimental treatments detailed in the previous
section. Even those treatments that only have transient effects on play in stimulus animals can
still have chronic effects on subjects in the right context. To illustrate, Einon et al. (1978) housed
juvenile rats in partial isolation – alone except for a short daily social interaction session – and
over three weeks, subjects were repeatedly paired with either drugged or undrugged stimulus
rats. Thus, all subjects had the same amount of social experience, but some had consistently
more playful partners than others. While habituation, tolerance, and sensitization may be a
concern in such experiments, they could potentially be avoided by replacing or rotating stimulus
animals as needed.
Another potential way to chronically affect partially isolated subject play is to schedule
subject-stimulus interactions at times when stimulus animals should be least or most motivated
to play. Play may show diurnal rhythms (e.g. my subjects in Chapter 2; Fagen, 1981) and may
peak around feeding or when animals are hungry (e.g. my subjects in Chapter 2; Barrett and
Bateson, 1978; Norscia and Palagi, 2011; Palagi et al., 2004; Pellis, 1991) or anticipating other
rewarding events (Dudink et al., 2006; Reimert et al., 2013). These rhythms could be exploited
by, for example, holding some subject-stimulus interactions before feeding, and others after.
Treatment and control stimulus animals could even be kept under different light cycles or
feeding schedules, but paired with subjects at the same time of day. Another potential avenue
would be to subject stimulus animals to different degrees of social isolation before pairing them
with subjects. Rats show dramatic spikes in rough-and-tumble play when reunited after
temporary isolation, compared to non-isolated controls (Einon et al., 1978; Panksepp and
Beatty, 1980). Longer periods of isolation cause larger rebounds in play (Panksepp and Beatty,
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1980; Thor and Holloway, 1983b), and the effect is most pronounced shortly after reunion: after
one hour of social interaction, former isolates may be satiated and no longer play more than
non-isolated controls (Hole, 1991). Thus, subjects in different treatment groups could be
exposed to stimulus animals deprived for different lengths of time, or instead sequentially to the
same stimulus animal, in which case the latter’s motivation to play with the second subject
would be reduced. This method could potentially be used in other species that show rebound
effects: after social isolation in golden hamsters (Mesocricetus auratus) (Guerra et al., 1999),
northern grasshopper mice (Onychomys leucogaster) (Davies and Kemble, 1983), and horses
(Equus caballus) (Christensen et al., 2002), as well as immediately after physical confinement
or bad weather in various species (Fagen, 1981; Mintline et al., 2012; Rushen and de Passillé,
2014). To my knowledge, no one has yet adopted any of the methods suggested in this
paragraph.
The approaches discussed above are suitable for contexts in which subjects do not live
with stimulus animals full-time, e.g. in partial isolates, because they rely on temporary
fluctuations in stimulus animal play. To induce chronic differences in play where subjects and
stimulus are consistently co-housed, one could use experimental treatments with longer-term
effects on stimulus animals, such as cerebral lesions or perinatal hormonal treatment. For
example, Sharpe et al. (2002) tested whether provisioning juvenile meerkats with extra food
daily would increase not only their own play, but also that of their littermates. Juvenile male rats
pair-housed with stimulus males neonatally treated with testosterone propionate both received
and initiated a higher number of rough-and-tumble play attacks than males housed with oiltreated controls (Pellis et al., 1992a). With a similar rationale, “play robots” could potentially be
used to provide subjects with more or less playful social partners; in one early experiment,
infant/juvenile rhesus macaques directed approximately ten times more rough-and-tumble play
behaviour toward their artificial “cloth mothers” if these moved around the cage at unpredictable
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intervals than if they were stationary (Mason and Berkson, 1975). Alternatively, stimulus animals
may be selected according to pre-existing individual differences in play. High and low players
could be identified in preliminary observations, before assignment to social groups. This might
be rapidly achievable using a procedure like Thor and Holloway's (1983b), in which individual
differences in play solicitation by rats in a 6-minute session with an unresponsive (scopolaminetreated) stimulus animal predicted of social play frequency in a subsequent session with an
untreated partner. In one exciting development, 3-day-old rhesus macaques who readily
imitated a human experimenter’s facial expressions later (at 4-6 months) engaged in more
social play than non-imitators (S.J. Suomi, public lecture, Stanford University, April 2015). As an
alternative to individual screening, stimulus animals may be selected according to other
characteristics predictive of play.
Age is one strong determinant of play, and animals in mixed-age groups preferentially
play with individuals in certain age classes (Byers, 1980; Cheney, 1978). Bell et al. (2010)
exploited this effect by housing juvenile rats either with their peers, or with an adult female, in
order to give them differential play experience. Somewhat similarly, male domestic kittens who
lived with their mother and a same-aged brother engaged in more social play than kittens living
with only their mother (Mendl, 1988). Single kittens played with their mothers more frequently
than did those with brothers, but not as much as brothers played with each other. In another
study, juvenile chacma baboon (Papio ursinus) play peaked at 27-30 weeks and then declined,
but peaked again at 51-54 weeks, when a number of later-born individuals were themselves
reaching their first peak (Cheney, 1978). It is tempting to attribute this second peak to the
availability of young play partners, but it could instead be a result of having more play partners.
As discussed in Chapter 2, there are also strain or breed differences in play in several
species (Malmkvist et al., 2007b; Reinhart et al., 2004; Saetre et al., 2006; Strandberg et al.,
67
2005; Walker and Byers, 1991). While this suggests that stimulus animals of different strains
could successfully affect subject play, I know of no studies that have done this.
Sex differences in play were discussed at length in the previous section. In addition to
performing more social play than females, as a general rule, males are often preferred play
partners for other males in mixed-sex groups: in ferrets (Biben, 1982), Belding’s ground
squirrels (Nunes et al., 2014, 2004), chacma baboons (Cheney, 1978), Siberian ibex (Capra
ibex sibirica) (Byers, 1980), rats (Poole and Fish, 1976) and, in my own currently unpublished
research, in domestic lambs (Ovis aries) (see also Vázquez et al., 2014). In some cases, as in
squirrel monkeys (Biben, 1986) and domestic piglets (Sus scrofa domesticus) (Dobao et al.,
1985), both males and females prefer same-sex play partners. One study of children also found
that this was the case, though interestingly, girls with congenital adrenal hyperplasia, who
produce low levels of cortisol and high levels of androgens, showed no preference for playing
with other girls (Hines and Kaufman, 1994). That these preferences exist, however, does not
necessarily mean that play frequency would differ if stimulus animals of only one sex were
available: it is possible that such preferences only emerge when animals have the luxury of
choice, and that they otherwise “make do” (Cheney, 1978). However, there is direct evidence
that stimulus animal sex can indeed modulate play frequency: juvenile male-male rat pairs seem
to play more than male-female pairs, and these more than female-female pairs (Pellis and
Pellis, 1990), while the opposite is true in the sex-role-reversed spotted hyena (Pedersen et al.,
1990). Male and female rats in another study both solicited more play from scopolamine-treated
males than from scopolamine-treated females (Thor and Holloway, 1983b). Additionally, female
domestic kittens housed with their littermates play more if at least one of their littermates is a
male (Barrett and Bateson, 1978); and the sex ratio of males to females in litters of wild
Belding’s ground squirrels – who play almost exclusively with littermates – positively predicts
rough-and-tumble play frequency (Nunes et al., 2014, 2004).
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These last two results, however, could be effects of having male siblings, rather than of
living with male siblings. This is because prenatal androgen exposure (e.g. from male twins) can
elevate levels of rough-and-tumble play (see previous section). Even when housed in the same
large groups, and not just with siblings, lambs with male twins tend to perform more butting,
mounting, and nudging play than lambs with female twins in my own work (unpublished) and in
a previous study (Vázquez et al., 2014). Further, rough-and-tumble play in female yellow-bellied
marmots (Marmota flaviventris) is positively correlated with anogenital distance (Monclús et al.,
2012), a marker of male-biased sex ratios and early androgen exposure (Hotchkiss and
Vandenbergh, 2005; Monclús and Blumstein, 2012; Swan et al., 2005; vom Saal, 1979). Though
this technique has not been adopted as far as I know, it might be possible to affect play in
subjects by choosing stimulus animals according to birth sex ratio or anogenital distance.
Even where stimulus animals do not inherently differ in playfulness, their relationship
with the subject animal may affect its play. Fagen (1981) predicted that if play has fitness
benefits, siblings should play more with each other than with unrelated animals. Preferences for
playing with siblings have indeed been found in several group-living species including baboons
and other primates (Cheney, 1978), piglets (Dobao et al., 1985), and lambs (my unpublished
research). In contrast, Belding’s ground squirrels did not play more often with full than with half
siblings within their litters (Nunes et al., 2014). Familiarity often coincides with relatedness, and
may have its own effect. Children engaged in more social play in short sessions with unrelated
children they knew from day-care than with children who were strangers (Doyle et al., 1980). As
discussed in the previous chapter, strangers may elicit play in polecats (Mustela putorius),
ferrets, and their hybrids, but may also be more likely to be greeted with aggression than
familiar animals (Poole, 1978, 1973, 1967, 1966; Shimbo, 2006). Because unfamiliar animals
rapidly become familiar, however, manipulating subject play using partner familiarity might
require a rotating cast of stimulus animals.
69
The body mass of social partners may also affect the degree to which they play. For
example, heavier juveniles may engage in rough-and-tumble play more frequently than lighter
ones (e.g. Nunes et al., 2004). Play partner preferences may also depend on body mass: for
example, monkeys may prefer to play with individuals smaller than themselves (Thompson,
1998), while Belding’s ground squirrels chose partners similar in weight to themselves, and
engaged in longer bouts of rough-and-tumble play with them (Nunes et al., 2014, 2004). Some
training-based hypotheses, meanwhile, predict that animals should seek to play with larger
individuals for maximal challenge (Fagen, 1981; Špinka et al., 2001).
There are a few distinct, non-mutually exclusive, ways in which stimulus animals may
affect the play of subjects. First, they may do so via their own behaviour: by being more or less
responsive to the subject’s solicitation; by soliciting at lower or higher rates themselves; and/or
by terminating play bouts more or less quickly. Second, their identity may modulate the subject’s
own motivation to play, for example because of their age, sex, or relatedness. Finally, there is
some evidence suggesting that a stimulus animal’s behaviour may also impact the subject’s
motivation. For example, scopolamine and methylphenidate decreased rough-and-tumble play
in rat dyads, not only because of decreased solicitation and responsiveness in stimulus animals,
but also because untreated subjects gradually reduced their rate of play solicitation (Pellis and
McKenna, 1995, 1992; Vanderschuren et al., 2008). In a later experiment using scopolamine,
however, the untreated subjects instead solicited play at increasingly higher rates (Deak and
Panksepp, 2006). Untreated rats whose partner had suffered a mild traumatic brain injury were
less likely to initiate play with them, and, like subjects with decorticated partners, less likely to
respond playfully to their initiation attempts (Mychasiuk et al., 2014; Pellis et al., 1992b).
Rather than use stimulus animals with different characteristics, it might be possible to
manipulate play simply by varying the number of stimulus animals. For example, Bell et al.
(2010) housed juvenile rats in groups of 2 or 4 in order to give them differential play experience.
70
Unfortunately, it seems they did not assess play frequency, but simply assumed that the latter
would play more. Evidence that having more potential play partners makes social play more
frequent is surprisingly hard to find. Juvenile rhesus macaques with more same-age peers
reportedly began playing at an earlier age (Kaufmann, 1966, cited in Cheney, 1978). Other
studies found no difference in play between individually-housed piglet litters and larger groups
(6-12 litters) in larger enclosures (Šilerová et al., 2010), nor in locomotor play between single
and group-housed calves (Jensen et al., 1998). Sub-adult mink (Neovison vison) housed in
groups of 4 to 10 similarly did not play more than pairs in smaller cages (Pedersen et al., 2004).
Finally, groups of 8 juvenile rats played less than groups of 4 rats, likely due to space restriction
in the larger groups (Klinger and Kemble, 1985), and larger litters played less in Belding’s
ground squirrels, probably due to their decreased body weight (Nunes et al., 2004).
Finally, it might be possible to manipulate play by changing the availability of relevant
social signals. As previously mentioned, rats can be rendered unresponsive to play solicitation
by anaesthetizing their napes (Lawrence et al., 2008; Siviy and Panksepp, 1987). Conversely,
Wilson (1973) stimulated social play in field voles (Microtus agrestis) by applying the scent from
the back of playful voles’ heads to the heads of non-playful voles. A number of olfactory ,
auditory, tactile, and visual signals might similarly promote play in various species (Wilson and
Kleiman, 1974). Held and Špinka (2011) suggested doing this using playback of 50-kHz
ultrasonic vocalizations, emitted by rats during anticipation of rough-and-tumble play and before
play attacks (B. T. Himmler et al., 2014; Knutson et al., 1998), or using the chuckle vocalization
of mink (see Chapter 2). This would represent a modification not only of the social environment,
but also of the physical one.
Manipulating the subject’s physical environment
The last way in which play can be manipulated is by altering a subject’s environment.
The amount of space available is one potentially important characteristic. For example, calves
71
showed more locomotor play when housed in larger pens or tested in larger or longer arenas,
as well as a rebound in play when released from confinement, especially from particularly small
pens (Jensen and Kyhn, 2000; Jensen et al., 1998; Mintline et al., 2012; Rushen and de
Passillé, 2014). Play can additionally be suppressed without changing an enclosure’s overall
size, by adding in obstacles to movement: metal partitions reduced play by forcing piglets into
single-file positions, preventing manoeuvres like pivots or pushing from the side (Donaldson et
al., 2002). In practice, however, many studies make it difficult to disentangle the effects of space
itself from those of environmental enrichment or increasing environmental complexity. For
instance, laboratory mice (Mus musculus) in large enriched cages played more than controls in
small barren cages (Marashi et al., 2004; but see also Gaskill and Pritchett-Corning, 2015 for
same effect without enrichment confound). Similarly, as reviewed in Chapter 2, where space
allowance has an apparent effect on play in mink and other mustelids, it is confounded with the
presence or absence of naturalistic stimuli (Erlebach, 1994, 1993; Jonasen, 1987; Vargas and
Anderson, 1998) or of swimming pools (Vinke et al., 2005). In preliminary analyses across 38
species of carnivores at the same zoo, enclosure size was found to have a positive effect on
play, as were the number of substrates and the presence of water features, though whether
these effects were independent of each other was not specified (Miller et al., 2013).
Indeed, the substrate on which animals are housed might also affect their play. Young
Siberian ibex locomotor play was preferentially performed on sloped rather than flat surfaces
(Byers, 1977); calves engaged in more vigorous galloping on bedded flooring than on bare
cement (Brownlee, 1984); and red-bellied tamarins (Saguinus labiatus) performed most of their
wrestling on broad flat surfaces, but most of their chasing on branches, tree trunks, and other
vertical structures (Caine and O’Boyle, 1992). I have also informally observed that some of my
pets, a common rabbit (Oryctolagus cuniculus) and a small dog, play much more readily on a
towel or carpet than on a smooth floor.
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Novelty or mild changes to the environment seem to temporarily stimulate play. Shortly
after exposure to a novel object, piglet locomotor and social play spiked as exploration
decreased (Newberry et al., 1988; Wood-Gush and Vestergaard, 1991). Provision of fresh hay
or bedding similarly stimulates play in several species (Pedersen et al., 1990; Špinka et al.,
2001). Access to novel areas also stimulated play in calves (Mintline et al., 2012), river otters
(Lutra canadensis) (Beckel, 1991) and polecats (Poole, 1978, 1966); some species may have
opposite reactions, however, as rats placed in an novel arena played less than in a familiar one
(Vanderschuren et al., 1995). Sudden changes, such as wind gusts, also appeared to stimulate
play in piglets (Newberry et al., 1988).
Weather and environmental conditions can themselves influence play. Fagen (1981)
reviews a number of such factors that can suppress play, including inclement weather, biting
insects, and the presence of a human observer. Desert-dwelling bighorn sheep (Ovis
canadensis), for example, did not play in extreme heat conditions, and seemingly learned to
avoid playing near dangerous cacti (Berger, 1980). Zoo common chimpanzees spent more time
looking at people and less time playing when large crowds were present (Wood, 1998). Snubnosed monkeys (Rhinopithecus roxellana) played at very low rates during the winter, though
here temperature is confounded with other season factors (Li et al., 2011). Intense light,
meanwhile, completely suppressed play in juvenile rats (Vanderschuren et al., 1995).
Finally, provision of environmental enrichments can itself modulate play. This was
reviewed extensively in the introduction to Chapter 2. In my own research, I found that juvenile
mink with two plastic balls and a hanging length of hose did play with these, but did not differ
from mink without objects in time spent in rough-and-tumble play.
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Project overview
The aim of this chapter is to identify experimental manipulations that have chronic
effects on the frequency of rough-and-tumble play in juvenile farmed mink. The effects must be
chronic, affecting play over weeks or months, if they are to be useful in testing hypotheses
about the long-term functions of play (Chapters 4 and 5). I focus on rough-and-tumble play
(R&T) because it is by far the most common type of play in farmed mink (see Chapter 2), and
because it is specifically involved in one of the long-term hypotheses to be tested.
The research described in this chapter spanned three summers, and three cohorts of
young mink (2012 to 2014). These will be known as Experiments 1 through 3, respectively.
Experiment 1 was the pilot, in which I tested whether a number of experimental modifications to
standard housing conditions had chronic effects on rough-and-tumble play. Recognizing the
elevated risk of type I errors inherent in this process, the purpose of Experiment 2 was to repeat
successful pilot treatments, in order to test whether their effects could be replicated. While this
had not originally been planned, it became necessary to run Experiment 3 in order to try to
elucidate the cause of discrepancies between the first two experiments.
Experiments were conducted on litters of mink (“litter phase”) between weaning and litter
separation, and on older pair-housed mink (“pair phase”). Litter and pair phase tests were
conducted at Millbank Fur Farm in Rockwood, Ontario (the same farm as in Chapter 2);
additional pair phase tests were also conducted at RBR Fur Farm in St. Marys, Ontario, in
Experiments 1 and 2. Hereafter, a letter added to the experiment number will denote the life
stage and farm (a: Millbank litters; b: Millbank pairs; c: RBR pairs).
Because my eventual aim is to test hypotheses about the function of play, I avoid
experimental treatments intervening directly on subjects themselves wherever possible, as
these are the most likely to introduce confounds. In addition, the range of experimental
74
treatments that can be tested on working commercial mink farms is necessarily limited: while
the setting afforded me the chance to work with thousands of subjects, this came at the price of
constraints on permissible modifications to cages or social groupings. I therefore rely primarily
on treatments that exploit spontaneous differences between stimulus animals (e.g. providing
subjects with male vs. female cagemates). My ideal, desired outcome is to identify multiple
experimental treatments with chronic, repeatable effects on rough-and-tumble play. This would
allow me to seek convergent evidence for or against functional hypotheses in subsequent
chapters.
General Methods
Subjects and housing
Subjects were born in late April and early May of each year, artificially weaned between
early and mid-June, and separated from their littermates (generally into pair-housing) in late
June and early July. As a general rule, farmers aimed to wean each litter at 42 days of age, and
to separate them at 60 days. However, they chose to delay weaning or separation for
particularly small kits, and also sometimes fell behind. In addition, I separated most Millbank
litters myself to impose experimental treatments, without following this exact 42/60 day
schedule. Specific ages and dates for these events are given for individual experiments and
treatments where available.
Housing and husbandry in the pair stage were as described for non-enriched mink in
Chapter 2, except that RBR Fur Farm (Experiments 1c and 2c) fed mink only once daily, in the
afternoon. Details of litter housing, and of pair stage experimental treatments that deviated from
the norm, will be given in the corresponding experiments.
Constraints associated with working on commercial farms sometimes prevented me from
using ideal experimental designs. For example, different cage types could not be mixed within
75
the same row, and farmers typically insisted on housing only similar pairs (e.g. same
combinations of sex or strain) in the same row. Thus, in some cases, there were inevitable
confounding factors associated with experimental treatments. I sought to avoid these where
possible, but recognizing this as a problem where I could not, potential confounding factors are
listed alongside treatments in each experiment.
Animal numbers were determined on the basis of three factors. First, I performed power
analyses to determine how many animals would be needed to detect treatment effects on subadult or adult outcome measures of interest. Wherever possible, I assigned at least this many
subjects to each treatment. Second, however, because this procedure typically produced a
larger number of subjects than I could feasibly observe as juveniles, I restricted my observations
to a subset of animals within each treatment. I chose subsample sizes that would allow me to
perform approximately as many total behavioural scans per subject as I had during the
August/September period in Chapter 2, because this amount of data had shown itself to be
sufficient to produce reliable estimates of rough-and-tumble play frequency in split-half
analyses. Finally, there were some treatments for which farmers could not provide as many
subjects as I had hoped to observe, and in these cases all were observed.
Behavioural observations
The observation protocol and ethogram were as in Chapter 2, with several exceptions.
First, I switched from instantaneous scan sampling to one-zero scan sampling (Martin and
Bateson, 2007), with a five second observation window, due to this method’s potential for
increased reliability when quantifying play. That is, I recorded whether or not each subject
performed a given behaviour or not during this window. If all animals within a cage were inactive
for the first second, the observation was terminated immediately.
76
Second, some modifications to the ethogram were required. Because some cages
contained more than two mink, these were considered to be engaged in mutual rough-andtumble play even in non-dyadic “daisy chains”. That is, if mink A directed R&T at mink B, and
mink B directed R&T at mink C, rough-and-tumble play was considered mutual for A and B. I
chose to classify behaviour this way during pilot observations when I realized that at this age,
juveniles frequently engage in R&T while lying in a pile, often with each animal apparently biting
whomever happens to be nearest to their face. My impression is that they often appear to be
either unaware that they are not targeting the same individual who is biting them, or indifferent
to that fact. Further, rough-and-tumble play was considered mutual if those animals involved
performed R&T at any time during the 5 second window, even if not simultaneously. When one
mink initiated R&T less than one second before the end of the observation window, the ‘target’
mink was watched for one extra second after the end of the window to see if it would respond
with R&T, in which case it was considered mutual for both.
Third, litters were observed from the aisle directly in front of their cage, rather than from
one aisle away, as pairs had been in Chapter 2. Mink at this age are quite small and spend
most of their time in a deep nest box with an opaque front, and thus could not be seen from a
distance. Pairs in some experiments (Experiments 1b and 1c, and parts of 2b and 2c) also had
to be observed from the aisle containing their cages because their cages faced a wall; in any
treatments for which this was the case, animals in corresponding comparison groups were also
observed in this manner. Unless otherwise specified, each row within an experiment was
observed in succession, one at a time, with order of observation switched daily.
Data analysis
In litters, behavioural frequencies were averaged across all littermates, as individuals
could not be reliably sexed without handling, nor individually identified, in the group context.
Unlike in the pair stage, litters in which kits died or otherwise went missing were not dropped
77
from the experiment, as rough-and-tumble play was still possible between remaining littermates.
These litters’ sizes were recalculated as the average number of kits present per scan (e.g. a
litter of 7 that lost one kit a quarter of the way through would have an average size of 6.25), with
the arbitrary assumption that kits had been missing for the entire day on which I noticed they
were gone or dead. Counting kits in situ at every scan would have been too time-consuming
and disruptive, particularly as they typically sleep in a pile – it then becomes very difficult,
particularly under dim light, to determine where one of these immobile, dark-furred animals ends
and where another begins.
In pairs, behaviour was recorded for each sex separately whenever possible; mink were
identified using sexually dimorphic body size and facial features or, in some cases, coat colour.
For observations where mink could not be reliably identified (in same-sex pairs, and
occasionally in mixed-sex pairs, e.g. due to positioning or rapid movement), behaviour was
averaged across cagemates. Other changes or variations in observation methods are specified
later for individual experiments.
The primary dependent variable of interest in these experiments is mutual rough-andtumble play as a proportion of overall time. While Chapter 2 illustrated the importance of
correcting for levels of general activity when testing certain hypotheses about play, this was not
a concern here. The goal was to identify treatments that affect overall levels of R&T, because
total time spent playing – regardless of time spent in other activities – is what can most readily
be expected to contribute to the long-term, developmental effects tested for in Chapters 4 and 5.
In any case, I did reanalyze the data in this chapter while correcting for activity levels, and found
that this did not alter any major conclusions.
Statistical analyses were conducted using ANOVAs, general linear models (GLM), and
general linear mixed-effects models (GLMEM) run with R statistical software and the “nlme”
package (Pinheiro et al., 2015; R Core Team, 2015). Separate statistical tests were run to test
78
for effects of each type of treatment on rough-and-tumble play. Cage row was included as a
random factor in all GLMEMs, but not in GLMs testing for effects of treatments which were
perfectly confounded with location (see Tables 1-3).
Where necessary to meet assumptions of parametric testing, behavioural frequencies
were arcsine-square-root transformed or, where this was insufficient, subjected to a box-cox
transformation calculated using the “MASS” package for R (Venables and Ripley, 2002).
Assumptions of parametric testing were verified by visual inspection and using the Shapiro-Wilk
test of normality. Sample and subsample means are presented below +/- standard error. All
tests are two-tailed and conducted at alpha = 0.05.
Experiment 1a
The aim of this experiment, in the summer of 2012, was to test whether the frequency of
rough-and-tumble play could be elevated or decreased by four different experimental treatments
in farmed mink litters.
Methods
Subjects and housing
Subjects were juvenile black Millbank Fur Farm mink litters of between 3 and 8 kits.
Shortly before weaning, I selected experimental and control litters housed in close proximity
while attempting to balance the following factors across treatments: birth and current litter sizes
(except in the “number of kits” treatments), kit age, dam parity (dams had between 0 and 2
previous litters), and incidence of fosterlings. To minimize the inclusion of cross-fosterlings –
such that littermates could more confidently be assumed to be siblings when moved to pairhousing for Experiment 1b – I only selected litters with 3 or less added cross-fosterlings
(avoiding cross-fosterlings entirely would have too severely limited the number of available
litters). These dam and litter data were obtained from farmers’ records. Treatment and
79
corresponding control group sizes differed where no appropriate litters could be found. All litters
were born between the 25th and 27th of April 2012, except for the “bunk and balls” litters and
their controls (see below), with birthdates between April 28th and May 4th. Observed litters were
distributed over seven rows of standard whelping cages (see below) and one row of extra-large
cages, in four different sheds. In each treatment and control group, all animals served both as
subjects and as stimulus animals for their littermates. Animal numbers for each treatment, as
well as potential confounds due to prior experience or to inevitable differences in housing, are
listed in Table 3–1. Experimental treatments were as follows:
Extra-large cage: Experimental litters in this treatment were housed in wire mesh cages
measuring 90 cm L x 30 cm W x 45 cm H with an external wooden nest box with internal
dimensions 23 cm L x 28 cm W x 20 cm H. Control litters lived in standard whelping cages
measuring 61 cm L x 36 cm W x 36 cm H, with an external wooden nest box with internal
dimensions 25 cm L x 23 cm W x 25 cm H. Thus, total floor area was 0.334 m2 vs. 0.277 m2,
respectively. However, extra-large cages also contained an elevated 13 cm L x 30 cm W wire
mesh shelf on the wall furthest from the nest box, 46 cm above the cage floor. For mink old
enough to climb (see results), this increased total floor area to 0.373 m2. Extra-large cages were
separated from their neighbours by a double mesh wall, with an inch of empty space, while
standard cages were separated by an opaque plastic partition. In both treatments, as in all
those below, most but not all cages (at the farmers’ discretion) were also furnished with a metal
or plastic bowl regularly filled with drinking water. As this was the first year farmers used the
extra-large cages, they chose to use them only for dams who had previously had small litters, or
who were difficult to breed. I therefore only selected controls for this treatment from the row of
standard cages directly across from the row of extra-large cages, in which farmers had placed
more of these low-performing dams. I predicted that litters in extra-large cages would perform
80
more rough-and-tumble play due to extra space, added structural complexity, and/or extra visual
or auditory cues from neighbouring cages.
Bunk and balls: Experimental and control litters were housed in standard whelping
cages, as described above. Experimental cages additionally contained semi-cylindrical, singlewalled wire mesh “get-away bunks” affixed to the ceiling, with a closed end against the wall
farthest from the nest box (35 cm L x 16.5 cm W x 10 cm H at the apex), which were installed
approximately four weeks after the start of whelping. These bunks were part of another
experiment in which dams were given elevated areas they could use to prevent unwanted
suckling. Overall, bunks had no significant effect on nursing frequency or kit growth, but did
decrease kit mortality (Buob et al., 2013; Dawson et al., 2013). Note, however, that the
conclusions of the cited papers are based on the same experimental litters as in my study, but
with different control litters, as they were selected according to different criteria. In addition to
the bunks, I added two plastic golf (“whiffle”) balls (the type of ball most frequently used in
Chapter 2) a few days before the start of observations. This was done in order to maximize the
qualitative difference between this and the extra-large cage treatment. Though toys were found
not to change the overall frequency of rough-and-tumble play in Chapter 2, it was unknown
whether this could be generalized to younger mink. I did not make a directional prediction as to
the effect of this treatment on rough-and-tumble play: extra space and complexity, and possibly
increased kit vigour, could promote R&T; but balls could either inhibit or stimulate R&T.
Sex ratio: Male-biased and female-biased litters, all housed in standard whelping cages,
were selected after visual inspection of genitalia in all surviving kits in a litter. These were
compared to each other, rather than to a sex-unbiased control group. To qualify for this
treatment, a litter had to contain at least 4 individuals of one sex, and at most 2 of the other.
This was decided upon as the most stringent criterion likely to yield a sufficient number of litters.
I predicted that male-biased litters would perform more R&T, considering the widespread sexual
81
dimorphism in the behaviour across other species. Instead of the immediate social environment,
increased prenatal androgenization in male-biased litters could provide an alternative
explanation for any observed difference. Sex ratios of these litters at birth are unknown,
however, due to frequent perinatal mortality (Malmkvist et al., 2007a) and cross-fostering.
Number of kits: Large and small litters were all housed in standard whelping cages.
Large litters had contained 8 kits (the maximum allowed by these farmers) when litter size was
recorded by farmers (after the early peak in mortality and cross-fostering), and still contained at
least 7 when I inspected them. Small litters contained between 3 and 5 kits at both times. These
criteria were chosen as the most extreme that seemed feasible. I predicted that large litters
would play more due to the availability of more partners, as well as the possibility that being
around playing cagemates might itself elevate play motivation (e.g. through vocalizations).
Behavioural observations
I conducted behavioural observations one row at a time, between June 12th and 24th
2012. Thus, subjects were between 46 and 60 days old, except for “bunk and ball” subjects and
their controls, ranging from 39 to 57 days. A few litters of various ages had not yet been weaned
when observations began; this decision was up to the farmers. All mothers were removed by the
second observation day. I conducted behavioural observations as close to hourly as possible
(some scans took longer) between approximately 7:30 and 20:30, for a maximum of 13 scans
per day, and a total of 118 per litter. Breaks were scheduled such that scans were balanced with
respect to time of day across the entire observation period. The order of observation between
treatment and corresponding control litters was more or less random, according to where
appropriate subjects happened to have been found, except for extra-large caged litters,
observed in one block before or after controls in the other row.
82
Data analysis
The individual GLM models comparing each treatment and corresponding control
included whelping (birth) date as a covariate, as well as the presence of the mother (for the first
day or two of observation) as a binary, fixed factor. These factors and covariates were chosen
on the basis of a model selection procedure which identified them as having a significant
impact: details are given in Experiment 1b, as the procedure had been used to identify the most
and least playful litters for use as pair stage stimulus animals. An additional global GLM was run
to test for effects of litter size and sex ratio (both averaged over all scans as described above)
over the entire sample of litters, controlling for cage type (standard, bunk and ball, or extralarge) rather than treatment.
Results
Results of tests for treatment effects are given in Table 3–1. Litters in extra-large cages
played significantly more than controls in standard cages, as predicted. Neither the presence of
a bunk and balls, nor litter sex ratio, had a significant effect. Finally, small litters unexpectedly
tended to spend more time playing than large litters. The global model supported the results of
the individual tests: sex ratio still had no significant effect (F1,335 = 0.01, r = .006, p = .929) while
litter size was significantly negatively correlated with R&T frequency (F1,335 = 8.48, r = -0.133, p
= .004).
Table 3–1: Treatment effects on rough-and-tumble play in Millbank litters (Experiment
1a).
Treatments
Extra-large cages
Standard cages
Bunk and ball
Standard cages
Male-biased
Female-biased
Large litters
Small litters
N
47
49
40
43
44
40
43
43
Potential Confounds
Location
Maternal care
Birth sex ratio
Maternal investment
and care
Mean (%)
8.99
7.21
5.77
6.09
6.37
6.51
6.35
7.16
83
SE (%)
0.30
0.26
0.27
0.26
0.27
0.26
0.22
0.34
Test Statistics
F1,92 = 17.44
p < .0001
F1,74 = 1.83
p = .180
F1,75 = 0.83
p = .366
F1,76 = 3.60
p = .062
Elevated shelves and bunks saw limited use, especially in very young kits. The earliest
observed use of shelves by kits in extra-large cages was on the first day of observation (June
12th); half of the litters had been seen using shelves by the seventh day; and 18 of 49 litters
were never seen using them. Bunks were first used on the fifth day, half had used them by the
ninth day, and 17 of 40 were never seen using them.
Farmers additionally noted an unexpected series of differences between cage types:
they reported that kits in extra-large cages grew more slowly, and appeared to be abnormally
likely to suffer from live cannibalism by littermates (see Introduction to Chapter 2). My own data
partially bore this out. Body mass measured 1-2 weeks after the end of behavioural
observations was significantly lower in extra-large caged mink than in their controls among
females (n = 83 vs. 88; 757 +/- 11 g vs. 823 +/- 8 g; F1,50 = 15.12, p = .0003), though not among
males (n = 69 vs. 54; 1003 +/- 20 g vs. 1049 +/- 19 g; F1,48 = 1.59, p = .213). The number of
cages with evidence of live cannibalism, when counted on the last day of behavioural
observations, did not differ between extra-large (8/47) and control (7/49) cages. However, the
incidence of mortality was higher in extra-large cages (9/47 vs. 2/49; Χ2 = 3.99, p = .046), so
evidence of extra cannibalism therein may have been destroyed. The incidence of live
cannibalism was not significantly higher in extra-large cages and controls combined than in all
other rows (15/96 vs. 29/241; Χ2 = 0.50, p = .481).
Experiment 1b
The aim of this experiment, in the summer of 2012, was to test whether the frequency of
rough-and-tumble play could be elevated or decreased by seven different experimental
treatments on Millbank Fur Farm mink in the pair stage.
84
Methods
Subjects and housing
I moved all animals into pair phase cages between June 27th and July 9th 2012, roughly
in order of whelping date. All subjects were drawn from litters observed in Experiment 1a,
except for one male transferred to an extra-large cage from a nearby unobserved extra-large
cage, because no previously observed animals were available. Not all mink observed in the litter
stage were used in the pair stage; I expressly chose subjects to maximize the number of
animals in each of the treatments below. Where individuals from within the same litter were to
be put into different pair stage treatments, I assigned them to these at random, after having
removed all kits from the litter cage.
Farmers required that animals they had identified as potential breeders (based on litter
size, maternal care, fur quality, and absence of chewed kits) be housed separately from nonbreeders, and that (except for those in extra-large cages) animals be housed in mixed-sex
littermate pairs whenever possible. Thus, pairs that deviate from this standard use surplus
animals (e.g. a male from a male-biased litter, or an animal from a litter of potential breeders,
individually disqualified from breeding due to undesirable white patches of fur). Wherever
possible, I chose control groups such that their status as potential breeders or as definite nonbreeders would match those of corresponding treatment groups.
In each treatment below, all animals served as both subjects and stimulus, except in the
“partner playfulness” and “partner litter cage” treatments, which had explicitly-defined subject
and stimulus roles. Sample sizes for each treatment, as well as potential confounds due to prior
experience or to inevitable differences in housing, are listed in Table 3–2. Experimental
treatments were as follows:
85
Extra-large cage: Experimental animals lived in groups of three (1 female and 2 males, a
composition decided upon by farmers) in the same extra-large cage in which they had
previously lived. A small number of trios included one or two animals taken from other litters,
where one female and two males could not be drawn from the original litter. Control animals had
also lived in extra-large cages during the litter phase, but were then moved as female-male
pairs to standard pair cages in three different rows in two other sheds. When control animals
were moved, experimental animals were also removed from the cage and handled, then put
back. Even prior to analyzing the results of Experiment 1a, I had predicted that extra-large
caged trios would perform more rough-and-tumble play than controls, for a number of potential
reasons: extra space and structural complexity; visual and auditory stimuli coming through wire
mesh from neighbouring cages (standard cages had opaque plastic sides); presence of an extra
social partner; and male bias.
Partner litter cage: Experimental and control animals had previously lived in standard
litter cages and were all pair-housed with opposite-sex animals in standard cages. Stimulus
animals were taken from extra-large litter cages for experimental subjects, and from standard
litter cages for controls. I predicted that, if there are any carry-over effects of early juvenile play,
subjects housed with partners from extra-large cages should perform more rough-and-tumble
play.
Partner playfulness: In this treatment, subjects in standard pair cages (all combinations
of sex) were housed with stimulus animals drawn from litters in either the top or bottom quartile
for a measure of playfulness, described below. Subjects themselves were drawn from the two
middle quartiles. The measure of playfulness used was not simply the frequency of rough-andtumble play in each litter; rather, it was the result of a process meant to strip out the effects of
external and demographic factors, such as housing or sex ratio, on play. Thus, ‘playfulness’ was
operationally defined as the residual of a GLM model that controlled for all of these factors – in
86
effect, attempting to estimate how much each litter would play if conditions were standardized.
The model used was that selected as the best fit according to Akaike’s Information Criterion,
which strongly penalizes the inclusion of extra independent variables with little predictive power,
using a negative stepwise multiple regression in which the most weakly predictive independent
variables were removed one by one until the most parsimonious model remained. This was
performed using R statistical software with the “lme4” package (Bates et al., 2015; R Core
Team, 2015). The final model controlled for whelping date, presence of the dam at the start of
the observation period, number of kits, and cage type (extra-large, bunk and ball, or standard). I
predicted that subjects housed with stimulus animals from the most playful litters would perform
the most rough-and-tumble play, especially given that R&T was shown to be somewhat
consistent across littermates and ages in Chapter 2.
Partner sex: Male-male pairs were housed in one section of one row, female-female
pairs in two rows in a different shed, and female-male pairs scattered throughout several sheds.
Female-female pairs were housed in slightly smaller old cages (25 cm vs. 30 cm W) that
farmers held to be inappropriate for larger-bodied males. I predicted that, due to sexual
dimorphism in play, male-male pairs would show the most rough-and-tumble play, followed by
mixed-sex pairs, then female-female pairs.
Partner anogenital distance (AGD): As previously mentioned, anogenital distance,
corrected for body mass, is a sexually dimorphic indicator of androgen exposure (Hotchkiss and
Vandenbergh, 2005; Monclús et al., 2012; Swan et al., 2005; vom Saal, 1979). Around the time
when they were moved into standard pair cages, I measured body mass in a random subset of
female-male pairs, and an assistant measured the distance between the centre of the anus and
the centre of the penis or vulva while I held the animal. I predicted that subjects whose partners
had the largest AGDs, relative to their body mass, would perform the most R&T due to
increased androgenization.
87
Partner body mass: In the same subset of female-male pairs, I predicted that subjects
housed with heavier stimulus animals (weighed at the time of pairing) would play the most, due
to the presumably good nutritional status of the latter. In addition, without a directional
prediction, I tested whether the difference in body mass between partners would affect play.
Partner relatedness: Subjects in standard pair cages (all combinations of sex) were
housed either with a littermate (possibly a full sibling, half sibling, or fosterling) or with an animal
from a different litter. I predicted that littermate pairs would perform more rough-and-tumble play
due to a preference for playing with siblings and/or familiar individuals.
Behavioural observations
I conducted behavioural observations between July 14th and August 18th 2012, when
subjects were between 71 and 105 days old. I conducted behavioural observations as close to
hourly as possible (some scans took longer) between approximately 7:30 and 20:30, for a
maximum of 13 scans per day, and a total of 248 scans per cage. I was asked to stop
observations on two very hot afternoons, in order not to disturb possibly heat-stressed animals.
Data analysis
To test for differences in means between particular treatment groups, I used either ttests or, following a significant ANOVA main effect, planned comparisons between those
specific group pairs hypothesized to differ, run using the “multcomp” package for R (Hothorn et
al., 2008), with Holm-Bonferroni p value adjustment. Litter was included as a random factor in
analyses that matched for litter, where paired t-tests could not be used. For treatments where
both experimental and control subjects were housed in more than one type of pair (“partner
playfulness” and “partner relatedness” with male-male, female-male, and female-female pairs)
or came from different types of litter cages (“partner anogenital distance” and “partner mass”
with subjects from extra-large and from standard whelping cages), this was included as a fixed
factor, along with its interaction with the treatment being tested.
88
Results
Results of main tests for treatment effects are given in Table 3–2.
Table 3–2: Treatment effects on rough-and-tumble play in Millbank and RBR pairs
(Experiments 1b and 1c).
Treatments
N
Extra-large cages
Standard cages
Partner from extra-large cage
Partner from standard cage
Partner from playful litter
Male-male
Female-male
Female-female
Partner from non-playful litter
Male-male
Female-male
Female-female
Male-male pairs
Female-male pairs
Female-female pairs
Partner AGD
Female subject, male stimulus
Male subject, female stimulus
Partner body mass
Female subject, male stimulus
Male subject, female stimulus
Absolute difference in mass
Littermate pairs
Male-male
Female-male
Female-female
Non-littermate pairs
Male-male
Female-male
Female-female
Black female + black male
Black female + pastel male
Pastel female + pastel male
Pastel female + black male
43
21
41
173
11
69
9
14
62
12
31
173
41
Potential
Confounds
Location
-
-
Location
Cage size (females)
Birth or litter sex ratio
Mean
(%)
4.44
3.80
3.50
2.83
SE
(%)
0.20
0.46
0.29
0.11
3.78
2.82
3.24
0.37
0.18
0.47
4.36
2.66
2.43
3.97
2.83
2.60
0.33
0.18
0.34
0.23
0.11
0.18
r
Test Statistics
F1,62 = 3.82
p = .055
F1,205 = 2.32
p = .129
F2,206 = 0.01
p = .907
F2,242 = 10.13
p < .0001
63
63
-
.026
-.098
F1,52 = 0.13, p = .721
F1,52 = 0.22, p = .641
63
63
63
-
.104
.067
-.110
F1,53 = 0.002, p = .990
F1,53 = 0.81, p = .372
F1,53 = 0.12, p = .735
6
40
20
-
25
133
21
49
48
52
50
Location
Food rations
Partner relatedness
Family origin
89
3.40
3.03
2.42
0.63
0.19
0.23
4.10
2.78
2.78
2.13
2.27
2.63
1.88
0.25
0.12
0.29
0.19
0.15
0.15
0.17
F2,231 = 0.23
p = .630
F3,195 = 4.54
p = .004
Mink in extra-large cages tended to perform more mutual rough-and-tumble play than
those in standard cages. Increasing the latter sample to include not only the 21 standard cages
where both mink had grown up in extra-large cages, but all 62 where at least one had (mean
3.60 +/- 0.24%) made this difference significant (F1,102 = 4.55, p = .013). In contrast, limiting the
samples to the 20 cages per treatment within which all animals were from the same litter cage,
and which could be matched across treatment, made the difference a trend (4.59 +/- 0.29% vs.
3.88 +/- 0.47%; paired t(18) = 1.45, p = .082). This treatment effect did not appear to be due
simply to extra play between male dyads in the extra-large cages (with females not playing
more than those in standard pair cages): there was no significant effect of sex on play in these
cages (females: 4.41 +/- 0.24%; males: 4.46 +/- 0.20%; paired t(41) = 0.27, p = .786). To ensure
that this was not due to misidentification, I conducted this analysis on the subset of cages in
which body mass sexual dimorphism was most pronounced: those with females at least 150 g
(arbitrary threshold) lighter than either of the males. Again, there was no significant difference
(females: 3.86 +/- 0.46%; males: 3.95 +/- 0.30%; paired t(11) = 0.25, p = .810). Thus, the trend
for more frequent mutual rough-and-tumble play in extra-large cages was not limited to males.
Subjects in standard cages whose partners had been raised in extra-large cages did not
perform significantly more rough-and-tumble play than subjects whose partners had been raised
in standard whelping cages, as all these subjects themselves had been.
Pair-housed subjects across all three possible combinations of sexes did not play more if
housed with subjects from the most playful litters than with subjects from the least playful litters.
There was no significant interaction between type of pair (sex combination) and partner
playfulness (F2,206 = 1.23, p = .296).
Partner sex had a highly significant impact on mutual R&T in paired housing. Multiple
comparisons tests showed that while male-male (MM) pairs played significantly more than
female-male (FM) pairs (p < .0001), the latter did not significantly differ from female-female pairs
90
(p = .379). To eliminate the potential confound of differential birth or litter sex ratios, I analyzed a
subset of 9 MM and 14 FM pairs in which one of the MM males (or both, for littermate pairs)
could be matched to a male FM littermate. These represented 9 different litters, with 1 to 3 FM
pairs per litter. MM pairs tended to spend more time in mutual R&T than litter-matched FM pairs
(3.91 +/- 0.48% vs. 2.61 +/- 0.40%, F1,13 = 4.58, p = 0.052).
The anogenital distance of the stimulus animal, controlled for body mass, did not have
an effect on mutual rough-and-tumble play in female-male pairs. This was true whether the male
was treated as the subject and the female as the stimulus, or vice versa.
Similarly, neither the body mass of either sex, nor the absolute difference between the
two (the male almost always being heaviest) had any significant effect. The type of whelping
cage in which animals had been raised had significant interactions with the mass of the male
(F1,53 = 5.98, p = .018) and the absolute difference in mass (F1,53 = 4.78, p = .033), in both cases
due to non-significant negative relationships in mink from extra-large whelping cages, and nonsignificant positive ones in mink from standard whelping cages.
Finally, pairs of littermates did not perform significantly more mutual rough-and-tumble
play than did non-littermate pairs, and there was no significant interaction between this and type
of pair (combination of sexes).
Experiment 1c
The aim of this experiment, in the summer of 2012, was to test whether the frequency of
rough-and-tumble play could be elevated or decreased by co-housing RBR Fur Farm mink of
different strains in the pair stage.
91
Methods
Subjects and Housing
Juvenile mink were separated into female-male pair housing by farmers in late June.
Exact whelping, weaning, and pairing dates were not recorded. Mink were vaccinated between
July 20th and 24th 2012, though the exact date is unknown. Each mink in this experiment served
as both subject and stimulus animal, and was either of the black or pastel (beige) colour type, or
genetic strain. I predicted that mixed-strain pairs would show levels of mutual rough-and-tumble
play intermediate between those of the two types of same-strain pairs. Any strain difference in
rough-and-tumble play (thus far untested) should have this effect on mixed-strain pairs. In a
similar experiment in laboratory rats, strains which differed in their use of different evasive
manoeuvres in rough-and-tumble play converged in their evasive behaviour when housed
together (S. M. Himmler et al., 2014).
All subjects were housed in four rows within the same shed at RBR Fur Farm. One row,
adjoining the outer wall of the shed, was filled with black-black pairs. The row along the opposite
wall contained pastel-pastel pairs. Two inner rows were each divided between black female –
pastel male pairs (all in a block, nearest the shed doors) and pastel female – black male pairs
(all in another block, further from the shed doors). Housing treatments were spatially segregated
at the request of farmers.
The section of the shed in which these mink were housed contained approximately 114
cages per row. Because farmers usually housed pairs from the same litter in adjacent cages, I
chose 50 cages per treatment that were as widely separated as possible, in order to sample
from all litters. Unfortunately, I subsequently lost the information I collected on the provenance
of each pair, and I therefore cannot tell which pairs were littermates and which were not.
Farmers also unfortunately tended to place animals from the same breeding lines (sharing
ancestors two or more generations back) within the same row, and this is therefore inevitably
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partially confounded with treatment. Treatment is also confounded with feeding regime: farmers
soon noticed that mixed-strain pairs appeared to be consuming more food than same-strain
pairs, and therefore began feeding all mixed-strain pairs larger rations.
Behavioural Observations
I conducted behavioural observations two rows at a time, alternating between cages in a
same-strain row and those in the facing mixed-strain row, for two days per week between July
18th and August 16th 2012, scanning them every 30 minutes between approximately 7:00 and
20:00, for a maximum of 26 scans per day, and a total of 253 scans per cage (having missed a
few due to uncontrollable circumstances).
Data Analysis
Statistical analyses were conducted as in Experiment 1b, using an ANOVA followed by
planned comparisons with Holm-Bonferroni correction.
Results
Pair type had a significant global effect on the frequency of rough-and-tumble play
(Table 3–2). Multiple comparisons tests showed that pastel female – black male pairs played
significantly more than either pastel-pastel (p = .002) or black-black controls (p = .038), but
black female – pastel male pairs did not significantly differ from pastel-pastel (p = .112) nor from
black-black pairs (p = .357).
Unfortunately, I do not know which litters each of the observed pairs of animals in this
experiment came from. Based on the typical number of kits produced per litter, and on my count
of the number of different litters contributing kits to the cages in the same area in the following
year (Experiment 2c), however, there were undoubtedly several cases in which I observed more
than one pair from a single litter. I observed 50 pairs per section of cages in Experiment 1c, but
these sections contained mink from an average of only 37 different litters in Experiment 2c.
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Assuming that there was a similar number of independent litters in Experiment 1c, I therefore
recalculated p values after reducing degrees of freedom accordingly, while keeping the same
test statistics. The main effect of pair type remained significant (F3,144 = 4.54, p = .004), as did
the pairwise comparisons (p = .002; p = .039). This suggests that I would have found group
differences even had I sampled only one pair per litter in Experiment 1c.
Discussion – Experiment 1
The data from this series of experiments suggested that the frequency of mutual roughand-tumble play could be effectively increased by four experimental housing treatments: small
rather than large litter groups; extra-large cages both for litters and older trios; male-male rather
than female-male pair-housing; and mixed-strain pair-housing, specifically for pastel females
and black males.
Small litters performed more rough-and-tumble play than large litters. This was a trend in
the individual treatment effect test, and significant in the global model. One possible explanation
for this, discussed in the introduction, is that mink from smaller litters received higher levels of
prenatal and perinatal investment from their mothers (Hansen, 1997; Nunes et al., 2004).
Another is that young mink are generally successful at soliciting rough-and-tumble play from
their cagemates when motivated: if so, mink in small groups might more often find themselves
being roused or goaded into playing by playful cagemates, while those in large groups can more
readily find another individual who is already motivated to play. Unfortunately, I believe that the
true explanation is more mundane. While conducting observations, I began to anticipate this
result, and grew increasingly suspicious of it. This was because large numbers of active mink
could be difficult to track – especially when they were climbing or rolling over each other in a
single large pile – and my estimates of the number actually engaged in rough-and-tumble play
tended to be conservative. This was rarely a problem in small litters. It is quite possible that I
systematically underestimated the frequency of rough-and-tumble play in large litters.
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The increase in play in extra-large cages was as predicted on the basis of extra space,
complexity, stimuli from juveniles in neighbouring cages coming through the transparent cage
wall, and the presence of an extra male cagemate. It is unlikely that any of these alternative
explanations singlehandedly accounts for the effects seen in both phases. First, extra space
and physical complexity likely had little effect on very young juveniles, who largely spent the
early part of the litter phase clustered in the nest box, during which time they also could not yet
climb onto the shelf. Even by the end of the litter phase, a third of litters had never been
observed using the shelf. Second, because nest boxes in all treatments were opaque, stimuli
from neighbours are unlikely to have exerted a large effect until the latter part of the litter phase.
Third, the extra male cagemate was only present during the pair phase. Not only did the cage
effect pre-date this, but females also played as much as males in extra-large cages during the
pair phase, and there was no indication, in litters, that larger groups played more. Finally, extralarge cages also unexpectedly differed from controls in their slow growth and high mortality (and
possibly live cannibalism – an effect that was non-significant but may have been obscured by
increased mortality). Farmers thought that these effects were due to nutritional deficiencies in
extra-large cages, in which it may have been more difficult for very young kits to access solid
feed (D. Broadfoot, personal communication, June 2012). This is unlikely to also explain the
difference in rough-and-tumble play, given the typically positive relationship between food intake
and play, reviewed in the introduction. The cage effect would also then not have been expected
to follow through into the pair phase, as both experimental and control subjects were drawn
from extra-large litter cages.
Male-male pairs performed more rough-and-tumble play than female-male pairs, as
predicted on the basis of sexual dimorphism in other species. The difference is unlikely to be
due to birth or litter sex ratios, confounded with pair phase housing treatment, because it
persisted (as a trend) in a litter-matched subset. However, it is puzzling that other effects
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predicted on the basis of sexual dimorphism in rough-and-tumble play were not observed. For
example, there was a very clear lack of correlation between rough-and-tumble play and sex
ratio in litters. One possibility is that sexual dimorphism in rough-and-tumble play only develops
in older juveniles: we have previously seen that androgens continue to have positive activational
effects on rough-and-tumble play at age 10 weeks in domestic ferrets (Stockman et al., 1986).
Further, neither female-female pair housing (even in especially small cages) nor partner
anogenital distance affected rough-and-tumble play. It may be that the former has only a weak
effect, perhaps obscured by a floor effect, and that in the latter case, any causal link between
anogenital distance, androgenization, and later rough-and-tumble play is too tenuous to
generate an easily discernible effect, particularly considering that anogenital distance was
measured on live, wriggling animals.
Finally, pastel females and black males housed together at RBR Fur Farm played more
than black and pastel same-strain control pairs. Black females with pastel males also played at
high frequencies, though not significantly higher. These results were particularly surprising,
given that we expected mixed-strain pairs to play at intermediate levels, if strain differences
existed. In this case, there are also confounding explanations that cannot be ruled out. Mixedstrain pairs were fed larger rations, which would indeed be expected to stimulate extra play.
Note, however, that farmers only increased their rations because mixed-strain pairs appeared to
be eating more than controls, which may mean that any difference in vigorous activity (e.g. play)
pre-dated the change in ration. Further, animals of the same strain tended to be more closely
related within than between treatments. While litter or maternal effects are unlikely to explain
our results, as treatment effects persist even when degrees of freedom are reduced accordingly,
different breeding lines still predominated in each treatment. One more potential confound – that
same-strain pairs tended to come from the same litter, while mixed-strain pairs obviously never
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did – is unlikely to explain our results, as partner relatedness was not found to have any effect in
a separate sample at Millbank Fur Farm.
Puzzling over the above results any further may be futile. It may be possible to account
for them entirely using one previously-unmentioned factor. With a single exception, every
treatment found to have a significant or trend effect on rough-and-tumble play suffered from the
same unfortunate, inevitable confound: location.
Location could plausibly affect the behaviour of juvenile mink through its correlations
with subject provenance, environmental stimuli, and/or management procedures. The
correlation of housing location with breeding line in RBR Fur Farm pairs has previously been
discussed. Environmental stimuli experienced by male-male and extra-large caged trios may
have differed greatly from those of their controls, housed in entirely different sheds. For
example, mink in these treatments may have been exposed to different microclimates or
ambient levels of noise or light. Mixed-strain pairs and extra-large caged litters, meanwhile,
were housed in rows immediately adjacent to their controls, c. 1.5-2 m away, so environmental
stimuli were likely quite similar. However, in every case, farmers carried out feeding and other
management procedures row by row, at separate times, meaning that the experience of
treatment and control animals may have differed. With the exception of weaning and separating
extra-large caged litters slightly later than others, management procedures were not carried out
in a way that would necessarily introduce systematic bias, but it is possible that bias arose by
chance. For example, while rows were not fed in the same order every day, neither was feeding
order systematically varied, so it could be that farmers developed habitual routes.
Perhaps even more tellingly, treatments that were not confounded with location almost
universally had no significant effect on rough-and-tumble play. The sole exception, the small vs.
large litter effect, was likely an artefact of systematic bias in my observations, as previously
discussed. Again pointing to location’s importance, rough-and-tumble play was higher in the
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standard litter cages in the row directly opposite the extra-large cages than in any other row I
observed.
Besides differing in location, it should also be noted that treatments with significant
effects were those that I, as the observer, could not possibly be blind to. Treatment and controls
were housed in different types of cages, in different numbers, or in different combinations of sex
and coat colour. It is possible that subconscious bias, on my part, fed into these significant
results.
Thus, the experimental manipulations that successfully affected rough-and-tumble play
in Experiment 1 also happen to be those affected by location-based confounds, and for which
observer blinding was not possible. While I had already planned to attempt replication in
Experiment 2, this suggests a special need for it.
However, many of those experimental treatments that did not produce significant effects
are relatively free of confounds, and are thus likely to represent a genuine failure to support the
alternative hypothesis. I will briefly discuss these in turn before moving on to Experiment 2.
The sex ratio of litters was uncorrelated with rough-and-tumble play. The only plausible
confound – prenatal androgenization of male-biased litters – would not have counteracted the
predicted positive effect of living in male-biased groups, but instead reinforced it. Especially
considering the elevated statistical power of the correlational analysis (n = 349), this appears to
represent a genuine lack of sex differences in mink rough-and-tumble play. As previously
discussed, it is not out of the question that sexually dimorphic play appears only in older mink.
Bunks and balls also had no significant effect. While improved health or vigour of
experimental kits (Buob et al., 2013; Dawson et al., 2013) may have had an effect, counteracted
by (presumably negative) effects of the furnishings themselves on kit rough-and-tumble play,
there is no evidence to suggest that this is the case. This result is a partial replicate of that
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obtained in Chapter 2, in which older enriched and non-enriched pairs did not differ in roughand-tumble play. The lack of effect here additionally suggests that the most important difference
between extra-large and standard cages was not the presence or absence of an elevated shelf.
In the pair stage, I failed to find any evidence for effects on rough-and-tumble play of
several experimental manipulations that had no clear confounds. First, while there was a sizable
difference in rough-and-tumble play frequency between subjects paired with subjects raised in
extra-large cages and those paired with standard-raised stimulus animals, it was not significant.
With 41 pairs in the former treatment, and more in the latter, sample sizes were such that any
reasonable effect size should have been readily detectable. Second, being paired with stimulus
animals from the most or least playful litters disappointingly had no effect on subject rough-andtumble play. Put another way, there was no stability in levels of rough-and-tumble play in
stimulus animals between the litter phase and being paired with a new, unrelated juvenile. This
is somewhat surprising given that I had found stability over an equally long time period (though
later in the juvenile period) in Chapter 2. However, there are several points to remember: the
effect identified in Chapter 2 was rather weak; rough-and-tumble play was not quantified for
individuals within litters, but rather for litters as a whole and stimulus animals selected at
random from within these; while Chapter 2 also showed genetic or maternal effects on roughand-tumble play, these were also rather weak; and, finally, the procedure here involved a
change in housing and social context, which may itself have had unpredictable effects (if, e.g.,
certain dyads are more compatible than others). Pre-screening for playfulness remains an
intriguing possibility, which may work better over short time periods or in other species (Thor
and Holloway, 1983b). Third, anogenital distance, corrected for body mass, had no effect. As
previously discussed, it may be that the measure was too noisy to realistically apply to live
animals. Fourth, neither partner body mass, nor the difference in body mass between partners,
significantly predicted rough-and-tumble play. These variables, which unlike anogenital distance
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could be measured quite easily, could theoretically have affected rough-and-tumble play in any
direction. Finally, it made no significant difference whether paired juveniles came from the same
or from different litters. It is important to remember, however, that some unknown proportion of
littermate pairs consisted of unrelated individuals, due to the prevalence of cross-fostering.
While such pairs would still have been familiar with each other, this is unlikely to have an effect
over the timespan of the pair phase observation period, during which previously-unfamiliar
individuals had ample time to become familiar. In this, as for many of the above ‘failed’
experimental manipulations, it is possible that experimental and control animals differed with
respect to whether they were paired with preferred or non-preferred playmates, yet that their
baseline levels of playfulness were robust to this. In other words, they may have “made do” with
partners whom, in group settings, they would have ignored in favour of preferred individuals.
One final observation is worth noting: the frequency of rough-and-tumble play in the pair
stage was surprisingly low. Millbank Fur Farm pairs, at 10-16 weeks of age, spent about as
much time in R&T as 16- to 20-week-old mink had in Chapter 2, and only about a third as much
as they had at 10-11 weeks. Furthermore, the one-zero sampling regime used here should yield
behavioural frequencies that are as high or higher, but not lower than, those produced by
instantaneous sampling, as used in Chapter 2. I suspect that the reason rough-and-tumble play
was so rarely seen in this experiment is that I observed mink from the aisle directly in front of
their cage, rather than from one aisle away, and was therefore more likely to disturb play bouts,
or prevent them from starting. This is an issue that will be returned to in Experiments 2b and 2c.
Given the confounded nature of manipulations that affected rough-and-tumble play, and
the generally more experimentally sound nature of those that did not, the focus of Experiment 2
will be replications of the former, namely extra-large caging and male-male and mixed-strain
pairing.
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Experiment 2a
The aim of this experiment, in the summer of 2013, was to replicate the test for an effect
of extra-large cage housing on rough-and-tumble play in the litter phase. This effect had been
found to be positive in Experiment 1a.
While it would have been ideal to conduct this test without a location confound, this was
impossible because extra-large and standard cages were installed in separate rows. I therefore
used cages in different extra-large and standard cage rows than in Experiment 1a, such that
any effects of location would not be constant between replicate experiments.
Methods
Subjects and housing
Subject litters of 5 to 8 kits were selected as in Experiment 1a, 46 per treatment,
balanced in terms of birth and litter size, fosterlings, dam parity, and whelping date. Subjects
were born between April 26th and May 2nd 2013. As in Experiment 1a, extra-large cages had
been stocked with litters from mothers who were either difficult to breed, or had previously had
small litters. Controls were selected from standard whelping cages containing litters from similar
mothers.
Behavioural observations
I conducted behavioural observations as in Experiment 1a, one row at a time, between
June 13th and 24th 2013. Thus, subjects were between 42 and 59 days old. All litters had been
weaned when observations began. I was unable to observe them from the 17th to the 21st due to
illness, and found one extra-large cage to be empty when I returned. I do not know whether
these kits died or were moved by farmers, but this litter was excluded from analysis. I conducted
behavioural observations every twenty minutes (some scans took longer) between
approximately 6:40 and 20:00, for a maximum of 40 scans per day, and a total of 156 per litter.
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Some observations were conducted after mounds of fresh bedding had been placed on the
mesh on top of some nest boxes (to gradually fall in, or be pulled in by kits); observations from
these rounds were later discarded for all litters, because the bedding obscured behaviour of kits
in the nest box.
Data analysis
Housing treatments were compared using a GLM model that included whelping date as
a covariate.
Results
There was no significant treatment effect on rough-and-tumble play frequency (Table 3–
3). As in Experiment 1a, the incidence of mortality was much higher in extra-large than in control
cages (11/45 vs. 1/46; Χ2 = 8.01, p = .005), but there was no significant difference in the
incidence of live cannibalism, recorded during behavioural observations starting on the 16th
(12/45 vs. 9/46; Χ2 = 0.31, p = .579). Body mass was not recorded at the end of the observation
period.
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Table 3–3: Treatment effects on rough-and-tumble play in Millbank litters and in Millbank
and RBR pairs (Experiments 2a, 2b, and 2c).
Treatments
Litter phase
Extra-large cages
Standard cages
Pair phase
Extra-large cages
Standard cages
Close observation
Male-male pairs
Female-male pairs
Far observation
Male-male pairs
Female-male pairs
Close observation
Black female + black male
Black female + pastel male
Pastel female + pastel male
Pastel female + black male
Far observation
Black female + black male
Black female + pastel male
Pastel female + pastel male
Pastel female + black male
Male-male demi pairs
Female-male demi pairs
N
Potential Confounds
Mean (%)
SE (%)
Test Statistics
45
46
Location
8.89
8.89
0.22
0.23
F1,88 = 0.001
p = .974
F1,115 = 4.56
p = .035
55
62
Location
Age (?)
Family origin
5.99
6.77
0.20
0.27
14
15
Location
Family origin (far only)
5.10
5.89
0.53
0.68
7.58
8.63
0.32
0.29
5.44
4.20
2.86
3.16
0.38
0.30
0.25
0.23
6.27
6.48
7.74
6.25
5.22
4.92
0.26
0.26
0.30
0.35
0.39
0.25
42
45
21
24
37
29
41
36
31
31
29
60
Location
Partner relatedness
Family origin
Location
F1,112 = 6.48
p = .012
F3,242 = 2.72
p = .045
F1,87 = 0.48
p = .490
Experiment 2b
The primary aim of this experiment, carried out at Millbank Fur Farm in the summer of
2013, was to replicate the tests for effects of housing mink in trios in extra-large cages, or in
male-male pairs in standard cages, during the pair phase. In Experiment 1b, R&T had been
found to be more frequent in extra-large than in standard cages, and more frequent in malemale pairs than in female-male pairs. The effect of mixed-strain housing, previously seen in
Experiment 1c, could not be evaluated here because all Millbank animals are of the same strain.
There was no way to avoid confounding cage type (extra-large vs. standard) with
location. Farmers also declined to house male-male and female-male pairs in alternating cages
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within the same areas. Again, then, I resorted to housing these treatments in different locations
than in Experiment 1b, so that location would differ across replicate experiments.
A subsidiary aim of this experiment was to test the prediction that observing juvenile
mink from up close suppresses observed rough-and-tumble play and increases vigilance toward
the observer. This was tested opportunistically, because the location of some subject cages
precluded observation from afar, my preferred method.
Methods
Subjects and housing
Farmers agreed to let me house a limited number of males they had identified as
potential breeders in male-male pairs (see hypothesis in Chapter 5), but a larger sample was
needed to adequately test for an effect of housing type on juvenile rough-and-tumble play. I
therefore observed two separate groups each of male-male and female-male pairs: nonbreeders and potential breeders, with mink allocated to each by farmers. Additionally, farmers
chose to house groups of 3 non-breeder males in extra-large cages (rather than 2 males and 1
female, as in Experiment 1b). Therefore, the appropriate control group for this treatment was
considered to be non-breeder male-male pairs. Thus, females serve as stimulus animals for
male subjects in female-male pairs, while males in other treatments simultaneously serve as
subject and stimulus.
I moved potential breeder pairs as well as non-breeder female-male pairs into pair
housing myself, on July 2nd and 3rd. Pairs of potential breeders were taken from litters born
between April 26th and 30th 2013, containing at least 3 males and 1 female, such that one malemale and one female-male same-litter pair could be formed from each. These were housed in
the same row, with male-male pairs in the 15 cages nearest the door of the shed, and femalemale controls in the next block of 15. One of the purported male-male pairs was later found to
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actually be female-male. Another female-male pair in this row was discarded because one of
the mink died. Non-breeder female-male pairs occupied two different rows in the same shed
and were composed of subjects from standard whelping cages used in Experiment 2a, mostly in
same-litter pairs, all born between April 26th and 28th. Non-breeder male-male pairs had already
been moved into a different row within the same shed by farmers shortly before July 2nd (actual
date unknown), and were all born April 28th or earlier. Because the farmers prioritized the
pairing of littermates, and this year had chosen to wean most non-breeder litters into femalefemale and male-male pairs (as opposed to female-male pairs, as in Experiment 1b), these
were mostly same-litter pairs, though each individual’s litter of origin was not recorded. Subjects
were chosen from within this row such that they would be the same distance from the shed
doors as their female-male controls. Finally, pairs of males from standard whelping cages were
moved into extra-large cages on July 10th, along with another female juvenile. On the morning of
July 12th, farmers removed the females from extra-large cages and replaced them by a third
male, due to a farm-wide male surplus. Extra-large caged male whelping dates are unknown,
and while the first two males in each cage may or may not have been littermates, the third
almost certainly was unfamiliar to them. At this point, I selected additional pairs from the nonbreeder male-male row, to enlarge what had just become the most appropriate control group for
this treatment.
Behavioural observations
I conducted behavioural observations between July 8th and August 16th 2013, when
female-male pairs were 69 to 110 days old (exact ages for other treatments unknown). A
different observer, whom I trained, conducted observations between August 6th and 9th. I
conducted behavioural observations as close to half-hourly as possible (some scans took
longer) between approximately 7:00 and 20:00, for a maximum of 26 scans per day, and a total
of 256 scans per cage. Extra-large cages and extra male-male cages added on July 12th were
105
observed 225 times each. At the farmers’ request, I did not observe the mink during two
afternoon periods when the ambient temperature was 30 °C or over.
The row containing female-male and male-male pairs of potential breeders faced a wall,
and therefore had to be observed from the aisle directly in front of these cages. All other
subjects were observed from one aisle over. Because this was likely to affect their levels of
vigilance, I added an item to the ethogram for Experiments 2b and 2c: mink were classified as
“alert” if they oriented toward the observer, with their eyes open, for the duration of the one-zero
observation period. Mink were also classified as alert if they fled from the observer at the start of
the observation period, then oriented toward him/her for the remainder of the interval.
Data analysis
I used separate ANOVAs to test for effects of male-male and extra-large cage housing
treatments. In the former case, the test included observation method (close vs. far, perfectly
confounded with non-breeder vs. potential breeder status) and its interaction with treatment. In
the latter case, for both the treatment and control groups, I used only data recorded after male
trios were moved into extra-large cages. For this reason, the mean frequency of R&T differs for
non-breeder male-male pairs when used as an experimental group (vs. female-male nonbreeders) or as a control group (vs. extra-large cages). I compared the subset of male-male and
female-male pairs that could be matched for litter using a paired t-test.
Results
Both treatments had unexpectedly negative significant effects on rough-and-tumble play
frequency (Table 3–3). Trios of males in extra-large cages played less than pairs of males in
standard cages, who in turn played less than female-male pairs. Within the litter-matched
subset of potential breeders, the difference in rough-and-tumble play between male-male and
female-male pairs was not significant (5.16 +/- 0.57% vs. 6.20 +/- 0.73%; paired t(12) = 1.35, p
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= .202). In addition to housing treatment, observation distance (or potential breeder status) had
a highly significant effect, with male-male and female-male pairs observed from the aisle directly
beside their cage performing less R&T than those observed from afar (F1,112 = 33.90, p < .0001).
As expected, males in the latter treatment were alert and oriented toward the observer
significantly less often than those observed from nearby (male-male: far 0.71 +/- 0.07% vs.
close 2.46 +/- 0.24%; female-male: far 0.20 +/- .04% vs. close 1.10 +/- .18%).
Experiment 2c
The primary aim of this experiment, carried out at RBR Fur Farm in the summer of 2013,
was to replicate the tests for effects of housing mink in mixed-strain or in male-male pairs,
during the pair phase. In Experiment 1b, conducted at Millbank, R&T had been found to be
more frequent in extra-large than in standard cages. In Experiment 1c, R&T was more frequent
in pastel female – black male pairs than in either type of same-strain pair. RBR Fur Farm does
not have extra-large cages, so their effect could not be evaluated here.
Unfortunately, farmers again declined to place mink from different housing treatments in
alternating cages, preferring to spatially segregate them by treatment. Mixed- and same-strain
pairs were kept in the same shed as in Experiment 1c. However, the location of animals from
each treatment was different than in that experiment.
A subsidiary aim of this experiment was to again test whether observing juvenile mink
from up close makes them more vigilant and temporarily suppresses their rough-and-tumble
play. As in Experiment 2b, this was tested opportunistically, because circumstances prevented
observation of all mink from afar.
107
Methods
Subjects and housing
Farmers separated litters in late June and early July. Mixed-strain pairs and same-strain
control pairs were formed on June 25th and 26th 2013. Black mink in these pairs had whelping
dates between April 24th and 30th; the range of birthdates for pastel mink is unknown, but likely
similar. Male-male pairs and their female-male controls were of the demi colourtype, chosen by
farmers because of a farm-wide male surplus in this strain. Male-male pairs were moved into
pair housing on July 2nd, 3rd, and 6th, while female-male controls had been moved prior to July
2nd (exact date unknown). While their range of whelping dates is unknown, farmers stated that
both demi groups contained mink of similar ages, likely at least as old as the black mink above.
Black and pastel mink occupied one entire shed, filling up four rows of cages. Each row
was separated in half, such that each of the four unique treatments (black-black, black female –
pastel male, pastel female – black male, and pastel-pastel) occupied half of one of the two rows
along the outer wall of the shed, and half of an inner row, in a different part of the shed. Demi
mink, meanwhile, were housed in entirely different sheds: male-male pairs in one row, femalemale controls in two adjacent rows in a different shed.
In same-strain pairs (including demis), cagemates were almost universally from the
same litters, while this was obviously not true of mixed-strain pairs. In all treatment groups, I
selected subject pairs such that no two observed pairs contained individuals from the same
litter. While this was an improvement on Experiment 1c, it was unfortunately the case that as in
that experiment, breeding lines were not evenly distributed between rows, and therefore were
partially confounded with location and treatment.
At the beginning of the observation period, no pairs had access to any environmental
enrichments. On July 17th, farmers spontaneously decided to furnish some cages with a ball
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(golf or whiffle). On the same day, I therefore added a whiffle ball to cages in corresponding
treatments so that enrichment would not differ between treatments. All demi pairs received a
ball, as did five cages within each mixed-strain or same-strain treatment in inner rows.
Behavioural observations
I conducted behavioural observations for four days per week between June 30th and
August 1st 2013. I initially conducted 18 rounds of observations per day, at 40-minute intervals,
but switched to 53-minute intervals (15 scans daily between 6:55 and 20:10) after I began
observing demi mink. The latter were observed a total of 213 times vs. 271 for blacks and
pastels. In one mid-July week, two observation days were cut short after 12 scans due to
extreme heat (on previous days, mink had been profoundly inactive in the heat).
Cages were observed row by row. Demi mink as well as blacks and pastels in rows
along the outer edge of the shed were observed from one aisle over. Blacks and pastels in inner
rows (which faced the shed wall), had to be observed from within the aisle directly adjacent to
them.
Data analysis
I used separate ANOVAs to test for effects of mixed-strain and male-male housing
treatments. In the former case, the test included observation method (close vs. far) and its
interaction with treatment. I then performed planned comparisons between those specific mixedand same-strain pair groups hypothesized to differ, run using the “multcomp” package for R
(Hothorn et al., 2008), with Holm-Bonferroni p value adjustment.
To test for an effect of breeding lines on rough-and-tumble play, I used ANOVAs
controlling for pair type, observation method, and their interaction, with the addition of breeding
line, nested within strain, of either the female or male cagemate. Females’ and males’ breeding
lines had to be tested separately because they were highly correlated, and so that male-male
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pairs could be included. Because these tests included all cages, they used only data taken after
all cages had started being observed. A few pairs were not included due to unknown breeding
line (1 female, 4 male).
Results
Among black and pastel mink, there was a significant main effect of housing treatment
(Table 3–3). Because of a significant interaction between housing and observation method
(F3,242 = 16.84, p < .0001), I examined differences between pairs of housing treatments
separately within each observation method. The significant main effect of housing was still
present within each of these subsets (close: F3,107 = 14.74, p < .0001; far: F3,135 = 5.58, p =
.001). Within subjects observed up close, female pastel – black male pairs played less than
black-black pairs (p < .0001) and did not significantly differ from pastel-pastel pairs (p = .264);
while black female – pastel male pairs also played less than black-black pairs (p = .032), but
more than pastel-pastel pairs (p = .001). In pairs observed from afar, however, pastel-pastel
pairs played more than either black female – pastel male pairs (p = .009) or pastel female –
black male pairs (p = .003), while black-black pairs did not differ from either mixed-strain group
(p = 1.000 in both cases). Thus, multiple comparisons tests showed that main effects of housing
treatment were not the same as in Experiment 1c, and differed according to observation
method.
There was no significant different in rough-and-tumble play between male-male and
female-male demi pairs.
Female breeding line, overall, had a significant effect on rough-and-tumble play (F20,280 =
1.61, p = .049), while the male’s did not (F18,329 = 1.22, p = .246).
As in Experiment 2b, mink observed from close up performed less R&T than those
observed from afar (F1,242 = 203.58, p < .0001), and also spent more time alert. For example,
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black males in outer rows (observed from afar) oriented toward the observer on less than 1% of
scans (with black female: 0.98 +/- 0.12%; with pastel female: 0.92 +/- 0.13%), while those in
inner rows (observed from nearby) did so much more often (with black female: 5.04 +/- 0.41%;
with pastel female: 4.54 +/- 0.39%).
Discussion – Experiment 2
This series of replications completely failed to bear out any of the results obtained in
Experiment 1. Housing treatments previously found to have positive effects on rough-andtumble play frequency now either had no significant effect, or the opposite effect.
While some of the experimental manipulations were somewhat different than they had
been in Experiment 1, none of these changes could have been expected to negate the apparent
play-eliciting effects of these treatments. Extra-large cages contained three males rather than
two males and one female; if anything, this might have been expected to reinforce the positive
effect of extra-large cages, rather than to counter them. Mink of different strains were used to
test male-male housing on both farms in Experiment 2; there was no a priori reason to expect
that the effects of this treatment would differ between black and demi mink. Further case-bycase speculation about such differences does not appear to lead anywhere.
One factor that might more satisfactorily explain the divergence in results between pair
phase tests in Experiments 1 and 2 is their differing observation methods. Mink in Experiment 1
were observed from the aisle directly outside their cage, while this was mostly not the case in
Experiment 2; the data from the current experiment suggest that standing very close to the
subjects temporarily suppresses their rough-and-tumble play, instead motivating them to look at
the experimenter. This not only means that close observation underestimates the actual
frequency of R&T, but that this procedure could introduce systematic bias, if mink in some
treatments are more or less likely to detect and/or be affected by the observer’s presence than
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in others. For example, female mink may be more reactive to the presence of the experimenter
(see Chapter 4), and therefore more likely to remain vigilant rather than play during close
observation. This could be responsible for the apparent positive effect of male-male housing on
R&T, in Experiment 1. However, this does not explain why the same effect was not seen in
potential breeder male-male and female-male pairs in Experiment 2b, also observed from up
close. Trios in extra-large cages may have shown higher rough-and-tumble play than standard
pairs only when closely observed because they were able to stay further away from the
observer than mink in smaller cages and/or because individuals tend to be less vigilant in larger
groups (Roberts, 1996). In the future, then video observations by a blind observer could
alleviate these problems. In the present case, this mode of explanation may offer some
answers, but it is clear that it cannot explain everything.
Finally, another potential explanation for the inconsistency of these results, though
specific to Experiments 1c and 2c, lies in the predominance of different breeding lines (i.e.
families) in each treatment. In Experiment 2c, the breeding line of the female cagemate was a
significant predictor of R&T frequency, independently of housing treatment and observation
method. Therefore, apparent effects of housing treatment may instead be breeding line effects.
These could not be expected to be consistent between experiments, as the relationship
between treatments and breeding lines was not held constant. Unfortunately, the effect of
breeding line could not be verified in Experiment 1c because the necessary data was lost.
Ultimately, the results of Experiment 2 are weak because I was unable to resolve the
principal problem I had faced in Experiment 1: treatments were still confounded with location.
However, the actual locations of mink in every treatment changed between experiments.
Therefore, I suspect that the environmental or management-based correlates of location have
previously unmeasured effects on rough-and-tumble play. These are the focus of the following
exploratory analyses and of Experiment 3.
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Exploratory Analyses
The aim of these analyses is to retrospectively investigate whether cage location affects
the frequency of rough-and-tumble play, and the mechanisms by which it might do so. This will
be done using exploratory analyses of some of the data already presented thus far. Any
apparent relationships between environmental or management factors and R&T emerging from
these analyses will then be formally investigated in Experiment 3.
The sources and quality of the data used for the tests below were different for each
exploratory hypothesis. Accordingly, the statistical methods used also varied as appropriate,
and will be explained case-by-case. While I explored a variety of different hypotheses, I only
present these that led to interesting conclusions, and/or are useful for the design of Experiment
3.
To begin, I simply sought to determine whether there are any regularities in the relation
between cage location and rough-and-tumble play, without yet speculating as to their cause. To
aid in the reader’s understanding, Figures 3–1 to 3–3 show the layout of Millbank Fur Farm and
the inside of a typical shed on this farm. This farm is the one at which most previous work was
conducted, as well as the sole site of Experiment 3, which focuses on effects of location. The
internal layout of sheds at RBR Fur Farm is largely similar, though most sheds there have
whelping cages in the middle rather than in the outer rows.
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Figure 3–1: Satellite image of Millbank Fur Farm. The image was obtained using Google
Earth. Mink sheds are numbered 1 through 8. Each shed is divided into quarters, the first
(Q1) and last (Q4) of which have been labelled on Shed 8. The feed kitchen is labelled ‘A’.
The main farm building, which houses equipment, the lunchroom, and washrooms, is
labelled ‘B’. Employees typically park their vehicles in front of the main farm building and
in the sandy area between this building and the feed kitchen.
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Figure 3–2: Diagram of a typical mink shed at Millbank Fur Farm. The shed is divided into
four aisles (a-c), eight rows of cages, and four quarters (Q1-Q4). Areas shaded in light
grey represent blocks of pair cages; areas in light grey represent blocks of whelping
cages. Dimensions are not to scale (compare to Figure 3–1). The Q1-Q2 and Q3-Q4 gaps
are absent in some rows/sheds.
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Figure 3–3: The inside of a Millbank Fur Farm shed late on a sunny afternoon.
Translucent skylights can be seen at regular intervals along the middle of the ceiling.
Doors at both ends of the aisle have been closed for the night. My dog is begging to go
home.
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To test whether play is systematically higher in certain types of cage rows than in others,
I used a dataset (Experiment 1b) which included several rows of identically-housed subjects:
173 female-male pairs of black mink from standard whelping cages, all observed up close,
spread over 8 rows. Tests were conducted using ANOVAs including row as a random factor.
First, I found that mink in cages that faced roughly south-east (4/8 rows) tended to perform more
R&T than those in north-west facing cages (n = 130, 2.94 +/- 0.12%; n = 43, 2.53 +/- 0.24%; F1,6
= 4.54, p = .077). I then tested whether the same orientation effect could be found in the only
other suitable dataset, Experiment 1a, which included litters in standard whelping cages in 7
different rows. In a GLMEM with the same covariates and factors as standard in Experiment 1a,
with the addition of litter size, and row as a random factor, litters in south-east facing cages (3/7
rows) did not significantly differ from those facing north-west (n = 127, 6.88 +/- 0.16%; n = 135,
6.39 +/- 0.15%; F1,5 = 0.81, p = .410). An additional finding from Experiment 1b was that mink in
rows located in the two middle aisles of a shed (4/8 rows) also tended to perform more R&T
than those in the aisles adjacent to a shed’s outer wall (n = 30, 3.40 +/- 0.33%; n = 143, 2.72 +/0.10%; F1,6 = 4.18, p = .087). This effect could not be tested for in any other dataset: all litter
cages except for extra-large ones are located directly along outer shed walls, and other pair
phase experiments confounded edge/middle rows with treatment and/or observation method. It
should also be noted that mink pairs in rows near the outer wall, but not those in inner rows,
tended to be potential breeders, so maternal quality was partially confounded with location.
Thus, while these results hint at the possible role of location or environment type, they are far
from conclusive.
The timing of feeding and related events might plausibly explain why mink in some areas
engage in more or less rough-and-tumble play: mink are fed once or twice daily, row by row,
and it can take over two hours to feed all the mink on a single farm the size of RBR or Millbank,
allowing for a great deal of spatial variation. As shown in Figure 2–1, mink become very active
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and playful around feeding and related events, peaking most clearly at the afternoon feeding. I
hypothesized that late feeding leads to high levels of R&T by extending the duration of food
anticipation: my prior observations (Chapter 2) suggested that mink play in anticipation of being
fed, and continue to do so after feed is delivered, but soon (c. 30-60 minutes) sink into
postprandial inactivity. In order to test this hypothesis, I examined my field notes from
Experiments 1 and 2 for data on the timing of feeding and related events. Because these often
took place while I was observing mink in a different shed, some data points are missing, and
most are only approximations. In particular, I often failed to notice or note when old feed was
spread (redistributed between cages) or scraped off, unless I happened to be present when this
was done. Overall, the data were not of sufficient quality to test whether overall differences in
R&T between cage rows were related to consistent differences in feeding/spreading/scraping
time. Instead, I used data from Experiments 1b and 2c specifically (those with the highest
number of observation days) to test whether between-day differences in feeding time were
related to between-day differences in R&T. The unit of replication was considered to be the row
(Experiment 1b) or block of cages experiencing the same treatment (Experiment 2c), with R&T
frequency averaged across all subjects therein. Each data point represents the average level of
R&T in a row/block on a given date. I analyzed the relationship between daily R&T and feeding
time using a GLMEM that corrected for row/block as a random factor and for date as a
covariate, to account for age- and season-related changes in mink play and in farmer efficiency.
Days on which observations had to be interrupted due to extreme temperatures were excluded.
The two experiments were analyzed separately due to differences in feeding regimes between
farms (RBR mink – Experiment 1c – are not given fresh feed in the morning).
As expected, there was a positive relationship between afternoon feeding time and R&T
in Experiment 1b (F1,202 = 4.16, p = .043, r = .117). R&T was also positively related to the time at
which old feed was scraped off cage tops in the early afternoon, prior to feeding (F1,66 = 4.16, p
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= .036, r = .233). Scraping and afternoon feeding times were correlated, and neither quite
remained significant when both were analyzed concurrently, though there remained a trend for
an effect of scraping time. Morning events – feeding and spreading of old feed – were not
significantly related to daily R&T levels. Experiment 2c produced quite unexpected results,
however. Here, afternoon feeding time was significantly negatively correlated with daily R&T
(F1,188 = 21.17, p < .0001, r = -0.242), as was morning spreading time (F1,136 = 8.98, p = .003, r =
-0.205). Both these effects remained significant when analyzed concurrently.
Thus, these data suggest that the timing of feeding and related events may influence the
overall amount of rough-and-tumble play performed across days, though the direction of the
effect differed between experiments. It remains unclear, however, if consistent differences in
feeding time between rows would result in corresponding differences in play. While rows are not
fed in the same order every day, there could still be important average differences between
rows. While my data are somewhat patchy, I did run ANOVAs to test whether average
feeding/related times differed between rows/blocks. In Experiment 1b, only afternoon feeding
time was affected by row (F1,11 = 7.73, p < .0001), with subsequent multiple pairwise
comparisons showing that rows in sheds located near the middle of the farm were generally fed
later than rows at the extremes. In Experiment 2c, only morning feed spreading was affected by
cage block (F1,11 = 2.11, p = .023), with no significant pairwise comparisons. This is not
conducive to further analysis.
Finally, ambient temperature is quite likely to affect the frequency of rough-and-tumble
play. Certainly, mink do appear to become extremely inactive on very hot afternoons. In
addition, as shown in Chapter 2, rough-and-tumble play tends to peak around dusk and dawn,
potentially due in part to cooler temperatures. Different sheds, and rows within sheds, could
potentially show differences in ambient temperature, e.g. due to differences in ventilation or
exposure to sunlight. While I did not have the data to test for differences between specific areas,
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it was possible to again test for differences between days using historical temperature records.
For each day of observation in each pair phase experiment (1b-c, 2b-c), I calculated the farmwide average rough-and-tumble play frequency, and obtained peak daily temperatures from
Environment Canada (http://climate.weather.gc.ca/) for the weather stations nearest the farms
(Millbank: Guelph, ON; RBR: Stratford, ON or, when unavailable, London, ON). Days on which
observations were stopped early due to high heat were excluded. All the data were analyzed in
a single GLM controlling for date as a covariate and for farm and year (Experiment 1 vs. 2), and
the interactions of farm and year with temperature. As expected, peak daily temperature was
significantly and strongly negatively correlated with daily rough-and-tumble play (Figure 3–4).
The effect was visible across all experiments, with no interaction between farm or year and
temperature, and the effect remained significant when play was counted as a proportion of
active time only (F1,46 = 15.23, p = .0003, r = -0.393). Thus, day-to-day differences in
temperature strongly affect rough-and-tumble frequency, though it remains to be seen how large
location-to-location differences in temperature are, and what their effects might be.
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Figure 3–4: Mutual rough-and-tumble in relation to peak daily temperature. R&T and
temperature were negatively correlated across days of observation (F1,47 = 16.12, p =
.0002, r = -0.394). These are partialled data, corrected for the effects of date on play and
temperature, representing Experiments 1b (filled black circles; solid black line), 1c
(hollow black circles; dashed black line), 2b (filled grey circles; solid grey line), and 2c
(hollow grey circles; dashed grey line).
Mutual rough-and-tumble play (% total scans)
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Peak daily temperature (°C)
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Experiment 3a
The aim of this experiment, in the summer of 2014, was to test whether the overall
frequency of rough-and-tumble play in juvenile mink litters is affected by location and by
variation in environmental factors across locations.
Methods
Subjects and housing
Millbank Fur Farm consists of 8 sheds, each of which contains two rows of standard
whelping cages along opposite walls, each row being divided into four quarters or blocks of
cages of approximately equal length (Figures 3–1 and 3–2). Within each of these 64 blocks, I
selected the first 5 litters (nearest one end of the shed) that met a list of criteria intended to
balance birth and litter size, fosterlings, dam parity, and whelping date across blocks. Subject
litters contained between 6 and 8 kits and were born between April 28th and 30th 2014.
Behavioural observations
Behavioural observations were conducted as in Experiments 1a and 2a, one row at a
time, between June 13th and 27th 2014. Thus, subjects were between 44 and 60 days old. All
litters had been weaned when observations began. Two litters were excluded from analysis
because the kits were moved to a different location partway during the observation period. A
second observer (not the same as in Experiment 2b), trained by me, performed the
observations in my place on some days. Observations took place every eighty minutes (some
took longer or were missed) between approximately 6:40 and 20:00, pausing between 10:40
and 13:20, a period during which mink in previous experiments played very little. Thus, we
performed a maximum of 8 scans per day (sometimes less), for a total of 65 observations per
litter.
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During some observation rounds, environmental measures were also recorded c. 0.5 m
from the front of each cage, where the observer stood. We measured ambient temperature,
relative humidity, and light intensity using a Shimana SHSEHY002 Environmental Meter. To
allow time for the reading to stabilize at each location, the measure was taken at the end of the
5 second behavioural observation window. To avoid delaying observations, we avoided
switching modes on the meter and in each round recorded only one of temperature, humidity, or
light intensity. There was no set schedule for these different environmental measurements; they
were taken when time permitted (always for every cage within a round of observation) while
attempting to measure each environmental variable at least once per day, and at different times
across days. Ultimately, we performed 11 rounds of light intensity, and 9 each of temperature
and humidity measures. The light meter’s photoreceptor was kept horizontally flat and strapped
to the top of a bicycle helmet worn by the observer, to avoid casting shadows on it. Temperature
and humidity probes were placed horizontally on the observer’s notepad, at the level of the
upper abdomen.
Data analysis
In order to test the relationship between overall levels of rough-and-tumble play and
average levels of different environmental measures (temperature, humidity, or light intensity), I
combined all measurements into a single score per cage per type of environmental measure. An
average of raw values would be inappropriate, as it would put undue weight on high values (e.g.
light intensity measurements taken at noon). Using the median of raw values, meanwhile, would
render any measurements taken at times that produce extreme values across all cages (e.g.
noon and dawn/dusk) useless. Instead, for each individual round of measurement, I computed a
Z score for each cage, i.e. the number of standard deviations above or below the average
measurement for that round. Thus, each measurement of brightness, temperature, or humidity
taken for a given cage is given equal weight, by being translated onto the same scale (zero-
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centred normal distribution). I then took the median of Z scores for temperature, humidity, or
light intensity for each cage as their individual single score, thus minimizing any effect of
abnormally high or low readings attributable to measurement error, rather than to prevailing
conditions at a certain time of day. Unlike temperature and humidity, light intensity measures
were not normally distributed, and were therefore log-transformed before Z scores were
computed. In addition, I visually examined histograms from each round of measurement for
each environmental variable, and found two that showed a clear bimodal distribution. Both were
for light intensity measures, and examination of informal notes taken during these measurement
rounds showed that cloud cover changed partway through and dramatically altered light levels.
These rounds were excluded from the median calculation because Z scores were less reflective
of spatial effects than of temporal ones.
First, to test for effects of location on rough-and-tumble play, I used an ANOVA with
factors orientation (north-west or south-east facing), quarter (1 through 4) and row (1 through
16), the latter nested within shed (1 through 8). Subsequent multiple comparisons tests were
run using the “multcomp” package for R (Hothorn et al., 2008), with Holm-Bonferroni p value
adjustment. Then, to test for correlations between environmental covariates (one by one) and
R&T, I used a GLMEM controlling for quarter (fixed) and row within shed (random). Finally, to
test for consistency of each type of environmental measure, I used an ANOVA with cage (fixed)
and observation round (random), with within-round Z scores as the dependent variable.
Results
Location was correlated with overall rough-and-tumble play frequency. While row
orientation did not have a significant effect (F1,299 = 0.68, p = 0.411), there were significant
effects of quarter (F3,299 = 4.24, p = 0.006) and shed (F7,299 = 4.92, p < 0.0001). Row did not
have an effect independently of shed (F7,299 = 0.94, p = 0.473). Multiple comparisons tests
showed that there was significantly less R&T in the first quarter than in either the third (p = .025)
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or fourth (p = .011) quarters, and that R&T in shed 6 was significantly lower than in sheds 1 (p =
.0002), 3 (p = .006), 4 (p < .0001), and 8 (p = .007), and tended to be lower than in shed 2 (p =
.081). Interestingly, the areas in which R&T was least frequent are those likely to experience the
most disturbance. The first quarter of each shed is the one adjacent to the parking lot, feed
kitchen, and the main farm building, in which employees use the washroom and take lunches
and breaks (Figure 3–1). Thus, it would be most closely exposed to noises from vehicles,
machinery, and staff; at the other end of the sheds lies an empty field. Shed 6, meanwhile, is
located directly in front of the main door used by employees for the main farm building.
The frequency of R&T was also correlated negatively and rather weakly with light
intensity (F1,298 = 4.19, p = .042, r = -0.116) and positively with relative humidity (F1,298 = 6.52, p
= .011, r = .141), and was not significantly related to temperature (F1,298 = 1.51, p = .221, r = 0.068). However, repeated measures analyses suggested that only with respect to light intensity
were individual differences between cages consistent: cage had a significant effect on the Z
score of the log of light intensity (F318,3175 = 9.93, p < .0001), but no significant effect on Z scores
for relative humidity (F318,2541 = 0.47, p = 1.000) or temperature (F318,2544 = 0.97, p = .649).
Average differences in humidity or temperature between areas are either inexistent or too minor
to be detected, considering the amount of measurement error inherent in my method. Thus,
observed between-cage variation in humidity likely consists largely of noise, and its significant
relationship with rough-and-tumble play likely a type I error.
Light intensity was relatively high at cages in the first quarter, because many of them
were near the shed doors, which remain open for most of the day and are one of the main
sources of light inside the sheds. Within this section, light intensity was therefore confounded
with distance from the shed door, and from the associated sources of disturbance. I therefore
reran the test of whether light intensity is correlated with rough-and-tumble play excluding the
cages in the first quarter, leaving only those further away, where this confound was not at play.
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Within this subset, the negative relationship between light intensity and rough-and-tumble play
was no longer significant (F1,219 = 1.51, p = .220, r = -0.080). In contrast, adding light intensity as
a covariate into the prior analysis for an effect of quarter did not change the main result (F3,298 =
4.17, p = 0.006), with the first quarter still playing significantly less than the fourth quarter (p =
.011) and tending to play less than the third (p = .063).
Experiment 3b
The aim of this experiment, in the summer of 2014, was to test whether the overall
frequency of rough-and-tumble play in pairs of juvenile mink is affected by location and by
variation in environmental factors across locations.
Methods
Subjects and housing
I randomly selected female-male pairs from among subjects used in Experiment 3a and
moved them into pair-housing between July 2nd and 5th 2014. I then moved all of them to
different pair cages on July 9th, from which time they were then housed in four different rows,
each in a different shed. While it would have been ideal to use more rows to investigate
location-based effects, other rows were unavailable. At the request of farmers, in order to
minimize disturbance of other mink within these sheds by the observer, all subject pairs were
housed within the first quarter of each row. Wherever possible, several litter-matched pairs were
formed so that these could be compared across treatments. This was done either by drawing
animals from litters containing at least two males and at least two females, or by combining
several surplus females from one female-biased litter with several surplus males from a malebiased litter.
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In order to test whether rough-and-tumble play differs between cage rows, I housed
between 56 and 60 pairs within each row. This included 8 sets of pairs matched for litter across
all four rows, and 14 sets litter-matched across three of the four rows each.
In order to test whether pairs housed very close to the shed doors differ from those
housed in the rest of the row, the first fifteen cages in each row contained subject pairs with a
litter-matched pair further down the row. The latter were distributed relatively evenly over the
remaining ~95 cages in the first quarter. I chose to use the first fifteen cages because this
specifically mimicked the location of the male-male pairs in Experiment 2b, who unexpectedly
played less than female-male controls housed further from the shed doors.
In order to test the effect of light intensity on play, each row also contained five pairs
housed directly under translucent skylights set into the roof – the main sources of light besides
the open shed doors – litter-matched to five pairs housed in the gap between skylights (Figure
3–3). Preliminary measurements had confirmed that areas under skylights are considerably
brighter than those in between.
Finally, in order to test whether feeding time affects rough-and-tumble play, two rows
(shed 6, NW-facing; shed 2, SE-facing) were consistently fed at the very start of the afternoon
feeding period, while the other two (shed 4, NW-facing; shed 7, SE-facing) were fed at the end
of this period between July 23rd and August 3rd, after which feeding times were reversed until the
end of my observations. However, on the first two observation days of the first period (July 23rd
and 25th), equipment malfunctions in the feed kitchen meant that afternoon feeding was not
carried out until during or after the very last scan of the day. No special instructions were given
to workers regarding times of morning feeding, spreading, or scraping, which therefore varied
more or less randomly.
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Behavioural observations
Behavioural observations were conducted as in previous pair stage experiments, one
row at a time from one aisle away, between June 16th and August 19th 2014. Thus, subjects
were between 77 and 113 days old. Observations were performed by me and by two other
observers, both trained by me, including the second observer in Experiment 3a. Observations
took place every forty minutes (some took longer or were missed) between approximately 7:00
and 20:00, pausing between 11:00 and 13:20, a period during which mink in previous
experiments played very little. Thus, we performed a maximum of 16 scans per day, for a total
of 210 observations per pair.
Unlike in Experiment 3a, environmental measures were not recorded for individual cages
during behavioural observation rounds, but instead during separate measurement rounds
conducted during the mid-day break and in between behavioural observation rounds, when
sufficient time was available. Preliminary analysis showed that, as in Experiment 3a, individual
differences in temperature and humidity were not reliably measured. We therefore switched,
beginning July 30th, to taking a single measure of temperature and humidity per row of cages in
each round of behavioural observation. When entering the shed, the environmental meter was
placed near the door, in the shade, on the wooden beam to which the top of all the observed
cages in that section were attached. Data were then recorded several minutes later, when
exiting the shed after observation, such that the readings would have ample time to stabilize.
We obtained a total of 81 readings per row for each of temperature and humidity. We continued
to measure light intensity for each cage individually, as we had before, for a total of 15 readings
per cage. These environmental measures were taken by a total of four observers, including the
three who conducted behavioural observations.
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Data analysis
As in Experiment 3a, logs of light intensity measures were combined into a single
median Z score per cage. A single score per shed or row for each of temperature and humidity
was obtained as the residual of a repeated measures ANOVA. To test for consistency of each
type of environmental measure, I used an ANOVA with measurement location (fixed) and
observation round (random) as independent variables, with within-round Z scores as the
dependent variable.
I used a one-way ANOVA to test for differences in R&T between sheds, followed by
multiple comparisons with Holm-Bonferroni p-value adjustment (“multcomp” R package: Hothorn
et al., 2008). I also then used separate paired t-tests between each combination of rows, using
the subset of subject pairs that could be matched for litter. To test for an effect of being in the
first fifteen cages of a row, I used both an ANOVA controlling for row as a random factor in my
full sample, and a separate test additionally controlling for litter as a random factor in the subset
of litter-matched pairs. To check whether feeding time affects R&T, I used a repeated measures
two-way ANOVA, predicting a significant interaction between feeding block (rows 4 and 7 vs.
rows 2 and 6) and time period (before vs. after reversal of feeding order). I used a similar test to
compare pairs living beneath a skylight with their litter-matched pairs housed between skylights.
Finally, I tested for effects of environmental measures on R&T using GLMEMs controlling for
shed as a random factor.
Results
The frequency of rough-and-tumble play was significantly affected by shed (F3,226 = 4.22,
p = .006). Multiple comparisons showed that rough-and-tumble play was less frequent in shed 2
than in sheds 6 (p = .011) and 7 (p = .017). Paired t-tests among litter-matched pairs were only
partially consistent with these results (6 vs. 2: t(28) = 2.18, p = .047; 7 vs. 2: t(30) = 1.71, p =
.107; all others non-significant).
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Pairs in the first fifteen cages of a row (8.49 +/- 0.28%) and those further from the shed
door (8.43 +/- 0.16%) did not significantly differ in rough-and-tumble play frequency (F1,225 =
0.05, p = .831). Neither was this difference significant when they were compared only to littermatched pairs (8.63 +/- 0.29; F1,56 = 0.08, p = 0.780).
Feeding time did not alter the relationship between row and rough-and-tumble play. In a
repeated-measures analysis, there was no significant interaction between feeding block and
time period (F1,228 = 1.00, p = .318).
There was no significant difference between pairs located directly beneath a translucent
skylight and litter matches located between them (beneath: 9.00 +/- 0.44%; between: 8.10 +/0.41%; F1,56 = 2.29, p = 0.146), despite cages beneath skylights being considerably brighter
(F1,56 = 119.63, p < .0001). There was, however, an unexpectedly positive relationship between
light intensity and rough-and-tumble play over all individual cages (r = .240, F1,225 = 13.91, p =
.0002). This was largely due to the low level of R&T in shed 2, which was also very dark
because an attic-like storage area blocked much of the light coming from the skylights. When
shed 2 was excluded from the analysis, the relationship was no longer significant (r = .059, F1,169
= 0.61, p = .437). Individual differences in light intensity between cages were consistently
measured (F232,3248 = 132.56, p < .0001).
Finally, there were also trends for a relationship between rough-and-tumble play
frequency and temperature or relative humidity (measured for each shed/row as a whole). R&T
was unexpectedly positively related to temperature (r = -0.229, F1,2 = 12.61, p = .071), and
negatively to relative humidity (r = .231, F1,2 = 12.89, p = .070). This, again, appeared to be
entirely due to the low levels of play in the extremely dark, cold, and humid shed 2. However, as
in Experiment 3a, neither measures of temperature (F3,133 = 0.82, p = 0.485) nor relative
humidity (F1,133 = 1.05, p = 0.374) proved to be consistent.
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Experiment 3c
The aim of this experiment was to test whether the overall frequency of rough-andtumble play in litters and pairs of juvenile mink is affected by variation in environmental factors
across locations, across all previous experiments.
This was a retrospective analysis of subjects observed in Chapter 2 as well as in
Experiments 1 through 3 of Chapter 3. Environmental measures were taken during and after
Experiment 3b, with the assumption that relative differences across location had remained
consistent since the original experiments were carried out. There is no obvious reason to
believe that this is not the case – e.g. no renovations were carried out and no new installations
added to these areas that would have affected environmental measures.
Methods
Subjects and housing
Details of subject selection and housing for the litters and pairs used here can be found
in the corresponding section of previous experiments. A small number of subjects from
Experiment 1 were not included here, as the exact locations of their cages were unknown.
Subjects from Experiment 2a were not included at all for the same reason.
Behavioural observations
Details of behavioural observations can be found in the corresponding section of
previous experiments. Measures were taken by a total of five individuals, including the four who
did so in Experiment 3b.
Light intensity was measured between August 3rd and 19th at Millbank Fur Farm, and on
August 21st at RBR Fur Farm. This was measured individually for each cage, as in Experiments
3a and 3b, with all cages used in any given past experiment being sampled within the same
round of measurements. Thus, measures were most readily comparable across locations within,
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rather than between, experiments. Each location was sampled between 4 and 6 times at
Millbank, and 7 times at RBR Fur Farm. Again, there was no set schedule for these
measurements. For cages in Experiments 3a and 3b, I used the same data I have already
presented.
Relative humidity and temperature were measured separately, in rounds which included
measures taken at every sampling location on a given farm. At Millbank, there was one
sampling location for each aisle (between two rows of cages; see Figure 3–2) in which subjects
had been housed in any previous experiment: the experimenter walked about 10 metres into the
first quarter of the row and waited till the meter stabilized before recording a reading. While it
would have been ideal to obtain separate measures for other quarters of each row, this would
have increased the total time needed for each round, allowing more opportunity for measures to
be spoiled by sudden changes in temperature or humidity. The same procedure was used at
RBR Fur Farm, though because a smaller number of aisles were measured, I sampled four
different points within each (c. 1/8, 3/8, 5/8, and 7/8 of the way through), and took separate
measures for each side of the aisle, with each cage being assigned values from the nearest
measured location. While temperature and relative humidity were each measured 6 times per
location at RBR Fur Farm, at Millbank we obtained 13 measures of temperature per location,
but only two measures of relative humidity. This disparity was due to a miscommunication
between myself and the other observers.
Data analysis
Logs of light intensity measures were combined into a single median Z score per cage,
as in Experiments 3b and 3c. In some cases, separate rounds of measurement carried out for
cages from separate experiments may have included the same cages twice, in which case
these cages have different Z scores within each experiment. This is because each Z score is
computed relative to other cages measured in the same round (thus controlling for factors like
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time of day and cloud cover). As in Experiment 3b, a single score per sampled location was
calculated for each of temperature and humidity, as the residual of a repeated measures
ANOVA. To test for consistency of each type of environmental measure, I used an ANOVA with
measurement location (fixed) and observation round (random), with within-round Z scores as
the dependent variable.
I tested each experiment separately for a relationship between light intensity and mutual
rough-and-tumble play using a GLMEM that controlled for cage row as a random factor and, in
experiments where these varied, for housing treatment and/or observation method as fixed
factors. Then, I tested for an overall relationship across all experiments a GLMEM that
controlled for the factors above, nesting cage row within experiment (and thus farm and life
stage). For temperature and humidity, I only performed an overall test, across all experiments,
as between-cage variation was not measured. The GLMEM was the same as that used for light
intensity, except that the unit of replication was not the individual cage, but the cage row (at
Millbank) or cage row quarter (at RBR). Mutual R&T frequency was averaged over all subjects
within each of these areas. As in previous experiments, arcsine-square-root or box-cox
transformations were employed where necessary to satisfy the assumptions of parametric
testing.
Results
Within individual experiments, only two yielded a significant relationship between light
intensity and mutual R&T. These two have already been described – a negative relationship in
Experiment 3a, and a positive one in Experiment 3b. In the global model, across all
experiments, light intensity had no significant effect (r = -0.022, F1,2052 = 1.02, p = 0.312).
Individual differences between cages in light intensity were consistent (F1534,8661 = 19.87, p <
.0001).
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There was an unexpected trend for a positive relationship between temperature and
mutual R&T (r = .127, F1,52 = 2.82, p = 0.099). However, as in Experiments 3a and 3b,
differences in temperature across areas were not consistent (F47,409 = 1.08, p = 0.343). Relative
humidity, meanwhile, did not have any significant effect on R&T frequency (r = -0.112, F1,52 =
1.78, p = 0.188), though it was consistently measured (F47,128 = 3.11, p < .0001).
Discussion – Experiment 3
This experiment failed to reveal a general relationship between features of the
environment and the frequency of mutual rough-and-tumble play. Exploratory analyses
conducted after Experiment 2 suggested that location might somehow affect levels of R&T. In
line with this, subjects in Experiment 3 showed significantly different R&T frequencies across
areas of the farm, despite living in identical housing treatments. These location effects were
partially replicated even in litter-matched sub-samples in Experiment 3b, showing that location
can affect play even independently of the co-occurring differences in breeding line or maternal
quality that plagued some previous experiments in this chapter. Overall, however, the data do
not point to any particular explanation for such location-based differences, which remain
idiosyncratic and unique to each experiment.
Attempts to identify effects of light intensity on R&T did not produce consistent results.
Sheds, rows, and cages showed large individual differences in light intensity. In Experiment 3a,
I found the predicted negative effect of light intensity on rough-and-tumble play in litters.
However, I found the inverse relationship among pairs of mink in Experiment 3b, and no overall
effect when testing across all previous experiments. The observed effects in Experiments 3a
and 3b may have been artefacts of some other location-based effect, as in both cases play was
very low in notably bright (3a) or dim (3b) areas, and no significant relationship remained
outside of these areas. Furthermore, in Experiment 3b, pairs housed directly under skylights did
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not play any more or less than litter-matched pairs housed in the consistently dimmer areas
between skylights.
Meanwhile, not only did temperature and relative humidity have inconsistent
relationships with rough-and-tumble play, but I also mostly failed to obtain consistent individual
differences between areas for these variables. First, temperature had no significant effect on
litter R&T, a positive effect in pairs, and a trend for a positive effect across all previous
experiments. This was not the expected result, as previous exploratory analyses had shown
convincingly that pair-phase mink are less active and perform less rough-and-tumble play on hot
days. The obtained positive relationships are likely Type I errors, as I never managed to
measure consistent individual differences in temperature between areas, despite refining my
methods between experiments. Besides some component of noise in the measurements, this is
likely also due to any actual differences between areas being quite minor: for example, among
all aisles used in any previous experiment at Millbank Fur Farm, the largest average difference
between two aisles was just under half a degree Celsius. Extrapolating from the slope of the
inter-day relationship between temperature and play found in exploratory analyses (Figure 3–4),
this could account for a mere 0.1% absolute difference in R&T time budget between these
areas, hardly enough to generate the significant differences observed between areas and
treatments in this chapter. Relative humidity, for its part, was positively related to R&T in litters
and negatively in pairs, though individual differences between areas were not consistently
measured in either case. It is difficult to extract any meaning from these results. Across all
previous experiments and the areas used therein, measurements of relative humidity provided
consistent individual differences, and relative humidity had no significant relationship to R&T.
The results of Experiment 3a suggest a possible alternative explanation: R&T was least
frequent among litters housed in areas – shed 6 and the first quarter of cage rows – that tend to
be subjected to high levels of human disturbance. While this does square with the finding that
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observing juveniles from up close temporarily suppresses their play, this post hoc explanation
cannot be carried much further. First, other differences between locations have no obvious
relation to levels of disturbance: in Experiment 3b, it was in shed 2, not 6, that play was very
infrequent. Second, differences between locations are not necessarily consistent between
experiments: for example, mink in shed 2 showed rather high levels of play in previous
Experiments 1b and 3a, and in the former they were even housed in the exact same cage row
as in Experiment 3b. Third, in Experiment 3b, subjects housed nearest the door of the shed –
and therefore especially likely to be disturbed by farm staff and machinery – did not play at
particularly low levels. Finally, and most importantly, levels of disturbance were not formally
measured, and can only be guessed at based on general impressions, making statistical
analysis difficult, especially using data retroactively.
Another possible factor – differences in the times at which different cage rows were fed –
could account for differences in R&T, even if location effects vary from experiment to
experiment, as farmers might also have changed their feeding routines. Exploratory analyses
provided contradictory conclusions about the day-to-day effects of early or late feeding: across
observation days, afternoon feeding time was positively correlated to R&T at Millbank Fur Farm,
but negatively correlated to R&T at RBR. In Experiment 3b, I experimentally tested whether
such effects might also apply to chronic differences in feeding time between areas. Some cage
rows were consistently fed early while others were consistently fed late, and this order was
reversed partway through the observation period. This did not seem to have any effect on
rough-and-tumble play.
Finally, one of the tests in Experiment 3b was for a particular type of location effect: does
being housed in the first fifteen cages of a row, nearest the shed doors, depress play? I did not
run this test to find a general explanation for location-based effects on play, but rather as an
attempt to account for an inconsistency between Experiments 1b and 2b. In the former, male-
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male pairs played more than female-male pairs, while the opposite was true in the latter, which
included a sub-sample of male-male pairs housed in the first fifteen cages of a row. However, in
Experiment 3b, whether they were compared to all other subjects or only to litter-matched pairs
housed further up the row, subjects in the first fifteen cages did not differ in R&T. As with the
rest of Experiment 3, these data shed little light on my previous results, and do not help resolve
the inconsistencies between Experiments 1 and 2.
General Discussion
The experiments presented in this chapter did not allow me to fulfill my primary aim: to
identify experimental housing treatments that chronically elevate or depress the frequency of
rough-and-tumble play over the juvenile period of farmed mink. In Experiment 1, I identified
three candidate treatments with elevated levels of rough-and-tumble play: extra-large cages
(during both the litter and pair phase), male-male pair-housing, and mixed-strain pair-housing.
When I replicated these treatments in Experiment 2, however, I obtained largely opposite
results.
The most plausible explanation for these conflicting results is that at least some of them
were in fact Type I errors, false positives brought about by uncorrected multiple testing and/or
pseudo-replication (Hurlbert, 1984). For all three of these treatments, management constraints
prevented me from using appropriate experimental design, which demands that treatment and
control cages be interspersed or distributed randomly in space. Instead, treatment and control
subjects had to be housed in different rows of cages, or even in different sheds entirely. This
likely allowed external factors, varying between locations, to differentially influence the
frequency of rough-and-tumble play in different treatment groups, independently of the effects of
cage size or group composition I was actually attempting to measure. Unfortunately, however,
Experiment 3 did not allow me to identify which environmental influences might be responsible:
ambient light intensity had no consistent effect on rough-and-tumble play; neither did
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experimentally manipulating feeding times; and temperature and relative humidity differed
between areas in only minor and largely inconsistent ways.
That being said, I did obtain some interesting negative results. I was able to use proper
experimental design to test several housing treatments for their potential to modulate roughand-tumble play frequency, while avoiding location-based confounds: male-biased litters did not
play more than female-biased ones; neither did litters housed with balls and elevated bunks,
relative to those without (similar to my result in older mink in Chapter 2); there was no effect of
being pair-housed with a stimulus animal raised in an extra-large cage, nor with an animal from
a very playful litter, nor with one’s own littermate; and neither the stimulus animal’s body mass,
nor the difference between cagemates’ masses, had any effect. These results can not only help
guide future work, but some of them are interesting on a fundamental level: for example, given
the widespread sexual dimorphism in rough-and-tumble play across species, the complete lack
of a sex ratio effect is quite surprising. Two other treatments were tested without location-based
confounds, but likely suffered from other problems: mink in small litters appeared to play more
than those in large litters, but this may be due to observation error; and while anogenital
distance did not predict rough-and-tumble play, measurement error may have obscured some of
the true individual differences in this trait.
Some other housing treatments that could affect rough-and-tumble play were not tested
here. First, playback of juvenile mink chuckle vocalizations could potentially stimulate roughand-tumble play (Held and Špinka, 2011), but I was unable to attempt this because of concerns
by farmers that it might instead elicit aggression. Second, an experimenter could exploit agerelated variations in playfulness (e.g. Vinke et al., 2005) by pair-housing a subject with a series
of stimulus animals of particular ages. Given that the gap between the earliest and latest
whelpings on a mink farm can exceed two weeks, subject animals born early in the season
could initially be pair-housed with stimulus animals of their own age, then re-paired at c. 10
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weeks of age with younger 8-week-olds; thus, just as they neared the end of their age-related
peak in play, they would be introduced to a partner just entering theirs. Third, my exploratory
analyses demonstrated a strong effect of day-to-day variations in ambient temperature, with hot
days resulting in high inactivity and infrequent rough-and-tumble play. While spontaneous
differences in temperature between areas of the farm were minimal or inconsistent,
experimental manipulation of temperature could allow one to test whether the observed day-today effect can translate into a chronic one. Finally, Experiment 2 showed that mink inhibit their
rough-and-tumble play and increase their vigilance in the presence of a human observer
standing near their cage, as opposed to one row away. Similarly, in previous work on adult
mink, the frequency of stereotypic behaviour was lower when animals were observed by a
human standing in front of the neighbouring cage than in video recorded with no human
present, though this was only true for the subset of animals who were least stereotypic
(Svendsen et al., 2004). Some (though not all) of the results of Experiment 3 suggest that areas
which experience frequent disturbance from staff and farm machinery are also those in which
subjects play least often. An experimental program of frequent disturbance (e.g. via playback of
various noises) might depress the frequency of play, particularly if it was difficult for animals to
become habituated to it.
To conclude, the experiments in this chapter were meant to identify experimental
treatments that could be used as the first step in experimental tests of two hypotheses about the
function of rough-and-tumble play, detailed in Chapters 4 and 5. As this was not accomplished, I
must instead fall back on considerably weaker correlational tests of these hypotheses (see
Chapter 1).
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Chapter 4 – Decreasing Fearfulness through Play
Abstract
I tested the hypothesis that rough-and-tumble play, by repeatedly exposing juveniles to
surprising situations and temporary losses of control over their movements, prepares them to
cope with unexpected, potentially dangerous situations. Using subjects previously observed as
juveniles in Chapters 2 and 3, I tested the prediction that juvenile rough-and-tumble play (R&T)
would be negatively correlated with fearfulness toward human handlers in sub-adults/adults. I
assessed fearfulness using three different tests: approach to vs. withdrawal from an
approaching experimenter (stick/glove tests); screaming when handled (handling test); and fecal
corticosteroid metabolite production in response to capture and 30 minutes of holding in a
restrictive carrying cage (carrying cage test). The frequency of juvenile rough-and-tumble play
was not predictive of reactions in any of the three tests. While this correlational study provides
no support for the stated hypothesis, further experimental research will be required to properly
evaluate it.
Introduction
Much of what makes animal play so much fun to watch – and an alluring topic of study –
is the behaviour’s spontaneous, fluid, unpredictable, and frankly goofy nature. This quality was
eloquently, if hyperbolically, captured in Robert Fagen’s (1982) speech entitled “The perilous
magic of animal play.” Among the many hypotheses about play’s evolutionary functions, one
family in particular seems directly inspired by this. These propose that play movements are
awkward, exaggerated, or reckless because the role of play is to force animals into situations
that demand flexible behaviour, thus allowing them to learn how to react to future unpredictable
challenges. In this view, play is conceptualized as “training for the unexpected”, a mechanism
that promotes behavioural flexibility (Špinka et al., 2001).
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This is, in large part, based on an argument from design. By twisting their bodies in midjump, or by flopping onto their backs in the middle of a chase, playing animals are creating just
the types of situation that may help them learn – in a safe context – how to recover from a trip or
loss of balance in a future life-and-death situation (Špinka et al., 2001). Even reckless play that
carries a minor risk of injury may be beneficial: playfully leaping for a branch that is out of reach
may teach a monkey how to deal with the eventuality of grabbing onto a broken branch, for
example (Graham, 2011). Rapid head movements, such as rotations or jerks, are extremely
common in play across many taxa, and likely hinder visual perception and vestibular orientation
(Petrů et al., 2008). Playing animals regularly seem to be deliberately putting themselves into
difficult situations. In rough-and-tumble play, for example, young laboratory Norway rats (Rattus
norvegicus) often leave themselves vulnerable to sudden reversals by pinning their opponent
with all four paws, a highly unstable position, though they could pin them more solidly by
anchoring their hind legs on the ground, as older rats tend to (Foroud and Pellis, 2003; Pellis
and Pellis, 2009). These and other forms of self-handicapping may increase the difficulty or risk
of play movements, and make it more likely that flexible behaviour will be required to regain an
advantageous position. Social play is particularly likely to generate situations of uncertainty, due
to the unpredictability of each partner’s offensive and defensive manoeuvres (Pellis and Pellis,
2009). Considering all of this, Špinka et al. (2001) quipped that if the function of exploration is
“to learn how to avoid getting into trouble”, the function of play may be “to learn how to get out of
trouble”.
Human and animal researchers have put forward several variations on this theme,
differing in how and what individuals learn once they have successfully gotten themselves into
trouble (Bateson and Martin, 2013; Eibl-Eibesfeldt, 1982; Fagen, 1981; Fedigan, 1972; Pellis
and Pellis, 2009; Poirier, 1982; Špinka et al., 2001; Sutton-Smith, 1997; Vandenberg, 1981).
Primatologist Linda Fedigan (1972) proposes that play is an iterative cycle of probing and
receiving feedback from the environment, through which animals not only practice species141
typical behaviours, but also test the consequences of novel variations on these behaviours. The
frequent recombination of actions from different behavioural contexts in play may be particularly
conducive to generating such innovations (Fagen, 1982b; Špinka et al., 2001). Similarly, for
developmental psychologist Brian Sutton-Smith (1997, 2002), play is an action selection
mechanism, in which individuals physically and/or mentally simulate different courses of action
and their eventual outcomes in order to learn which are likely to be successful. While these
ideas are clearly applicable to the learning and refinement of new physical movements, they are
also compatible with previously-reviewed hypotheses (see Chapter 1) about short-term benefits
to learning or insightful problem-solving (Birch, 1945; Köhler, 1925; Schulz and Bonawitz, 2007).
Several authors have focused on the role of play in facilitating creative thinking and innovation,
or cognitive flexibility, which may even be transferable to contexts far removed from situations
experienced in play (Bateson and Martin, 2013; Dansky and Silverman, 1975). Animals may be
learning through play not only “how to get out of trouble,” but also how to take advantage of
novel opportunities.
While I have thus far emphasized the potential function of play in selecting and
practicing behaviours that aid survival and reproduction, some authors have stressed its role in
regulation of affect. Sutton-Smith (1997, 2002), foremost among these, explicitly states that it is
the emotional outcomes of simulated play actions that determine whether individuals discard,
repeat, modify, or adopt them as part of their behavioural repertoire. In his writing, the horror of
being eaten looms much larger than the attendant loss of reproductive potential. The secondary,
muted emotions experienced in play are sufficient for learning, while sparing us the raw
emotional brunt of the “real”, non-play experience (Eibl-Eibesfeldt, 1982; Sutton-Smith, 2002,
1997). At the time of his death, earlier this year, Sutton-Smith was reportedly working on the
aptly titled book “Play as Emotional Survival” (Sutton-Smith, 2008). Špinka et al. (2001) add
that, in addition to developing motor skills or “kinematic flexibility”, play allows animals to
experience surprise, fear, or disorientation in relatively safe contexts and successfully regain
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control of the situation. Thus, they propose, animals develop emotional resilience that allows
them to cope successfully with a wide variety of unexpected or frightening stimuli later in life.
This tempering of negative emotions may be adaptive, as acute stress can not only have
negative effects on cognitive performance (e.g. in terms of working memory: Roozendaal et al.,
2006; Shields et al., 2015; Taverniers et al., 2011), but also degrade the performance of learned,
skilled motor tasks (Metz et al., 2005; Pellis, 2002b).
Almost by definition, hypotheses that propose that play promotes behavioural flexibility
generate very wide-ranging predictions, which often are shared with competing hypotheses. For
example, developing the capacity to react quickly and adequately to another individual’s
behaviour could conceivably aid in predator-prey interactions, agonistic encounters, and
courtship – effects that are also predicted by hypotheses that play constitutes training for
specific types of behaviour. Thus, Špinka et al.'s (2001) exhaustive list of 24 predictions of the
“training for the unexpected” hypothesis is especially helpful. Most of the predictions that
usefully diverge from those of alternative hypotheses call for design-based evidence. For
example, social play between familiar individuals should be more frequently initiated by the
smaller partner, for whom the interaction is likely to be most beneficial due to the physical
disadvantage. Furthermore, play should be most frequent in animals most likely to face
dangerous, unpredictable situations necessitating a flexible response, for example: within
individuals, after encounters with novel or frightening stimuli or environments (e.g. Mintline et
al., 2012; Newberry et al., 1988); within species, in whichever sex is most likely to later
encounter unexpected situations, e.g. the dispersing sex; and, across species, in those that live
in less stable habitats. In terms of predictions requiring experimental evidence, play is expected
to result both in: more competent behaviour in response to unexpected or unpredictable events,
such as better self-righting when thrown off-balance, lower likelihood of conflict escalation with
rivals, and more successful predator evasion; and in less emotionally reactive behaviour, such
as more rapid exploration of and habituation to novel objects, and more muted behavioural and
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physiological responses to frightening stimuli. In addition, the hypothesis predicts that playing
should result in developmental changes in brain areas involved in flexible motor behaviour. The
predictions concerning kinematic or motor flexibility have not been extensively tested. While
some results do provide qualified support – for example, play frequency in juvenile Belding’s
ground squirrels (Urocitellus beldingi) was positively correlated with improvement in ability to
remain perched atop a thin wooden rod (Nunes et al., 2004) – these can as readily be explained
by hypotheses that play improves specific skills or provides all-around motor training.
In contrast, there does exist correlational and experimental evidence that play enhances
behavioural flexibility in terms of creativity, emotional reactivity, and coping skills. In humans
(Homo sapiens sapiens), playfulness as rated by subjects themselves (for adults) or by teachers
(for children) is positively correlated with ratings of creativity; in adults, it further predicts a
performance-based measure of creativity: the total number of uses subjects can devise for a
household object (Bateson and Nettle, 2014; Tegano, 1990). Children allowed to play with
objects shortly before a testing session, compared to controls who completed a non-play task,
similarly listed more creative uses for a household object (not one available during the play
session), and showed more varied, original approaches to solving open-ended problems
(Dansky and Silverman, 1975; Pepler and Ross, 1981), though methodological problems cast
some doubt on these conclusions (Lillard et al., 2013). A sizable body of work by developmental
psychologists and education researchers, usually using survey-based ratings by parents or
teachers, further indicates positive correlations between playfulness and ability to cope with
everyday stressors (Chang et al., 2013; Christian, 2012; but see also Staempfli, 2007). In one
study, notable for rating playfulness based on video observation, playfulness positively predicted
teacher ratings of preschoolers’ coping success in a variety of situations, for example when
another child took their toy away (Saunders et al., 1999). Corroborating evidence is available
from play-deprivation experiments in animals. Socially-isolated rats entered an open field more
slowly, manipulated novel objects less, and habituated more slowly than socially-reared rats
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(Einon et al. 1975, Einon and Morgan 1976). The behaviour of partial isolates, given one hour of
social contact per day, which was largely spent playing, was intermediate between that of
isolates and socially-reared rats, except if their companions were made non-playful using drugs,
in which case they resembled the isolates (Einon et al. 1978). In a separate series of
experiments, rhesus macaques (Macaca mulatta) were reared in isolation with either a
stationary or mobile “cloth mother” (a bottle covered in synthetic fur); in the latter condition, the
“mother” was attached to a robotic arm and moved around the cage in unpredictable directions,
at irregular intervals (Mason and Berkson, 1975). Infants with mobile mothers directed ten times
more rough-and-tumble play behaviour at these than did infants with stationary mothers; they
also showed a lower latency to enter a novel arena, were less likely to defecate during this test,
and tended to approach humans more readily. While the two groups did not differ in
manipulation of a novel object, in a later experiment where (with a similar rationale) infants were
reared either by an adult female dog (Canis lupus familiaris) or with an inanimate hobbyhorse
(with locked wheels), dog-reared animals were quicker to approach a novel puzzle-feeder, and
more strategic and successful in solving this and other tasks (Capitanio and Mason, 2000).
Finally, a currently unpublished experiment produced remarkable results along the same lines in
puppies: litters provided with toys and structures meant to elicit play performed more object and
social play than did litters deprived of toys and structures; after 18 months, dogs from enriched
litters more readily explored a novel environment, had less pronounced fearful reactions to
visual and auditory stimuli, sought less support from their owners in these situations, and
performed better in a cognitive task (Claudia M. Vinke, personal communication, December
2011). Thus, a number of correlational and experimental studies do suggest that playing is
associated with more cognitive and emotional flexibility.
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Project overview
The aim of this project was to evaluate the hypothesis that play trains juvenile American
mink (Neovison vison) to cope with unexpected, potentially dangerous or frightening situations. I
intended to compare various outcomes in subjects who, as juveniles, had been assigned to
housing treatments that elicit different amounts of play. To my knowledge, this was to be the
first such experimental test not utilizing a play-deprivation paradigm. Unfortunately, as detailed
at length in Chapter 3, none of my housing treatments had unequivocal effects on play. I am
therefore restricted to looking for correlational evidence, less convincing than experimental
evidence would be.
Here, rather than test the hypothesis of play as training for the unexpected writ large, I
restrict my focus to the potential of rough-and-tumble play to lessen emotional reactivity. I chose
rough-and-tumble play because, as seen in Chapter 2, it is by far the most frequent type of play
in farmed mink, and because the involvement of a playmate makes it inherently unpredictable
(Pellis and Pellis, 2009). I chose to focus on effects on emotional reactivity because, if the
predictions are supported, my findings could indicate practical solutions that would improve the
welfare of a species farmed in tremendous numbers. Thus, I used tests of fearfulness meant to
assess reactivity to handling or disturbance by humans, a frequent aversive experience of
farmed mink.
I also tested an alternative hypothesis that could account for any observed correlations
between juvenile play and adult fearfulness. As shown in Chapter 3, observing animals from
near their cages both decreased observed rough-and-tumble play and increased time spent
“alert”, attending to the observer. If this is especially true of the most fearful juveniles, and if
these then become fearful adults, the predicted negative relationship between play and later
fearfulness could be an artefact of the observation method. I therefore used alert behaviour
(recorded in Experiments 2b and 2c) as a proxy of play suppression in juvenile mink. Note,
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however, that this assumption could very well be false: Jeppesen and Falkenberg (1990) found
that juvenile mink who spent the most time in “curious” behaviour (looking at the observer from
near the door of the home cage) were those who performed the most social play, were most
active, and spent the least time in their nest box.
Methods
Subjects and housing
I conducted this research using a subset of the subjects from the experiments related in
Chapters 2 and 3. Those from Chapter 2 were tested for fearfulness as part of a wider
experiment on the effects of practical, inexpensive enrichments (Meagher et al., 2014). For
those from Chapter 3, because of my initial desire to compare animals from high- and low-play
housing treatments, the animals tested were drawn from treatments that differed in rough-andtumble play frequency. In some cases, in order to increase sample size in tests for treatment
effects, I added subjects within each treatment group who had not been observed as juveniles,
but who had been housed in the same manner and in the same area as those I had observed.
These are noted separately where they occur.
After the conclusion of summer behavioural observations, subjects typically remained in
pair-housing until pelting season, in late November and early December, at which time most
animals were killed. Those that remained, having been selected for breeding, were then moved
and singly housed in cages much like the ones they had previously occupied. For some
treatment groups at RBR Fur Farm, however, female-male pairs were separated in early
autumn, with males remaining in the original cage and females temporarily occupying a
separate, larger whelping cage until pelting.
The groups of animals used, and any differences in their autumn housing, will be listed
separately for each test below. Unless otherwise specified, all observed animals from within a
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treatment group were tested, except for any that were lost (e.g. died or were moved) between
the end of summer behavioural observations and the date of testing.
Stick and glove tests
As an indicator of fear of human handlers, I evaluated the reactions of subjects to an
experimenter approaching their cage, using either the ‘stick test’ (Chapter 2 animals) or the
similar ‘glove test’ (Chapter 3 animals). The stick test provides a widely-used and well-validated
measure of fearfulness in mink, known to correlate with fearfulness as measured in a variety of
other contexts (e.g. Hansen and Møller, 2001; Korhonen et al., 2002; Malmkvist and Hansen,
2002; Meagher et al., 2014). In the stick test, an experimenter silently approaches and inserts
the tip of a short wooden stick (e.g. a popsicle stick or coffee stirrer) through the metal mesh at
the front of an animal’s cage. The subjects’ reactions to the stick are then classified as
“aggressive”, “confident”, “fearful”, or “unresponsive” (see below). Because the incidence of
fearful reactions in this test tends to be fairly low in adult mink, particularly in males (e.g.
Andersen, 2013), the more aversive glove test, less prone to floor effects, involves inserting into
the cage the tip of one finger of a used leather handling glove, rather than a stick (Meagher et
al., 2011).
After insertion of the stick/glove stimulus, the animals within the cage were observed for
up to 30 seconds, until their initial reaction could be qualitatively scored using the following
categories: “aggressive” if the animal lunged at and bit the stimulus, “confident” if the animal
approached and sniffed or otherwise made contact with the stimulus, without a hard bite;
“fearful” if the animal either retreated from the stimulus or, if not in a position to retreat (already
at the back of the cage, or whole body including the head inside the nest box), stayed immobile
and oriented toward the stimulus for the duration of the test; or “unresponsive” if the animal
oriented toward the stimulus at least once but did not clearly show any of the above responses.
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Animals were given a minimum of 10 seconds to react after they first oriented toward the stick,
even if this prolonged the usual 30 second period.
Tests were conducted in the mornings, immediately after feeding (Millbank Fur Farm) or
redistribution of feed remaining from the previous day (RBR Fur Farm). This was done to
minimize the number of inactive or sleeping animals. Subjects housed in the same row of cages
were tested consecutively and in order, except for some RBR Fur Farm subjects housed in two
rows that faced each other across the same aisle, allowing the experimenter to alternate
between rows. Animals were tested repeatedly across two or more days (depending on the
experiment), with the order of testing (both between and within rows) being reversed from one
day to the next. Only for stick tests (Chapter 2 animals) was the order not reversed between
days. Some of the tested subjects lived in adjacent cages, and so would have had some
opportunity to habituate to the experimenter while their neighbour was being tested and/or may
have been affected by their neighbour’s reaction to the stimulus; to standardize the pre-test
experience, the stimulus was always inserted into the cage immediately preceding the cage
about to be tested for a minimum of 10 seconds. Animals sometimes either were sleeping or
otherwise did not orient toward the stimulus during the 30 second test, for instance because
they were in the nest box with their view and movements obstructed by their cagemate. In these
cases, they were not scored, and were instead re-tested once more later in the morning, after all
cages had been tested once.
In November 2011, I conducted two days of stick testing on the Millbank Fur Farm
subjects used in Chapter 2 (which included 88 remaining litter-matched pairs per treatment). In
November 2012, I conducted four days of glove testing on Millbank Fur Farm subjects: 43 malemale (26 of which were observed) and 62 (40) female-male pairs from Experiment 1b. Within
the female-male pairs, 29 (16) females and 18 (15) males had been raised in extra-large litter
cages during Experiment 1a, but had been pair-housed and now lived in regular pair cages. I
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additionally glove tested 41 trios and 2 pairs (each missing one male) who had lived (and still
lived) in extra-large cages during Experiment 1b. Based on pilot tests (on other animals), I
decided to test these animals from the backs of their cages rather than from the front, because
their nest boxes are at the front of their cages; this made it difficult to distinguish fleeing into the
nest box from approaching the experimenter, when testing from the front. I stopped testing them
after two days, however, when I concluded that their testing environment was still too different
from that of their controls to make comparisons worthwhile. Their results can, however, be used
to test for correlations within this group. In the same month, I conducted two days of glove
testing on a subset of the RBR Fur Farm subjects (Experiment 1c): 38 (25) pastel female –
black male pairs housed in the back half of one cage row, and the 40 (26) pastel – pastel and
40 (25) black – black pairs that were housed directly across from them. Subjects housed in the
other row of mixed-strain cages had already been separated, and so were not tested. Finally,
glove tests in 2013 were conducted by a separate experimenter, trained by me and blind to both
the hypothesis and the frequency of play in different subjects and treatment groups. This
experimenter had conducted a few days of behavioural observation at Millbank Fur Farm that
summer, as part of Experiment 2b. At both farms, she tested all available subjects from all
treatment groups, except for those in extra-large cages. She conducted two days of glove
testing at Millbank Fur Farm (Experiment 2b; late August and mid-September) and two at RBR
Fur Farm (Experiment 2c; late August and early November). All glove tests were recorded using
a handheld camera to allow for detailed analysis (e.g. recording starting location within the cage
and measuring latency to approach or withdraw), but were scored in real time by the tester.
Handling test
As an indicator of fearfulness during handling by humans, we recorded whether or not
individual mink screamed during a brief period of handling by farm workers (Meagher et al.,
2014). Screaming, in mink, is primarily shown by more fearful individuals (Malmkvist and
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Hansen, 2002). This test took place in November, prior to pelting, when farmers “graded” each
animal, evaluating their fur quality. All animals of one sex in a given aisle of cages were graded
in one session, with the other sex being evaluated separately. Several workers removed mink
from their cages by hand (using thick leather handling gloves) and took turns presenting them to
an experienced farmer, who briefly observed them under a bright light, occasionally stroking
their fur. Workers reported to us each animal’s identification number, listed on a card above its
cage. The animals were then returned to their cage, having usually been handled for under 30
seconds.
At RBR Fur Farm, I observed grading of a total of 187 females and 192 males from
Chapter 3’s Experiment 1c in 2012. I did not record screaming for animals from Experiment 1b
(Millbank Fur Farm) because those in apparently play-eliciting treatments had already been
excluded from breeding based on maternal and litter characteristics; farmers therefore did not
grade them. The previous year, another observer had recorded screaming in 185 Millbank Fur
Farm females from the experiment in Chapter 2.
Carrying cage test
As an indicator of physiological activation during handling and physical restriction, I
measured the corticosteroid hormone output of mink following handling by the experimenter and
a short period of physical restriction inside a small carrying cage. This test was inspired by a
previous study in which handling and immobilizing adult female mink in a carrying cage for 15
minutes produced an increase in fecal cortisol metabolites (FCM) detectable even 4 days after
the event (Malmkvist et al., 2011). The authors further investigated the time-course of the mink’s
adrenal response to different acute stressors. Handling and blood sampling produced a rapid
rise in plasma cortisol: samples collected 30, 60, and 120 minutes later all had higher-thanbaseline levels. Assessing FCM is a non-invasive alternative to blood sampling, but because of
the time taken for circulating hormones to be metabolized and pass into the feces, it is a
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delayed indicator of acute stress: radio-labelled cortisol metabolites injected into the
bloodstream were detected in the feces starting 2 to 3 hours after handling and injection, and
continued to be excreted at elevated levels until 6 to 7 hours after injection (Malmkvist et al.,
2011).
Here, I captured mink by hand and placed them inside a small carrying cage – different
sizes for males and females, in either case only slightly longer than the mink, and barely wide
enough for the animal to turn around – for a period of approximately 30 minutes before
transferring them individually to a collection cage. I then collected 3 samples from mesh nets
placed underneath each collection cage, representing all feces produced between 0-2 hours, 24 hours, and 4-6 hours after the time of capture. Any feces found beneath the carrying cage
were included in the 0-2 hour sample. Considering the lag inherent in fecal corticosteroid
metabolite measures, I took the 0-2 hour sample to reflect baseline (pre-capture) levels of FCM.
I predicted a clear increase in FCM in the 4-6 hour sample, with potential for a smaller increase
at 2 to 4 hours. At Millbank Fur Farm, I also recorded whether or not each mink screamed
during handling or while in the carrying cage.
The details of this test differed between farms. First, all male-male and extra-large caged
subjects were to be killed during pelting season, meaning it was necessary to carry out the tests
at Millbank Fur Farm in mid-November, while animals were still housed in pairs or trios. In
contrast, I tested the subset of animals selected as breeders at RBR Fur Farm in early
February, at which point they were singly housed. Second, after being captured and placed in
carrying cages, Millbank subjects were transported on a wheeled cart (in batches balanced
across treatments, with all cages held in the same wooden tray) to a separate shed, in which I
used whelping cages (as described in Chapter 3) as collection cages. RBR mink in their
carrying cages were similarly placed in a wooden transport tray, but after 30 minutes were
simply returned to their home cage, as they had no cagemates to contaminate their samples.
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Not transporting them allowed me to work with larger batches of animals at RBR, but this also
made it impossible to tell which specific individuals were screaming. Third, I captured Millbank
subjects around the time of morning feeding and held them for 6 hours (with food available in
the collection cage), following which I had to return them to their home cages for afternoon
feeding. Because RBR subjects were tested in their home cages, I was able to collect additional
samples: a 6 to 24 hour post-capture sample as well as 0-2, 2-4, 4-6 and 6-24 hour samples at
the same times of day on the day before capture – the only abnormal stressors on this day were
the placement of mesh nets under cages and the presence of the experimenter. This latter set
of samples was used to check whether any increases in FCM following capture could be
attributable to normal diurnal variation in FCM output.
I collected fecal samples in plastic bags and froze them on the same day, then later preprocessed them as described in several prior publications (e.g. Díez-León et al., 2013; Hansen
et al., 2007; Malmkvist et al., 2011; Meagher et al., 2014). Briefly, I thawed and homogenized
each sample; centrifuged a mixture of 0.5 g feces and 5 ml 80% ethanol; then collected and
dried down the supernatant. FCM levels were then quantified in Dr. Rupert Palme’s laboratory
via a previously validated 11β- hydroxyaetiocholanolone enzyme immunoassay (Frigerio et al.,
2004).
I thawed a subset of samples (the first of six batches processed) by letting them sit at
room temperature overnight before learning that this practice should be avoided, as bacteria
may continue to metabolize corticosteroids when not frozen (Möstl et al., 2005; Palme et al.,
2013). Therefore, I instead immersed subsequent samples in a warm water bath for rapid
thawing (c. 15-30 minutes). I processed one of the later batches twice, using first rapid and then
overnight thawing, in order to compare results of the two methods.
The subjects tested at RBR Fur Farm were all the potential breeder black males and
pastel females from the mixed- and same-strain treatment groups in Experiment 1c. I tested 17
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mixed-strain black males (9 observed as juveniles), 16 (11) same-strain black males, 17 (8)
mixed-strain pastel females, and 18 (9) same-strain females. All these were tested on the same
day. At Millbank Fur Farm, subjects had all been observed as juveniles and were a subset
chosen to avoid (wherever possible) testing multiple cagemates or littermates within a
treatment, and to maximize testing of pairs of littermates across treatments. Over four days, I
tested a total of 19 females and 19 males each from extra-large cages and female-male pairs,
as well as 19 males from male-male pairs. Of the latter group, there were 3 cases where both
cagemates were tested; they were distinguished based on previously-recorded physical
differences. Wherever one of two males residing in the same cage was to be tested, and these
males were littermates (i.e. where I was indifferent to which was tested), I chose which one
would be tested by flipping a coin prior to opening the cage. Thus, I avoided simply choosing
whichever subject I could most easily catch (quite possibly the least fearful one), which could
have led to systematic bias.
Data analysis
Statistical analyses were conducted using R statistical software and the “nlme”, “lme4”,
and “multcomp” packages (Bates et al., 2015; Hothorn et al., 2008; Pinheiro et al., 2015; R Core
Team, 2015). Wherever possible, global models were used to test for relationships across
experiments, and interactions between experiment and independent variables of interest were
included. All tests are two-tailed and conducted at alpha = 0.05.
I used mixed-effects logistic regressions (generalized linear mixed-effects models with a
logit link function) for tests with binary outcome measures (screaming when handled and
fearful/confident responses to the stick or glove), controlling for the following factors:
experiment; strain and treatment nested within experiment; sex; and, as a random effect, unit of
replication. Where screaming in the handling test was the dependent variable, the unit of
replication was cage at RBR Fur Farm, as multiple animals per cage were tested, and litter at
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Millbank Fur Farm, as multiple animals per litter were tested in separate cages. Here, I
additionally controlled for whether or not the animals were singly-housed when tested. For
stick/glove tests, which were performed more than once, the unit of replication was the
individual mink, nested within cage; in cases where more than one male lived in the same cage,
however, they were statistically treated as the same animal because they typically could not be
reliably distinguished. Here, I additionally controlled for juvenile observation type (close vs. far)
and test replicate (i.e. the nth day of stick/glove testing for this animal) – factors that had not
varied in the handling test. Only trials scored as fearful, confident, aggressive, or unresponsive
were considered valid; others (e.g. sleeping or unaware) were discarded. Because fearful and
confident responses are not the only possibilities, each outcome was tested for in separate
logistic regressions. To the basic models above, I added various independent variables of
interest in different models: these are reported on below.
To test for consistency of responding in stick/glove tests, I isolated the subjects (a total
of 1206) with valid responses on the two first days of testing. In other words, I excluded those
who slept or were unaware of the stimulus, even when re-tested, on either of those days. I also
excluded males in male-male or extra-large cages, as their individual identity could not be
reliably ascertained. I then used mixed-effects logistic regressions, as above, with binarized
responses on Day 1 (fearful vs. non-fearful; confident vs. non-confident) as an independent
variable, and binarized responses on Day 2 as the dependent variable.
For tests with continuous outcome measures (FCM; rough-and-tumble play (R&T) as
predicted by alert behaviour), I used paired t-tests and Pearson’s correlations where
appropriate, and ANOVAs, general linear models (GLMs), or general linear mixed-effects
models (GLMEM) for more complex models. In the latter tests, I controlled for farm or treatment
(and its interaction with the independent variable of interest) in addition to observation method
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and sex. Where multiple measures per animal were taken (e.g. FCM in different samples), I
additionally controlled for mink as a random variable.
Because FCM measures showed a highly non-normal, right-skewed distribution, they
were log-transformed for all analyses. For this measure, I either visually display the logtransformed means +/- standard error, or report back-transformed means and upper and lower
standard error boundaries, as the back-transformed standard error is not symmetrical around
the mean. Here and in other parametric models, I verified test assumptions via visual inspection
and the Shapiro-Wilk test of normality.
To test whether the pattern of changes in FCM with collection time differed between
days at RBR Fur Farm (Day 1: no handling; Day 2: handling and capture), I tested for an
interaction between day and collection time, followed by Tukey’s test for multiple comparisons
between each combination of day and time. In models testing for effects of certain variables on
change in FCM, concentration in one collection period (e.g. 4-6 hours) was used as the
dependent variable, while the baseline concentration was used as a covariate (Darlington and
Smulders, 2001; García-Berthou, 2001).
Results
Below, I report test statistics only for those variables that were of primary interest in each
analysis. For other covariates and factors included in each statistical model, outcomes are
presented separately in the appendix.
Test consistency and construct validity
The probability of fearful responding in stick and glove tests changed in predictable ways
with variations in protocol and with repeated exposure. First, the probability of fearful responses
differed between experiments (F4,823 = 50.80, p < .0001), notably being far lower in the 2011
(Chapter 2) stick tests than in 2012 and 2013 (Chapter 3) glove tests (odds ratio for stick vs.
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glove within the same farm = 0.13). Second, mink showed evidence of habituation, responding
most fearfully on the first day of testing (F3,823 = 25.07, p < .0001, odds ratios: Day 1 vs. 2 =
1.97, 1 vs. 3 = 2.84, 1 vs. 4 = 4.88). Furthermore, individual subjects were highly consistent,
with fearful responding on Day 1 predicting fearful responding on Day 2 (F1,228 = 7.85, p = .006,
odds ratio = 1.92), and confidence similarly predicting confidence (F1,228 = 5.18, p = .024, odds
ratio = 5.61). To illustrate this differently, 72.4% of mink rated as confident on Day 1 were again
rated as confident on Day 2, with only 10.2% becoming fearful; among fearful mink on Day 1,
50.3% were fearful on Day 2, and only 22.4% were confident (Table 4–1).
Table 4–1: Contingency table of responses of individual mink on the first and second
days of glove or stick testing, summed across experiments and sexes.
Day 1 / Day 2
Aggressive
Confident
Fearful
Unresponsive
Total
Aggressive
4
4
0
1
9
Confident
20
278
39
47
384
Fearful
5
129
290
153
577
Unresponsive
2
46
94
94
236
Total
31
457
423
295
I could not check the consistency of the handling test, as no animal was tested more
than once.
Within the subset of samples analyzed twice using different thawing methods, the
method used had no significant effect on mean FCM concentrations (overnight lower SE –
mean – upper SE: 114 – 150 – 197 ng/g; rapid: 124 – 133 – 143 ng/g; paired t(64) = 0.857, p =
.395). Furthermore, there was a strong correlation between overnight- and rapid-thaw FCM
measures: (r = 0.552, p < .0001). Therefore, I used data from all samples for subsequent
157
analyses, regardless of how they had been thawed; for the batch of samples analyzed both
ways, I used rapid-thaw values.
Most individuals unfortunately did not produce feces in all collection intervals, especially
in the baseline interval. Across both farms, I obtained samples for 76 individuals at 0-2 hours,
133 at 2-4 hours, and 138 at 4-6 hours. There was a main effect of collection time (0-2, 2-4, 4-6
hours post-capture) on FCM concentration (F2,185 = 16.21, p < .0001), along with an interaction
between collection time and farm (F2,185 = 21.99, p < .0001) and a significant effect of sex (F1,155
= 21.04, p < .0001; males: 143 – 154 – 167 ng/g; females: 63 – 71 – 80 ng/g). When each farm
was analyzed separately, there was no effect of collection time at Millbank Fur Farm (F2,90 =
0.72, p = .491), nor any other significant effects. At RBR Fur Farm, in contrast, there were
significant effects of both treatment (F2,62 = 6.32, p = .003) and sex (F1,62 = 30.74, p < .0001) –
note that as only black males and pastel females were tested at this farm, these may well reflect
strain rather than sex differences. At RBR, collection time did have a significant effect on FCM,
which surprisingly decreased with increasing time since capture (F2,87 = 37.27, p < .0001; Figure
4–1). Samples collected 6 to 24 hours post-capture (only at RBR) had near-baseline FCM
concentrations.
The change in FCM over time was different on the day when RBR Fur Farm animals
were captured and handled than on the previous day, when they were not (Figure 4–1). While
FCM concentrations followed the same overall U-shaped pattern on both days, the decrease
was less pronounced on the day when animals were captured and handled. While baseline and
2-4 hour FCM was lower on this than on the previous day, it was then higher but not significantly
different at 4-6 hours, and significantly higher at 6-24 hours.
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Figure 4–1: Fecal corticosteroid metabolites by collection period over two days at RBR
Fur Farm. Females (grey) and males (black) were not captured or handled on the first day
(solid), but only on the second day (diagonally hatched). There was a significant
interaction between collection period and day (F3,359 = 16.99, p < .0001). Collection
periods (for both sexes combined) that do not share a letter are significantly different.
2048
a
1024
b
b
512
bd
cd
c
256
e
128
e
FCM
(ng/g)
64
32
16
8
4
2
1
0-2 hours
2-4 hours
4-6 hours
6-24 hours
Collection period (time since capture or start of sampling)
Screaming during this test (while being handled or while in the carrying cage; recorded only at
Millbank Fur Farm) did not predict higher FCM at 4-6 hours, relative to the 0-2 hour baseline
(F1,29 = 0.35, p = .561).
Convergent validity
In the subset of animals subjected to both the handling test and the stick or glove test,
screaming when handled was not significantly correlated with the probability of fearful behaviour
toward the stick or glove (F1,252 = 2.71, p = .101, odds ratio = 1.18), though it did tend to predict
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a lower probability of confident behaviour in this test (F1,252 = 3.78, p = .053, odds ratio = 0.57).
It was impossible to test whether screaming in the handling test predicted FCM response
to the carrying cage test, as none of the animals subjected to both tests screamed.
FCM at 4-6 hours in the carrying cage test, controlled for FCM at 0-2 hours, did not
predict the probability of fearful responses in the glove test (F1,13 = 0.95, p = .347). However, it
tended to negatively predict confident responding (F1,13 = 3.89, p = .070). Animals most likely to
be confident toward the stick were those with the lowest FCM at 4-6 hours, relative to baseline.
Rough-and-tumble play and fearfulness
Rough-and-tumble play did not predict the probability of responding fearfully to the stick
or glove (F1,3273 = 1.10, p = .295), and had no interaction with experiment (F4,823 = 1.09, p =
.297). The probability of fearful responses was higher among females than among males (F1,823
= 35.41, p < .0001, odds ratio = 1.94) and differed between strains (F3,823 = 23.25, p < .0001),
being higher in black than pastel mink (odds ratio = 2.46). In addition to the significant effects
presented above (“Test Consistency and Validation”), the main effect of treatment was also
significant (F11,823 = 3.77, p < .0001), with the probability of fearful responses being: higher in
Experiment 1b subjects tested in extra-large cages than in those moved from these to standard
cages at the start of the pair phase (odds ratio = 3.89); lower in Experiment 1c mixed-strain
female pastel – black male than in single-strain black (odds ratio = 0.35) or pastel (odds ratio =
0.67) pairs; and to the contrary, lower in Experiment 2c single-strain pastel pairs than in mixedstrain female pastel – black male pairs (odds ratio = 0.38) or female black – pastel male pairs
(odds ratio = 0.36). R&T was no more predictive of confident than of fearful responses (main
effect: F1,823 = 0.40, p = .527; interaction: F4,823 = 0.14, p = .967). Here, I omit reporting effects of
other variables on confidence because they largely mirrored the effects on fearfulness, though
demi mink were more likely to be confident than black mink (odds ratio = 8.71), and treatment
effects in Experiment 1c were no longer significant. Finally, note that tests run using detailed
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data collected from video of Experiment 1 glove tests did not yield different results than the
simpler qualitative analyses above. For this reason, I do not present these results – of survival
analyses to predict latency to approach or withdraw from the glove, controlling for starting
position within the cage and other factors – and did not collect such data in Experiment 2.
Rough-and-tumble play did not predict screaming when handled (F1,551 = 0.19, p = .663),
and there was no significant interaction of play with experiment (F1,551 = 0.49, p = .484). The
model did produce two independently significant effects: females were more likely to scream
than males (F1,551 = 16.52, p < .0001, odds ratio = 4.13), as were black mink compared to
pastels (F1,551 = 20.08, p < .0001, odds ratio = 22.60).
Rough-and-tumble play did not predict FCM 4-6 hours after capture, controlled for
baseline FCM at 0-2 hours (F1,30 = 0.00, p = .966). Note that changing which measure was
considered the baseline (e.g. the 6-24 hour sample from the non-handled day at RBR Fur
Farm), or even removing the baseline measure from the model, in order to increase the sample
size available for testing, did not change this result. Because FCM is likely to covary with overall
levels of activity, I also ran these analyses using rough-and-tumble play as a proportion of active
time, rather than of total time budget. This did not change any of the results.
Play, fearfulness, and alert behaviour
There was a weak but significant negative relationship between time spent alert and
observed rough-and-tumble play in juveniles (F1,304 = 5.19, p = .023, r = -0.107). Even after
controlling for observation method, mink who spent time watching the experimenter spent less
time in R&T. There were significant interactions between time spent alert and both farm and
observation method; follow-up analyses showed that this relationship held for all subsets of the
data except for RBR mink observed from up close.
Only for glove and stick tests were data available to test for a correlation with alert
behaviour during the juvenile period. While contrary to my prediction, alert behaviour did not
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positively predict the probability of fearful responses (F1,243 = 0.51, p = .475), it was negatively
correlated with confident responses (F1,243 = 4.98, p = .027). When I restricted this analysis to
only those mink observed from up close (and therefore most likely to be vigilant during
observation), alert behaviour then tended to be positively correlated with fearful responding
(F1,67 = 3.78, p = .056), but no longer predicted confident responses (F1,67 = 0.05, p = .822).
Discussion
The evidence collected does not support a key prediction of the hypothesis that play
trains mink for the unexpected. There was no relationship between time spent in juvenile roughand-tumble play and adult/sub-adult fearfulness or confidence towards human handlers. Mink
who spent the most time playing were no less likely to withdraw from a stick or handling glove or
to scream when handled, and did not have a more limited corticosteroid hormonal response to
handling and holding in a small carrying cage.
My results suggest that, as assays of fearfulness, the stick/glove and carrying cage tests
possess some degree of construct validity. As in previous experiments, mink responded to the
stick or glove in a repeatable fashion (Meagher et al., 2011), became less fearful with
habituation (Malmkvist and Hansen, 2002; Meagher et al., 2011), were less frightened of the
stick than of the glove (Meagher et al., 2011), the latter actually being associated with handling
as well as reeking of alarm odours of other mink. Meanwhile, the lack of a rise in FCM following
capture in the carrying cage suggests that the acute stressor was not severe enough to cause a
detectable increase in fecal corticosteroid hormone metabolite excretion; in the previous study
that showed such an increase, mink were additionally physically restricted (dorsoventrally
squashed) in the carrying cage by raising a moveable floor (Malmkvist et al., 2011). However,
the likely explanation is that here, any such increase was counteracted by diurnal changes in
corticosteroid production: activity in farmed mink peaks in the early morning (e.g. Chapter 2;
Svendsen et al., 2007), around the time when subjects were captured, and corticosteroid
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production may therefore be expected to slow thereafter. The fact that a similar but significantly
smaller decrease in FCM over time was seen on a day when mink were not captured or
interfered with shows that the stressor did indeed lead to elevated FCM levels, but that these
were obscured by normal circadian rhythms. Thus, the data from this test are likely valid, but
extremely noisy, measures of physiological responsiveness to a stressor. To avoid such noise,
one solution may be to obtain baseline measurements on non-capture days, but this may
present some difficulties (e.g. where one needs feces from only one of two cagemates). FCM
may additionally constitute a less sensitive measure than plasma cortisol, and its inherent time
lag makes cross-period comparison difficult. Perhaps a better approach to assessing baseline
and capture-induced physiological activation might be to measure stress-induced hyperthermia
using ingestible data loggers (e.g. Carr et al., 2008; Harlow et al., 2010) (mink would likely
remove or destroy any wearable device).
I also obtained mitigated evidence for convergent validity in these tests, in the subsets of
animals tested in more than one way. Mink who responded confidently in the stick or glove test
tended not to scream when handled (Malmkvist and Hansen, 2002) and to have small FCM
responses to capture, and spent significantly less time being vigilant toward the experimenter
during juvenile observations. Oddly, the first two relationships did not hold with regard to fearful
rather than confident responses to the stick or glove. Within animals observed from up close as
juveniles, those who were most vigilant later did tend to respond fearfully in the glove test.
While my data do not support the stated hypothesis, neither do they provide strong
evidence against it. The carrying cage test produced very noisy data. The handling test is not
very sensitive, as it produces a binary outcome and was only performed once per mink. As
discussed in Chapter 1, correlational evidence for or against hypotheses about the functions of
play is fundamentally weak. In this specific instance, even if I had found that frequent juvenile
play predicts low adult fearfulness, this would not constitute clear support for the hypothesis, as
an alternative explanation would have been readily available. High levels of alert behaviour in
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juvenile observations appeared to interfere with observed rough-and-tumble play (but see also
contradictory findings from Jeppesen and Falkenberg, 1990), and were associated with reduced
confidence and more fearful behaviour in later glove tests.
In this experiment, I attempted to establish the consequences (or correlates) of roughand-tumble play with respect to emotional reactivity in mink. The relatively simple tests I used
were meant to compare the magnitude of minks’ reactions to aversive stimuli, but did not allow
me to assess whether diminished emotional reactions make certain animals more competent
than others in these situations. In future work, one potential way to address this question would
be to test the performance of a learned, skilled behaviour in stressful vs. non-stressful situations
(e.g. Metz et al., 2005). For example, male rats proficient in shelling sunflower seeds revert to a
much less efficient, unskilled, and time-consuming shelling technique in the presence of a social
stressor (Pellis, 2002b). Quantifying the negative effect of a standardized stressor on
performance in this or a similar task, in animals from high- and low-play experimental
treatments, could allow us to test whether playing makes animals not merely less fearful, but
also more competent in frightening situations.
Ultimately, then, more research is still required to properly evaluate this hypothesis. It
should, as I attempted but failed to achieve here, be both experimental in nature and yet not
based on a play-deprivation paradigm. The results will not only be fascinating on a fundamental
level, but will also have profound ethical implications for animal welfare. Housing treatments that
increase play in juveniles could represent a pro-welfare “double whammy” if, in addition to
eliciting a behaviour tied to positive welfare in the present (Boissy et al., 2007), they also make
animals more resilient in the face of future aversive situations in much the same way as
environmental enrichment can (Meagher et al., 2014). Such interventions in different species
could help mitigate the negative impacts of major events such as transport to slaughter.
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Chapter 5 – Increasing Sexual Prowess through
Play
Abstract
I tested the hypothesis that rough-and-tumble play prepares juvenile mink for adult
sexual behaviour, focusing particularly on males. Using subjects previously observed as
juveniles in Chapters 2 and 3, I tested the predictions that juvenile male rough-and-tumble play
(R&T) would predict shorter latencies to bite females’ necks and begin copulating, and longer
durations of copulation. I predicted the reverse in females. Frequent R&T, whether as a
proportion of total time or of active time only, was associated with long-lasting copulations in
males, and with longer latencies to copulate in females. This is, to my knowledge, the first
demonstration that juvenile rough-and-tumble play is correlated with adult sexual behaviour in
any species. However, juvenile social isolation beginning between 10.5 and 14.5 weeks of age
did not have adverse effects on sexual behaviour in either sex. While this does not support a
causal role for R&T in its relationship to sexual behaviour, given the low number of social
isolates available, this could easily be a Type II error. Among males, long-lasting copulation was
also associated with high output of fecal epiandrostenone. However, neither this nor any other
endocrine, morphological, or behavioural characteristics of males appeared to mediate the
relationship between rough-and-tumble play and sexual behaviour. Further research is required
to identify the causes of these correlations.
Introduction
Juvenile males reared in social isolation, and thus without rough-and-tumble play,
typically become sexually impaired adults (reviewed in Pellis and Pellis 2009). In addition, males
typically engage in more rough-and-tumble play than do females, particularly in promiscuous or
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polygynous species (Chau et al., 2008). As will be reviewed below, these observations have led
to the hypothesis that rough-and-tumble play is crucial to the development of male sexual
behaviour, thus providing long-term benefits in the form of a reproductive advantage (Missakian,
1972; Pellis and Pellis, 2009). Rough-and-tumble play could conceivably have this effect via a
number of different mechanisms: it may constitute direct practice for courtship and/or mating; it
may further masculinize the developmental trajectory of young males; or it may promote general
improvements in motor or social skills. In this chapter, I investigate the relationship between
juvenile rough-and-tumble play and adult sexual competence in mink (Neovison vison). While
my research here mainly concerns male mink, I also test for a relationship between play and
sexual behaviour in females.
Male sexual behaviour, social isolation, and rough-and-tumble play
Experimental evidence for sexual impairment in males socially isolated as juveniles
comes primarily from laboratory rhesus monkeys (Macaca mulatta) (Harlow et al., 1965; Mason
and Berkson, 1975; Mason, 1960; Missakian, 1969; Mitchell et al., 1966) and rats (Rattus
norvegicus) (Cooke et al., 2000; Gerall et al., 1967; Hole et al., 1986; van den Berg et al., 1999;
Ward and Reed, 1985; Zimbardo, 1958; but see also Beach, 1958; Gruendel and Arnold, 1969),
with similar findings in chimpanzees (Pan troglodytes) (Rogers and Davenport, 1969) and
domestic dogs (Canis lupus familiaris) (Beach, 1967). Males reared in isolation go on to show
disrupted, if not entirely absent, sexual behaviour in adolescence or adulthood, relative to wildborn or group-raised controls. In different species or experiments, isolates exhibit failures at
various appetitive or consummatory steps of sexual behaviour. Often, they act fearfully or
aggressively toward potential sexual partners (Beach, 1967; Deutsch and Larsson, 1974; Hole
et al., 1986; Rogers and Davenport, 1969), who may themselves also avoid the males or fail to
present to them (Mason, 1960). Isolated male rats in several studies show low sexual interest:
they were less likely to get an erection when presented with an estrous female across a mesh
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partition (Cooke et al., 2000); and showed delayed and less frequent anogenital investigation of
females (van den Berg et al., 1999). In other experiments, isolated males readily court females
but then clamber over or under them rather than mounting them (Gerall et al., 1967). In rats
(Hole et al., 1986), dogs (Beach, 1967), and rhesus macaques (Mason, 1960; Missakian, 1969),
isolates tend to fail to properly orient themselves when attempting to mount females,
ineffectually mounting their sides or legs rather than their rumps; isolated rhesus may succeed
in mounting an immobile wooden dummy yet struggle with a live partner (Deutsch and Larsson,
1974). Finally, isolated rats (Zimbardo, 1958) or rhesus macaques (Mason and Berkson, 1975)
may appropriately mount females from the rear, but then fail to clasp onto them with their paws
and to perform pelvic thrusts or achieve intromission. While the mechanism by which social
deprivation produces sexual dysfunction remains unknown, several authors have pointed to lack
of rough-and-tumble play as a plausible explanation, for example because these animals have
no opportunity to practice certain behaviour patterns (see below) (Beach, 1967; Missakian,
1972; Pellis and Pellis, 2009; Twiggs et al., 1978). Further hinting at the specific importance of
rough-and-tumble play are several partial isolation studies in which “social” rat or dog controls
outperform isolates despite themselves living alone with the exception of one brief (15-60
minute) daily peer interaction (Beach, 1967; Hole et al., 1986; Zimbardo, 1958). At least in rats,
subjects devote these short sessions almost entirely to rough-and-tumble play (Einon et al.,
1978).
Direct practice is one potential mechanism by which rough-and-tumble play may prepare
males for adult sexual behaviour. Particularly in mammals, in which sexual displays and
ornamentation are rare or muted, successful courtship and mating by males often hinge on
athletic performances requiring endurance and/or agility (Byers et al., 2010). At least in species
where skill or agility play a role, practice is likely to be beneficial. Sexually incompetent male
rats and rhesus macaques, deprived of social interaction as juveniles, have in some cases been
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partially rehabilitated by prolonged exposure to estrous females (Gerall et al., 1967; Missakian,
1972). Perhaps they were able to gain, via failed initial attempts to copulate, the experience they
had been denied while they were young. The case for rough-and-tumble play as practice can
also be made based on an argument from design. In many rodent species, playing individuals
compete to make contact with body parts that are also targeted in later sexual behaviour (Pellis
and Bell, 2011; Pellis and Pellis, 2009). In rats, for example, the nape is nuzzled by playing
juveniles and bitten by mating adult males; during aggressive fights, in contrast, adult males
typically attack the flank or rump, and accordingly use different evasive manoeuvres than in
rough-and-tumble play (Pellis and Pellis, 1987). Sexual dimorphism in the style of rough-andtumble play (e.g. in the evasive manoeuvres favoured) can also correspond with sex differences
in later sexual behaviour (Pellis and Pellis, 1990; Smith et al., 1998). Several species
additionally integrate mounting and thrusting into rough-and-tumble play, often most commonly
performed by males (Pellis and Bell, 2011; Vázquez et al., 2014). Thus, while the commonlyused term “play fighting” connotes a very specific function for rough-and-tumble play – that it is
an immature form of “real”, aggressive fighting – it may for some species be more appropriately
thought of as “play mating”. A major challenge for this hypothesis, however, is that some parts
of the sexual act, especially intromission, are rarely seen in rough-and-tumble play (Beach,
1967; Cooke and Shukla, 2011). Additionally, newly published research suggests that the
potentially proto-sexual behaviour seen in rough-and-tumble play does not itself necessarily
improve with practice: rats deprived of peer contact for the first 12 days of the playful juvenile
period, when later paired with other juveniles, performed nape attacks and evasive manoeuvres
with a frequency and in a manner nearly indistinguishable from socially-housed controls
(Himmler et al., 2015).
An alternative hypothesis is that rough-and-tumble play lays the ground for adult sexual
behaviour indirectly, by promoting male-typical neuroendocrine development (reviewed in
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Cooke and Shukla, 2011). While organizational effects of steroid hormones on later sexual
behaviour are traditionally thought to be restricted to prenatal and perinatal life, recent evidence
suggests that these sensitive periods may extend through the juvenile, pubertal, and even early
adult life stages (Curley et al., 2011; Schulz et al., 2009). For example, male golden hamsters
(Mesocricetus auratus) castrated shortly before the expected adolescent rise in androgen
production and implanted with testosterone capsules 1-2 weeks later – thus with normal
androgen exposure in infancy and adulthood – showed reduced sexual, aggressive, and flankmarking behaviour as adults (Schulz et al., 2009). In addition to behavioural effects, postweaning social isolation of rodents can also restrict the male-typical development of
neuroendocrine characteristics, such as adult plasma testosterone levels and the volume of
sexually dimorphic areas of the brain, and estrogen receptor density or aromatase activity
within, including the “sexually dimorphic nucleus” of the preoptic area, the posterodorsal
component of the medial amygdala, and the ventromedial hypothalamus (Cooke et al., 2000;
Dessì-Fulgheri et al., 1976, 1975; Lupo di Prisco et al., 1978; Ruscio et al., 2009). However, in
several of these isolation studies, differential housing was maintained beyond the onset of
sexual maturity, and the observed effects may have been due to adult social contact: sexual
activity, in particular, is known to affect characteristics such as estrogen receptor density (Borja
and Fabre-Nys, 2012; Hull and Dominguez, 2006; Phillips-Farfán et al., 2007). In terms of
plasma testosterone and estrogen receptor activity, isolates may come to resemble so-called
“duds”, or spontaneously non-copulating males, found with low frequencies in rats, rams (Ovis
aries), and other species (Antonio-Cabrera and Paredes, 2014; Borja and Fabre-Nys, 2012;
Clark et al., 1985; Damassa et al., 1977; Portillo et al., 2007, 2006). Some have suggested that
neuroendocrine effects of social isolation on males are mediated by rough-and-tumble play
deprivation (Cooke and Shukla, 2011; Cooke et al., 2000), perhaps via decreased acute
androgen release and its effects on expression of proteins involved in neurogenesis and
neuronal growth (Burgdorf et al., 2010). Thus, this provides an alternative explanation to direct
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practice for potential relationships between juvenile play and adult sexual behaviour, as the
sexually dimorphic traits reviewed above all participate in appetitive and consummatory aspects
of sexual behaviour (Balthazart et al., 2004; Baum et al., 1996; Cherry and Baum, 1990; Hull
and Dominguez, 2006).
The case for rough-and-tumble play having an impact on male sexual behaviour can
easily be made in mink, and either of the above prospective mechanisms could plausibly be at
work. First, as reviewed in Chapter 2, male mink socially isolated starting before 8 weeks of age
fail to achieve intromission in adulthood (Bassett et al., 1959; Gilbert and Bailey, 1969; Hansen
et al., 1997). Second, the direct practice hypothesis is very plausible, as courtship and mating
are oftentimes quite violent (sometimes even fatal for the female: Dunstone, 1993; Enders,
1952) and have been said to resemble rough-and-tumble play in this species (Kuby 1982, cited
in Vinke et al., 2005). Both involve attempts to bite the back of another individual’s neck –
though predation and aggressive fighting do as well. In one of the above experiments, isolated
males often failed to obtain a proper grip on females’ necks, instead biting them elsewhere on
their bodies (Gilbert and Bailey, 1969). Isolated males of a related species, the European
polecat (Mustela putorius), also failed to properly orient neck bites on females (Eibl-Eibesfeldt,
1982, 1963). In another experiment further illustrating the importance of practice, sexually
inexperienced male mink did not perform as well as experienced individuals, particularly after
biting the female’s neck: they typically either let go, dragged the female around the cage, or lay
beside her rather than proceeding directly to intromission (MacLennan and Bailey, 1972).
Another component of sexual behaviour that might be practiced in juvenile rough-and-tumble
play is the “chuckle” vocalization, which is at least superficially similar in both contexts (Enders,
1952; MacLennan and Bailey, 1969). Third, one experimenter noted that isolated males were
easily frightened by females, which likely interfered with sexual behaviour (Gilbert and Bailey,
1969). Finally, the ferret (Mustela putorius furo: domesticated polecat) has been extensively
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used as a model animal in the study of neuroendocrine masculinization and control of sexual
behaviour. This research suggests that sexual behaviour in male mustelids is regulated in a
similar manner as in other popular model species (e.g. rats, rams, Japanese quails (Coturnix
japonica)), with androgens and estradiol (aromatized from testosterone) acting on sexually
dimorphic areas of the brain, in which lesions impair sexual behaviour (Baum, 2003; Baum et
al., 1996; Carroll et al., 1988; Cherry and Baum, 1990; Tobet et al., 1986). While I know of no
studies relating play or social isolation to neuroendocrine development in mustelids, these same
structures and processes are affected by isolation in other species. Thus, existing research
suggests that early social isolation impairs male sexual behaviour in mink and related species,
and posits several plausible non-mutually exclusive explanatory mechanisms that invoke a
crucial role for rough-and-tumble play.
Female sexual behaviour, social isolation, and rough-and-tumble play
Less is known about the effects of early social deprivation on female sexual behaviour.
Isolated juvenile female rhesus macaques become overly aggressive toward conspecifics
(Kempes et al., 2008) and are less likely to present to adult males (Harlow et al., 1965; Mitchell
et al., 1966). Adult male rats castrated and chronically treated with estradiol benzoate – a
procedure which typically leads to performance of female sexual behaviour – were also less
likely to display lordosis to intact males if they had been raised in isolation (Ward and Reed,
1985). Other experiments on chimpanzees (Rogers and Davenport, 1969) and rats (Hole et al.,
1986), meanwhile, found that isolated females develop roughly normal sexual behaviour.
Finally, female mink isolated before the age of 8 weeks showed reduced agonistic behaviour
toward males and mated more readily (Gilbert and Bailey, 1969). Mink may be atypical in this
respect, perhaps because play is a potential precursor to the particularly violent courtship and
mating of this species.
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Female mink mate with multiple males, and birth litters with multiple paternity, both in the
wild and in captivity (Fleming, 1996; Shackelford, 1952; Yamaguchi et al., 2004). In mate choice
and self-paced mating experiments – where females can freely enter or exit a cage
compartment to which a male is restricted – female mink almost universally approach and mate
repeatedly with all available males (Díez-León et al., 2013; Fleming, 1996; Thom et al., 2004). It
then seems paradoxical that wild and captive females also usually appear to actively resist
copulation, even with males they have chosen to approach, by fighting them before intromission
and by struggling to escape during copulation (Dunstone, 1993; Enders, 1952). Partly as a
result of this, females often do not copulate when visiting a male or in forced or inescapable
mating situations – the standard on commercial farms – where they are confined in a single
cage with a male (Díez-León et al., 2013; Enders, 1952; Thom et al., 2004). Exceptionally,
females can even be so aggressive that historically, farmers sometimes resorted to taping their
mouths shut, giving them whiskey, or chasing them to exhaustion prior to introducing a male
(Enders, 1952).
Why should estrous female mink actively seek out males only to then avoid mating with
them? A likely explanation is that their aggressive coital behaviour is adaptive because it allows
them to exercise some degree of pre- and/or post-copulatory mate choice (or “paternity choice”)
(Andersson and Simmons, 2006). While they appear to mate indiscriminately, they may in fact
copulate with one male more frequently or for longer total durations than with another (DíezLeón et al., 2013; Thom et al., 2004). Further, in mate choice experiments, the number of
copulation bouts (Díez-León et al., 2013) and the total duration of copulation (Thom et al., 2004)
are predicted by male phenotypic characteristics like body size or the frequency of stereotypic
behaviour. Females may thus be promoting sperm competition and biasing paternity of their
litters by copulating more often or for longer in total with different males, which would
presumably confer genetic benefits to their offspring (Andersson and Simmons, 2006; Clutton-
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Brock and McAuliffe, 2012; Fleming, 1996). Male mink release sperm repeatedly or perhaps
continuously during copulations that can last several hours (Enders, 1952; Fleming, 1996), and
duration is correlated with likelihood of fertilization, at least for short copulations (Venge, 1956).
Copulation duration has been found to correlate with proportion of offspring sired in thirteenlined ground squirrels (Ictidomys tridecemlineatus) (Schwagmeyer and Foltz, 1990), though one
experiment on farmed mink failed to find such an effect (Fleming, 1996). Besides potentially
influencing paternity, controlling the timing of copulation may also be adaptive for females.
Female rats in a self-paced mating regimen copulated less frequently than force mated females
but required fewer copulations to induce pregnancy, and those that did get pregnant had larger
litters (Coopersmith and Erskine, 1994; Erskine, 1989). Limiting the number or duration of
copulations may additionally reduce the associated physical risk and energetic costs (Jennions
and Petrie, 2000).
Thus, it may be that female mink fight males as a way of testing their quality as potential
sires, and that it would be adaptive for them to limit the number and duration of copulations they
engage in, at least with less preferred mates and beyond a certain threshold. While it is clear
that not mating at all would be maladaptive, this danger is likely minimal in practice: according to
one previous estimate, no more than 1-3% of farmed females completely fail to mate (Sundqvist
et al., 1989) – a figure I estimate is well below 1% on the farms where I worked. While mating
only once is associated with reduced likelihood of fertilization (Fleming, 1996; Hansson, 1947),
and one study did find that more kits are born to females who mate thrice rather than twice
(Hansson, 1947), others have found litter size to be unaffected by matings beyond the second
(Fleming, 1996; Ślaska and Rozempolska-Rucińska, 2011). Finally, that females do fight back
does not necessarily imply that they offer all-out resistance and refuse to mate with any male
who cannot physically overwhelm them; it may be that they eventually reduce their efforts to
avoid or interrupt copulations according to each male’s perceived quality.
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To my knowledge, only one author has directly tested for a relationship between juvenile
female rough-and-tumble play and later fertility. Nunes (2014) found that wild Belding’s ground
squirrels (Urocitellus beldingi) who most frequently performed social play as juveniles had the
highest probability of successfully weaning a litter in their first breeding season. However, the
author concluded that this was likely mediated by their increased territorial aggression, and did
not examine their sexual behaviour in this study. If rough-and-tumble play allows female mink to
more effectively resist males during sexual encounters, as hypothesized, then it should be
expected to result in less frequent and/or shorter copulation, both of which would modulate the
amount of sperm gained from any given male. While I possess insufficient data to verify the
following prediction in this study, copulations should also be less evenly distributed between
males in number or duration, reflecting an increased degree of mate choice. Direct practice is
one plausible mechanism by which rough-and-tumble play could have these effects. However,
social isolation experiments also reveal changes in females that lessen the degree of sexual
dimorphism in some of the neuroendocrine traits, reviewed above, that are associated with
sexual behaviour (e.g. Balthazart, 1989; Dessì-Fulgheri et al., 1975; Ruscio et al., 2009).
Therefore, an increase in masculinization as predicted in males, but here with a corresponding
decrease in libido, is a competing mechanism that makes the same directional predictions
concerning relationships between rough-and-tumble play and sexual behaviour in females.
Causality in social isolation experiments
As discussed in Chapter 1, it is risky to ascribe a function to play based on deprivation
experiments alone. Several lines of evidence suggest that proper sexual development may
instead require non-playful forms of social contact. First, though guinea pigs (Cavia porcellus)
are not particularly known for their rough-and-tumble play, male isolates still showed reduced
anogenital sniffing and less overall social and sexual behaviour during encounters with females,
as well as problems orienting mounts and clasping females with their forepaws, resulting in low
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rates of intromission and ejaculation (Gerall, 1963; Valenstein et al., 1955). Second, some
experiments find that young male rats can develop normal sexual function despite not being
housed with peers, but instead with their mother or with a non-playful adult male (Cooke and
Shukla, 2011; Gruendel and Arnold, 1969). Finally, some experiments found that juvenile rats
(Hole et al., 1986) and guinea pigs (Gerall, 1963) housed individually but permitted limited nonplay contact with socially-housed juveniles through a wire mesh partition developed normal
sexual behaviour; some of the neuroendocrine effects of isolation can also be partially mitigated
by similar limited contact in rats (Dessì-Fulgheri et al., 1976). It therefore appears that, at least
in these cases, some aspect of social contact other than rough-and-tumble play is sufficient for
male sexual development. Experimental manipulations that more selectively target rough-andtumble play, without affecting other behaviours, are required to adequately test this hypothesis.
Project overview
The original aim of this project was to compare sexual behaviour in experimental groups
in which high or low levels of rough-and-tumble play (R&T) had been elicited. Unfortunately, this
was impossible because of my failure to identify experimental treatments that clearly affected
the frequency of R&T, as detailed in Chapter 3. Therefore, as in Chapter 4, I am again relegated
to relying mostly on correlational evidence.
I observed sexual behaviour in males previously used as juvenile subjects in Chapter 2
and in several experiments conducted on two farms in Chapter 3 (Experiments 1c, 2b, and 2c).
In females, I only studied the sexual behaviour of animals from Experiment 2c, for reasons
elucidated below. I predicted that males who performed the most rough-and-tumble play would
copulate with the largest number of females, show the shortest latencies to achieve neck-bites
and intromission, and the longest copulation durations. For females, the direction of these
predicted relationships should be reversed if rough-and-tumble play enables them to more
effectively limit the number or duration of copulations and/or reduces their overall sexual
175
receptivity. Differential copulation by females according to male quality could unfortunately not
be studied in this context. To my knowledge, any demonstration of correlations between juvenile
rough-and-tumble play and adult sexual outcomes in either sex would be novel, having not
previously been shown in any species.
There was an additional opportunity for a minor observational study of the effects of
early isolation. During Experiments 2b and 2c, I opportunistically selected new subjects as they
happened to become socially isolated partway through the juvenile period, due to their
cagemate having died or escaped. While previous research has demonstrated effects of social
isolation on sexual behaviour in mink, they did not necessarily use the same outcome measures
tracked here, and isolated their animals at much younger ages than in the present work. If
development of sexual behaviour is sensitive to the frequency of rough-and-tumble play in older
pair-housed juveniles (c. 10 weeks and up), then social deprivation of older animals should still
have observable effects.
In an attempt to test which explanatory mechanism could mediate any observed
relationships between rough-and-tumble play and adult male sexual behaviour, I also tested for
a role of other male characteristics in sexual behaviour. If increased masculinization plays a
role, then sexual behaviour should correlate with adult fecal androgen production or anogenital
distance, a marker of early androgen exposure (Hotchkiss and Vandenbergh, 2005; Monclús
and Blumstein, 2012; Swan et al., 2005; vom Saal, 1979). Effects of fearfulness or confidence
on sexual behaviour, meanwhile, would suggest the involvement of general social competence.
Should neither of these possibilities be supported, then direct practice or motor development
remain as likely fallback explanations.
176
Methods
Subjects and housing
Subjects, all in their first mating season, were individuals that farmers had selected as
breeders, in November of each year, on the basis of fur quality and body size. Male subjects
had additionally passed a testicle development check by farmers shortly before the start of the
mating season. Following selection as breeders at the age of c. 7 months, animals remained
single-housed in their pair-phase cages and were eventually moved (in some cases twice) to
similar home cages used for breeding. Males from Experiment 2b were housed singly in extralarge cages (as described in Chapter 3) between selection and the start of breeding. During the
mating season, males are segregated into different sheds or rows, and mated with different
pools of females, on the basis of either fur quality and body size (Millbank) or breeding line
(RBR). Among pastel mink at RBR Fur Farm, the small percentage of albinos is housed
separately as well.
For males from experiments in Chapter 3, I studied those from treatments that initially
appeared to have an effect on rough-and-tumble play. I did not study subjects from Experiment
1b because animals from extra-large cages or male-male pairings were not used as breeders by
the farms, having been ruled out early (and assigned to these treatments) due to their mother’s
poor reproductive performance or maternal care. In Experiments 2b and 2c, I additionally
observed males who had become single-housed at some point during the pair-stage
observation period. The male cagemates of all but one of the single-housed females were
known to have died, several of them during a July heat wave, but single-housed males’
cagemates all had unknown fates. Animals who went missing but were not confirmed dead
either escaped their cage or died, without this being noted by farmers (only RBR recorded
known deaths). I identified single-housed animals by checking every potential breeder cage on
the farm at least once per week during pair-stage observation, thereafter verifying weekly that
177
they had not been re-paired with a new cagemate. After farmers selected these males as
breeders, for each, I recruited the 3 nearest breeder males from the same row of cages as pairhoused controls. Because some controls later went missing or were moved to breeding areas
far removed from their single-housed counterparts, I then recruited other nearby males as
substitute pair-housed controls. Any of the males I had observed as juveniles who happened to
be housed in the same areas as single-housed males also served as additional pair-housed
controls.
Thus, over these four experiments, I observed 121 male mink with known levels of
juvenile rough-and-tumble play, as well as 6 black males single-housed starting during the pair
phase observation period, each with pair-housed controls sharing similar juvenile and/or
breeding environments (i.e. housed nearby). These males were first found to be single-housed
between July 14th and August 6th at the ages of c. 11 to 14.5 weeks (average c. 13 weeks).
Sample sizes for each experiment and treatment are given in Table 5–1.
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Table 5–1: Number of breeding males observed from each juvenile housing treatment
described in Chapters 2 and 3. Numbers in parentheses after plus signs represent males
observed as juveniles (thus already counted) additionally used as pair-housed controls
for single-housed males. For each experiment, the timing of fecal sampling is also given,
relative to the mating season.
Experiment
Chapter 2
(Millbank mated
2012)
Experiment 1c
(RBR mated 2013)
Experiment 2b
(Millbank mated
2014)
Experiment 2c
(RBR mated 2014)
Treatment
Far observation
Non-enriched
Enriched
Close observation
Black male + black female
Black male + pastel female
Pastel male + pastel female
Pastel male + black female
Close observation
Male-male pairs
Female-male pairs
Not observed
Single-housed
Pair-housed (juvenile match)
Pair-housed (adult match)
Pair-housed (match for both)
Close observation
Black male + pastel female
Far observation
Black male + black female
Black male + pastel female
Pastel male + pastel female
Pastel male + black female
Demi male-male pairs
Not observed
Single-housed
Pair-housed (juvenile match)
Pair-housed (adult match)
Pair-housed (match for both)
N
Fecal Sample
28
32
a few days
before
10
8
6
4
one month
before
6
6
during
4
6 (+1)
8 (+1)
6
4
during
6
1
2
4
4
2
2
5 (+9)
2
Turning to females, I only observed subjects from Experiment 2c. I had previously
avoided studying females because each individual female mates much less frequently than
each male, and is moved into a different male’s home cage for each encounter, while males
stay put. Thus, males can more readily be kept track of, and their performance can be more
precisely quantified, over a larger number of encounters. I studied a total of 39 breeding
females, checking at each session which, if any, had been paired with a male. Ten of them had
been observed for rough-and-tumble play as juveniles: 5 black females housed with pastel
179
males and observed from afar, 3 pastel females housed with black males and observed from
afar, and 2 pastel females housed with pastel males and observed from up close. Seven others,
5 black and 2 pastel, had been single-housed, having been first found alone between July 10th
and 29th, at ages c. 10.5 to 13 weeks (average c. 11.5 weeks). I selected pair-housed controls
for these in the same way as for males, but these controls were almost all lost when females
were moved to breeding sheds. I therefore used only controls selected from the same breeding
areas, 13 black and 9 pastel.
Farm mating regimen
Mating took place between late February and mid-March each year. During this 2-to-3
week period, farmers typically attempt to have each female mate 3 or 4 times in total. Because
they use a smaller number of male breeders, this results in each having the opportunity to mate
once or twice per day.
The two farms in this experiment used slightly different mating regimens. Farmers
typically attempt to have female mink mate on consecutive days because the first copulation
induces ovulation and may be less likely to result in fertilization on its own, and c. 8 days apart
to take advantage of two separate estrous periods (Enders, 1952; Fleming, 1996; Ślaska and
Rozempolska-Rucińska, 2011). Below, I refer to the first day on which any female mates
successfully to be her own “Day 1”.
Millbank Fur Farm attempted to have each female mate on the mornings of Days 1 and
8, and on the afternoon of Day 9, each time with a different male. Females who did not copulate
with a given male were re-paired with a new male several times consecutively, and again on the
following afternoon or next day, until copulation occurred. Neither males nor females were
allowed to mate with more than one partner per morning or afternoon, only being re-paired after
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failing to copulate. During the latter half of the mating season – with some females on Day 8 and
others on Day 9 – males could mate with different females in the morning and in the afternoon.
RBR Fur Farm aimed to have females mate on the mornings of Days 1, 2, 9, and 10. On
Days 9 and 10, females were paired with the same males they had mated with on Days 1 and 2,
respectively. When females did not copulate in the morning, they were only re-paired with one
additional male per day, in the afternoon. There was no set rule as to which males were used
for these afternoon pairings; this was at farm staff’s discretion. Thus, RBR Fur Farm males
could potentially mate up to twice per day, but the distribution of opportunities was uneven
between males. On both farms, a small number of male “duds” were removed from the breeding
pool due to health problems and/or to poor performance, having never or hardly ever mated
after the first several days.
Behavioural observations
Over several hours on each day of the mating season, several crews of c. 3-5 staff
worked in different areas, transporting females into and out of males’ home cages, monitoring
and noting the success or failure of each mating encounter. Depending on how busy they were,
they checked each cage every c. 5 to 25 minutes. In each case, they noted on the female’s
identity card whether the male had “caught” her (sustained bite on back of neck with the female
no longer fighting vigorously) and whether the two were copulating. Rarely, if they were fighting
extremely violently, pairs were separated after only a few minutes. Farmers eventually (after
delays of highly variable length) judged non-copulating encounters to be unsuccessful and
removed the female, typically allowing encounters to go on for longer if the female was caught.
Farmers recorded encounters as successful matings if the animals engaged in a bout of
copulation longer than a certain threshold (nominally 15 or 20 minutes, but again quite variable).
Copulations that had already gone past this threshold were often manually interrupted by
181
farmers. Millbank farmers avoided manual interruption of Day 1 copulations, and in subsequent
rounds typically waited for longer than RBR farmers before separating pairs.
I observed female-male pairs using instantaneous scan sampling with a variable interval
of between c. 3 and 15 minutes. The length of the interval was constrained by the number of
female-male pairs that were currently active – at certain times, only a small number of subject
males actually had females in their cages – and by how widely dispersed these were around the
farm. I recorded at each scan whether the male was maintaining a neck-grip on the female and,
if so, whether he was copulating with her. Using this, I calculated latencies from the start of the
encounter until neck-biting and until copulation, and the duration of each copulation bout. Given
that my sampling interval was variable, I estimated the actual time of events (e.g. the start of the
encounter) as midway between the current and last observation, except where direct knowledge
of farmers’ actions provided more information (e.g. if I saw farmers putting females into males’
cages in this area at a certain time). I also augmented my own observations with information
farmers recorded on females’ identity cards, for example by using the time at which they had
first reported seeing copulation as the estimated starting time if I had not observed copulation
first myself. Whenever I observed two mink copulating and returned to find that the female had
been removed from the cage, I assumed farmers had manually interrupted the copulation,
unless they indicated otherwise.
For each encounter, I recorded some information about the female, drawn from her
identity card and relayed to me by farmers. At Millbank Fur Farm, I recorded the female’s parity
and whether or not she had already failed to copulate with one or more other males during the
current round of mating (either immediately before being paired with the current male, or on the
previous day). Unlike at Millbank, RBR females have uniquely identifying numbers and detailed
mating records. I thus additionally recorded female ID numbers for each RBR female-male
encounter and, at the end of the mating season, examined each of their records to count the
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numbers of successful copulations and of failed mating days. The latter is not exactly
proportional to the number of encounters not resulting in copulation, as a morning failure
followed by afternoon copulation is simply recorded as a success, but may still provide a useful
index of how readily each female will mate.
In addition to the juvenile behaviour and adult sexual performance of males, I also
recorded their adult body mass, anogenital distance, and fecal androgen metabolite levels
(testosterone and epiandrostenone), as well as their stereotypic behaviour frequency in early
adulthood. Fecal samples were obtained and processed as described in Chapter 4, in all cases
from home cages and without handling or transport stress. Samples consisted of 24 hours of
feces, except for Experiment 1c animals, in which case I used the 16 hour sample from the day
before the carrying cage test. Table 5–1 specifies the dates at which samples were collected for
each experiment. I only measured anogenital distance at RBR Fur Farm, and then only in males
killed at the close of the mating season. I used a tape measure and measured AGD (distance
between the centre of the anus and the centre of the penis) to the nearest mm three separate
times for each male, keeping only the median result. I additionally measured their body mass to
the nearest gram, using an electronic scale. Excellent breeders were usually retained until the
next season, and females were kept at least until several months after they weaned kits, and
their AGD could not be reliably measured alive. Finally, I observed males in November (Millbank
2012 only; see Chapter 2) to quantify stereotypic behaviour as part of a wide-ranging project on
environmental enrichment (Meagher et al., 2014). Unfortunately, as I could not yet reliably
distinguish males and females from a distance, the frequency of stereotypic behaviour available
for each male is in fact an average for the two sibling cagemates.
I obtained behavioural data from nearly every day of the mating season at Millbank in
2012 (15 days of data, with help from another observer) and at RBR in 2013 (13 days) and 2014
(13 days). Given that both farms breed their animals simultaneously, my data are more
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fragmentary (missing more days and encounters) for Millbank in 2014 (Experiment 2b), with
only 7 days of data between myself and 3 other observers. I took all body mass and anogenital
distance measures myself, and collected all fecal samples except for those at Millbank in 2012,
which were collected by the other observer.
Data analysis
When testing for relationships between rough-and-tumble play and adult male sexual
behaviour, I analyzed data from each farm separately. I did this because a cross-farm analysis
could not be run without dropping some useful information available only on one of the farms. I
used a cross-farm model only when analyzing the effect of social isolation, as only a small
number of single-housed males were available at each farm. In this case, I did not include
variables not available for both farms. I did, however, include juvenile housing area or breeding
group as blocking factors in these models.
I used survival analysis, specifically mixed-effects Cox proportional hazards models, run
with the ‘coxme’ package for R statistical software (Cox, 1972; R Core Team, 2015; Therneau,
2015), to test for associations between juvenile male rough-and-tumble play and latency to
catch, latency to copulate, and duration of copulation, all two-tailed at alpha = 0.05. This
approach has the advantage of taking into account right-censored data (e.g. a copulation that
lasted at least 30 minutes but was manually interrupted by farmers, making it impossible to
know its true duration), which would otherwise either have to be discarded or have distorting
effects on measures of central tendency such as mean copulation duration for each male. The
Cox procedure models the hazard rate, or the probability that an event (e.g. the start or end of
copulation) will occur at any given time. While the hazard rate may change over time, the
multiplicative effects of factors and covariates are themselves assumed to be constant: for
example, belonging to a certain treatment group should have the same effect on the hazard rate
at all points in time. Therefore, in survival analysis, significant effects indicate both that the
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latency with which an event occurs differs between groups, and that the overall probability of
this event occurring differs between groups, assuming that they are observed for equivalent
lengths of time. I verified the so-called ‘proportional hazards’ assumption – that effects on the
hazard rate are constant over time – for each independent variable graphically, judging that it
was violated where survivorship curves for different subsamples (e.g. different treatment
groups) crossed each other (Ata and Sözer, 2007). I then used Ata and Sözer's (2007) nointeraction stratified correction procedure by entering these independent variables as strata,
which affect the baseline hazard rate rather than having constant multiplicative effects
(significance of these baseline effects is not tested). Cox models of latencies to catch females or
to begin copulating included every observed encounter, with right-censoring for those that did
not result in catching/copulating. Cox models of copulation duration included every observed
instance of copulation (sometimes more than one per encounter), with right-censoring for those
that were manually interrupted. Cox models of male sexual behaviour included the following
independent variables wherever possible:
Subject attributes: experiment (with Experiment 2c divided by juvenile observation
method, which had a large effect on observed rough-and-tumble play in Chapter 3); juvenile
housing treatment (which includes the male’s strain; despite having inconsistent effects from
one year to the next, treatments had large effects within each experiment, either on their own or
in combination with spatial confounds as described in Chapter 3); frequency of juvenile roughand-tumble play, as a proportion of total time; and subject identity (random factor in repeated
measures Cox models). I additionally included, in male models only, the interaction of R&T with
experiment (in farm-wide models) or with juvenile housing treatment (in single-experiment
models).
Attributes of the female paired with a male in each encounter: whether or not the female
is nulliparous (parous females generally mate more readily, if only because females who do not
185
produce litters in their first year are usually culled: e.g. Hansson, 1947); has failed to copulate at
least once during the current round of mating; and proportion of days on which she copulated
successfully (RBR only), out of all days during the mating season where she was paired with at
least one male, and that did not include an encounter with the present subject.
Circumstantial data: date (combined across years as days since March 1st; copulation
duration has previously been observed to increase later in the mating season: e.g. Elofson et
al., 1989; Fleming, 1996; Hansson, 1947); whether or not the male has already been paired with
one or more other females during the present morning or afternoon (Millbank males only); nth
copulation bout within encounter (copulation duration only); encounter (copulation duration only,
random factor nested within male identity).
Where survival analyses identified significant relationships between rough-and-tumble
play and a sexual outcome measure, I then tested alternative hypotheses about factors that
may mediate this relationship. First, I tested whether sexual behaviour is predicted by roughand-tumble play specifically, independently of overall activity levels, by re-running models using
rough-and-tumble play as a proportion of active time only, rather than of overall time. I had
already found activity levels to be related to rough-and-tumble play frequency, in Chapter 2.
Next, I also re-ran models with extra covariates, separately for each hypothesis. If a variable
does mediate the relationship between rough-and-tumble play and sexual behaviour, then its
inclusion in the statistical model should eliminate, or at least considerably weaken, that
relationship. To test for effects of adult androgen production, I used fecal concentrations of
testosterone and epiandrostenone metabolites. In each case, these were corrected for fecal
corticosterone metabolite concentrations (simultaneously included in the model) due to the
likelihood of cross-contamination of these metabolites in the assay we used (Andersen, 2013;
Díez-León et al., 2013; Rupert Palme, personal communication, September 2013). All hormonal
metabolite concentrations were log-transformed to achieve normality; while this is not required
186
for Cox models to produce valid results, it facilitates interpretation of hazard ratios for hormonal
effects on play. I also included the interaction of androgen concentration with experiment
because fecal samples were taken at different times of year in different experiments (Table 5–1)
and, at least in the case of testosterone production, individual differences may reverse
themselves over the course of the winter in mink (Sundqvist et al., 1984; Tahka et al., 1991).
Because hormonal concentrations are likely to be related to overall activity levels, I also ran
these tests using rough-and-tumble play as a proportion of active time. To test for effects of
early exposure to androgens, I used anogenital distance (AGD) corrected for body mass,
available only at RBR Fur Farm (Hotchkiss and Vandenbergh, 2005; Swan et al., 2005). Finally,
I tested for effects of stereotypic behaviour in early adulthood, given that in a previous mate
choice experiment, males from environmentally enriched cages copulated more often and were
less stereotypic than non-enriched controls, and stereotypic behaviour frequency predicted the
number of copulations obtained (Díez-León et al., 2013); although note, however, that a
separate study found no association between abnormal behaviours (including stereotypies) and
the proportion of female-male encounters resulting in copulation in a typical farm situation
(Andersen, 2013). There were other factors that could conceivably affect both rough-and-tumble
play and sexual behaviour, but that I did not test as potential mediators because I previously
found them to be unrelated to rough-and-tumble play. Body mass affected sexual outcomes in
previous mink experiments (Díez-León et al., 2013; Thom et al., 2004), but not rough-andtumble play frequency in Chapter 3. Socially isolated male mink were reported to be fearful of
potential mates (Gilbert and Bailey, 1969), but juvenile rough-and-tumble play did not predict
adult temperament in Chapter 4.
Below, I report the number of trials (male-female encounters) on which each survival
analysis test is based. Because statistical power in Cox models is a function of the number of
non-censored trials (Vittinghoff and McCulloch, 2007), I report this figure alongside the total
187
(non-censored + censored) number of trials. The number of trials is not constant between tests,
both because some encounters did not progress far enough (e.g. trials without copulation could
not be used to estimate copulation duration), and because some data were excluded due to
missed observations or faulty recordkeeping that made it impossible to estimate latencies or
durations, in some cases affecting only some outcome measures but not others. Below, effect
sizes are presented as hazard ratios, which represent the relative probability of an event
occurring in different groups of animals. The higher the hazard ratio, the shorter the latency or
duration estimated. I consistently present these hazard ratios such that quantities greater than
one represent increased probabilities, i.e. shorter latencies or durations, either in animals with
higher frequencies of rough-and-tumble play, or in those who are pair-housed as opposed to
single-housed. Thus, in these cases my hypotheses for males predict hazard ratios above one
(shorter) for latency to catch or to copulate, and below one (longer) for copulation duration. The
reverse is predicted for females. In analyses where R&T frequency or another continuous
variable is the predictor of interest, the hazard ratio compares subjects differing in frequency by
one standard deviation, calculated among the subset of animals used in that particular model.
Results
Below, I report test statistics only for those variables that were of primary interest in each
analysis. For other covariates and factors included in each statistical model, outcomes are
presented separately in the appendix.
Male sexual behaviour
The frequency of rough-and-tumble play was not correlated to latency to catch females,
i.e. to bite their necks (Table 5–2). The same was true for latency to begin copulating, whether
measured from the beginning of the encounter or from when the female was caught. This also
indicates that rough-and-tumble play did not predict how likely males were to catch females or
188
to copulate with them. In contrast, rough-and-tumble play was significantly correlated with the
duration of copulation on both farms2. Elevated frequencies of rough-and-tumble play were
associated with a reduced likelihood of copulations ending, i.e. with longer durations (Figure 5–
1). This effect was significant on each farm, with a significant interaction between R&T and
experiment at Millbank Fur Farm. Testing each Millbank experiment separately showed that the
effect was only seen in 2012 (Chapter 2) and not in 2014 (Experiment 2b). These effects
remained significant on both farms when double-checked using R&T as a proportion of active
time as a predictor, rather than of total time budget.
Table 5–2: Effects of juvenile rough-and-tumble play on adult male sexual outcome
measures in farm-wide survival analysis models. The interaction p value is for year or
treatment by rough-and-tumble play. For each model, I also list those female attributes
whose effects were tested, with an asterisk beside those that were significant and trends
in brackets. Where effects are not listed, they were not tested because they violated the
proportional hazards assumption. The effects are: (P)arity, (F)ailure to mate earlier in
current round of mating, and at RBR only, (S)uccessful mating percentage on days when
mating only with other males.
Outcome
Subset
Latency to
bite neck
Latency to
copulate
Latency to
copulate
after biting
neck
Copulation
duration
RBR
Millbank
RBR
Millbank
RBR
Millbank
RBR
Millbank
Millbank
2012
Millbank
2014
Number of
trials (noncensored/total)
564/695
642/937
465/695
522/938
465/564
521/642
Main
effect
p
.95
.44
.99
.93
.92
.97
Hazard ratio
(95% CI)
Interaction
p
0.94 – 1.04 – 1.15
0.80 – 0.98 – 1.19
0.70 – 1.00 – 1.43
0.85 – 0.99 – 1.16
0.70 – 1.00 – 1.43
0.85 – 0.99 – 1.16
.548
.330
.766
.572
.382
.827
Female
attribute
effects
P, F, S*
P*, F*
P*, F, S*
P, F*
P*, F, S*
P*, F
213/583
333/538
286/443
.047
.0003
.0013
0.49 – 0.69 – 0.99
0.63 – 0.74 – 0.87
0.61 – .073 – 0.88
.105
.022
.451
P(*), S*
-
47/95
.39
0.76 – 1.24 – 2.04
.999
-
2
While this was not the focus of this research, it is worth noting that as in several previous studies
(Elofson et al., 1989; Fleming, 1996; Hansson, 1947), copulation duration gradually increased over the
course of the breeding season. This effect of date was significant at Millbank Fur Farm and a trend at
RBR Fur Farm.
189
Figure 5–1: Survivorship curves comparing copulation duration in males according to
their levels of juvenile rough-and-tumble play. Males at Millbank Fur Farm (A) and at RBR
Fur Farm in 2012 (B) were classified into high (black), medium (dark grey), and low (light
grey) tertiles for R&T frequency using the residuals from an ANOVA in which juvenile
housing treatment predicted rough-and-tumble play, i.e. according to how much they
played compared to other similarly treated males. Circles represent right-censored
observations (manual interruption by farmers). Vertical dotted lines indicate the median
(A) or third quartile (B) within each group (too many observations were right-censored to
estimate median durations at RBR Fur Farm).
A)
190
B)
Next, I tested whether relationships between duration of copulations and rough-andtumble play frequency, the only ones that were significant, remained so after the inclusion of
various male characteristics that could mediate this relationship (Table 5–3). Fecal androgen
concentrations did not eliminate these significant effects on either farm, showing that androgens
did not mediate the link. Neither did fecal androgen concentrations, controlled for rough-andtumble play, themselves have any significant effects on copulation duration on either farm, and
nor did they interact with experiment at RBR Fur Farm. High epiandrostenone tended to be
associated with long copulations at Millbank farm only. Re-running these tests using rough-andtumble play as a proportion of active time changed no other results but made this trend
significant (286/443, p = .040, HZ = 1.01 – 1.21 – 1.45).
191
The relationship between rough-and-tumble play and copulation duration was no longer
significant when controlling for anogenital distance and body mass. However, this was the case
within the reduced subset of culled males with known AGD, even when not controlling for body
mass or AGD, presumably due to lower statistical power in this subsample. Further, anogenital
distance controlled for body mass (and rough-and-tumble play) did not itself predict copulation
duration.
Controlling for stereotypic behaviour did not affect the significance of the relationship
between rough-and-tumble play and copulation duration at Millbank Fur Farm, and neither was
stereotypic behaviour itself (controlled for rough-and-tumble play) predictive of copulation
duration.
I double-checked the lack of a relationship between rough-and-tumble play and latencies
to catch females and to begin copulating by re-running these analyses with each of the above
covariates or potential confounds in the model. This did not affect the significance of any of
these tests.
Table 5–3: Effects of including male attributes as covariates on the relationships between
rough-and-tumble play and copulation duration in RBR (both experiments) and Millbank
(2012) males. The p value and hazard ratio for the covariate itself is also given, to
indicate whether they had significant effects on copulation duration, independently of
rough-and-tumble play.
Potential
mediator
Farm
Testosterone
Epiandrostenone
AGD
Stereotypic
behaviour
RBR
Millbank
RBR
Millbank
RBR
Millbank
Number of
trials (noncensored/total)
117/392
286/443
117/392
286/443
80/182
286/443
R&T
p
Hazard ratio for
R&T (95% CI)
.042
.0021
.025
.0027
.51
.0048
0.19 – 0.43 – 0.97
0.61 – 0.74 – 0.90
0.14 – 0.35 – 0.88
0.62 – 0.75 – 0.91
0.47 – 0.83 – 1.45
0.60 – 0.74 – 0.91
192
Mediator
p
.38
.17
.21
.064
.11
.89
Hazard ratio for
mediator
(95% CI)
0.27 - .066 – 1.66
0.94 – 1.16 – 1.44
0.28 – 0.60 – 1.32
0.99 – 1.18 – 1.42
0.89 – 1.72 – 3.35
0.87 – 1.01 – 1.17
Finally, the sexual behaviour of males who became single-housed at some point during
the pair stage was not significantly different from that of pair-housed males in any way (Table 5–
4). This was the case whether the animals used as controls were those raised in similar juvenile
areas, or those who were part of the same breeding group (and thus were tended to by the
same workers and mated to the same pool of females).
Table 5–4: Effects of single-housing during the pair phase on adult male sexual outcome
measures, tested across both farms.
Outcome
Latency to bite
neck
Latency to copulate
Latency to copulate
after biting neck
Copulation duration
Matched
controls
Juvenile
Adult
Juvenile
Adult
Juvenile
Adult
Juvenile
Adult
Number of trials
(non-censored/total)
213/338
337/511
199/338
300/512
199/213
299/337
78/222
121/276
Main effect p
0.35
0.26
0.54
0.83
0.73
0.32
0.94
0.95
Hazard ratio
(95% CI)
0.84 – 1.18 – 1.65
0.83 – 1.28 – 1.97
0.71 – 1.16 – 1.90
0.44 – 0.92 – 1.94
0.54 – 0.91 – 1.54
0.40 – 0.73 – 1.34
0.35 – 0.96 – 2.65
0.47 – 1.03 – 2.26
Female sexual behaviour
First, female attributes had significant effects in many tests for relationships between
male rough-and-tumble play and sexual behaviour (Table 5–2). Having previously failed to mate
during the current round of mating, and having a low successful mating percentage throughout
the mating season, were both associated with increased latencies to be caught or to begin
copulating and, in the case of low success rates, with reduced copulation duration. Effects of
parity were less coherent, with parous females (who were also older) showing increased
latencies to be caught but reduced latency to begin copulating (on different farms), and tending
to have shorter copulations.
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Table 5–5: Effects of juvenile rough-and-tumble play and of single-housing during the
pair phase on adult female sexual outcome measures at RBR Fur Farm.
Outcome
Effect tested
Latency to bite
neck
Latency to copulate
Latency to copulate
after biting neck
Copulation duration
Main effect p
Juvenile play
Number of trials
(non-censored/total)
30/36
Single-housing
Juvenile play
66/75
20/36
0.89
0.0088
Hazard ratio
(95% CI)
0.545 – 0.99 –
2.15
0.49 – 1.05 – 2.27
0.10 – 0.27 – 0.72
Single-housing
Juvenile play
Single-housing
Juvenile play
Single-housing
53/79
20/30
50/66
7/23
17/60
0.38
0.0026
0.12
0.34
0.53
0.64 – 1.43 – 3.20
0.06 – 0.18 – 0.54
0.81 – 2.33 – 6.70
0.35 – 2.75 – 21.7
0.19 – 0.66 – 2.36
0.97
Turning to the effects of rough-and-tumble play itself in females, my correlational
analysis revealed no significant relationships between it and latency (or probability) to be caught
or duration of copulation (Table 5–5). However, females who played most frequently started
copulating significantly less rapidly than those who played the least, whether calculating latency
from the start of the encounter or from the time at which the female was caught (Figure 5–2). In
other words, the most playful females were those least likely to copulate with males. The
significance or lack of significance of these tests was not affected by using as a predictor roughand-tumble play as a proportion of active time, rather than of total time.
Females who were single-housed during the juvenile period did not significantly differ
from pair-housed controls (sharing an adult mating environment) on any measure (Table 5–5).
194
Figure 5–2: Survivorship curves comparing latency to copulate in females according to
their levels of juvenile rough-and-tumble play. Latency to copulate was measured from
the start of the encounter (A) or from the time at which the female’s neck was bitten (B).
Females at RBR Fur Farm were classified into high (black) and low (grey) R&T groups
based on a median split (not tertiles due to small sample size) using the residuals from
an ANOVA in which juvenile housing treatment predicted rough-and-tumble play, i.e.
according to how much they played compared to other similarly treated females. Circles
represent right-censored observations (separation without copulation having occurred).
Vertical dotted lines indicate the median latency within each group.
A)
195
B)
Discussion
To summarize my main findings, frequent rough-and-tumble play in pair-housed juvenile
mink, aged 10 weeks and up, was, as predicted, associated at adulthood with long-lasting
copulations in males, an effect that was not a by-product of overall activity levels. In addition,
among males, elevated fecal epiandrostenone concentrations were also associated with long
copulations, independently of rough-and-tumble play, but neither this nor any other measured
male attributes explained the relationship between R&T and copulation duration. In the smaller
sample of females observed at RBR Fur Farm in 2014, frequent rough-and-tumble play when
juvenile instead predicted high latencies to begin copulating. Social isolation starting between c.
10.5 and 14.5 weeks of age had no discernible effect on sexual behaviour in either sex.
196
The significant correlations between rough-and-tumble play and sexual outcomes
provide support for the hypothesis of play as preparation for adult sexual behaviour. Males with
the highest frequencies of R&T copulated for longer durations, possibly giving them an
advantage in sperm competition. Females who played the most, meanwhile, were slower and
less likely to even begin copulating. This is in line with my initial predictions, based on the
hypothesis that early rough-and-tumble play increases females’ later capacity to reject certain
males, thus exercising pre-copulatory mate choice. However, my data do not allow me to
distinguish between this and an alternative hypothesis, that the most playful females were less
sexually receptive overall due to enhanced masculinization, an issue I return to later in the
discussion. Within the large pool of females mated with RBR subject males, individual
differences were highly predictable and consistent – females who infrequently mated with other
males showed long latencies to be caught and to begin copulating, and short copulation
durations – showing that females exert a large degree of control over mating in mink, even in a
forced mating context. If rough-and-tumble play truly does provide a reproductive advantage in
both sexes, this could explain the unexpected lack of sex differences in R&T frequency
identified in Chapter 3 (in litters, where no spatial confounds confused the outcome). If the
benefits of play are not sexually dimorphic and male-biased in this species, then neither should
we expect to see the usual male-biased sexual dimorphism in play (Chau et al., 2008; Pedersen
et al., 1990).
Of course, my empirical support for the hypothesis that rough-and-tumble play has
sexual benefits must be qualified, for several reasons. First, only a few significant relationships
were identified: playful males did not catch females nor begin copulating faster than others, and
playful females did not take longer to be caught nor copulate for shorter durations. Second,
rough-and-tumble play frequency did not predict copulation duration among Experiment 2b
males; note, however, that this had the lowest statistical power of any of the statistical tests for
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copulation duration in males (Table 5–2), with the smallest number of subjects observed over
the smallest number of days (12 males with 47 non-censored copulations, vs. a minimum of 44
males and 213 non-censored copulations across both RBR experiments, which were tested in
tandem). The same criticism can be applied to my statistical tests of females (Table 5–5): the
number of times females were observed copulating was unfortunately very low (a mere 7 noncensored copulations split between 5 females), most likely unjustifiably so in the context of
survival analysis (Vittinghoff and McCulloch, 2007). Finally, as explained in this chapter’s
introduction and in Chapter 1, correlations are a fundamentally weak form of evidence for
functional hypotheses about play.
The lack of any significant effect of social isolation on sexual behaviour, whether in
males or in females, does not support the hypothesis that rough-and-tumble play is an actual
cause of effective sexual behaviour. That depriving animals of social contact did not impair their
sexual function may seem like a clear demonstration that play, at least within the range of ages
studied, is not crucial to sexual development. However, there are several reasons to interpret
these results very cautiously. First, this experiment does not discount the possibility that earlier
play experience may have served this function. Male mink in previous experiments became
sexually dysfunctional if isolated before 8 weeks of age (Bassett et al., 1959; Gilbert and Bailey,
1969; Hansen et al., 1997). Though none of these authors compared males isolated at ages
beyond 8 weeks to non-isolates, the more or less normal sexual behaviour of older isolates
(Bassett et al., 1959; Gilbert and Bailey, 1969) suggests that social experience between the
onset of rough-and-tumble play at c. 5.5 weeks and isolation at 8+ weeks may have been
sufficient. Ceiling effects could preclude isolation of older animals from affecting sexual
behaviour if only a small amount of R&T is required for proper development (Martin and Caro,
1985). That this could apply here is also suggested by previous findings that even brief playful
social contact leads to appropriate sexual behaviour in other species (Beach, 1967; Hole et al.,
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1986; Zimbardo, 1958). Second, the non-random assignment of subjects to the social isolation
condition may have obscured the true effects of social isolation on sexual behaviour. For ethical
reasons, I avoided deliberately isolating animals and instead only used spontaneously-isolated
subjects. While the former male cagemates of isolated females were almost all confirmed to
have died, I do not know how any of the male isolates lost their female cagemates. Isolates may
have differed from controls in relevant ways, for example if they were aggressive and injured or
killed their cagemates, or in the case of males, if they were housed with (and likely siblings of)
females capable of escaping their cage. Third and finally, the possibility of Type II error is also
quite real, given the very low numbers of male (6) and female (7) subjects who remained
isolated throughout the juvenile period after losing their partner and were subsequently chosen
as breeders. While I partially mitigated this problem by using at least 3 times as many pairhoused controls in each case, the fact remains that the presence of even just a few atypical
individuals in my isolate sample could easily obscure any real effects.
Despite testing several different endocrine, morphological, and behavioural
characteristics of males, I was unable to determine what factors might mediate the relationship
between rough-and-tumble play and duration of copulation. I found no evidence that highly
playful males copulated for longer durations because they had higher levels of androgen
hormones, or because they had been exposed to higher levels of these in early life (as indexed
by AGD). While controlling for body mass and anogenital distance did eliminate the significant
effect of play on copulation duration, this was only because doing so drastically reduced the
subject pool, and not because of the inclusion of these additional predictors. Additionally,
though fecal epiandrostenone levels were positively correlated with copulation duration,
controlling for these did not change the significant relationship between play and duration;
effects of these two variables were independent of each other. This relationship is in itself
somewhat novel. While plasma androgen levels (testosterone) and sexual behaviour have been
199
found to correlate positively in male rats (Damassa et al., 1977), it should be noted that there
was a ceiling effect starting near the lower end of the normal physiological range of testosterone
concentration in that study, and that other studies often find that sexual behaviour is related to
brain aromatase activity, rather than to circulating androgen levels (e.g. Balthazart, 1989;
Portillo et al., 2007). Here, I did not measure whether males differed in their sensitivity to given
levels of circulating androgens, and thus cannot rule out a correlation between this and roughand-tumble play or androgen exposure during the later juvenile period, with potential impacts on
sexual behaviour. The relationship between fecal epiandrostenone metabolite concentration and
copulation duration should be interpreted with caution, given that it only held on one of the two
farms, that fecal testosterone metabolites had no significant effects, and that one previous study
of over 300 male mink did not find any correlation between the proportion of mating encounters
resulting in copulation and fecal testosterone metabolites (controlling for fecal cortisol
metabolites, as in the present study) (Andersen, 2013). There was also no evidence that the
relationship between rough-and-tumble play and copulation duration was mediated by a
propensity for playful males to be generally more active, or less stereotypic. However, it is
important to note that stereotypic behaviour was only observed with low prevalence and very
low frequencies (25 of 60 males, with an overall mean of c. 0.1% of total time budget), and that
it was a particularly noisy measure, as males could not be reliably distinguished from their
female cagemates, and “stereotypic” males could in fact have been brothers of stereotypic
females.
Thus, none of the male attributes tested appear to be responsible for the relationship
between R&T and copulation. Environmental factors are unlikely to explain this relationship
within the standardized farm environment. Researchers working on wild animal play have taken
pains to control for factors such as differential food availability, which is likely to affect both
juvenile play and later outcome measures such as survival in brown bears (Ursus arctos)
200
(Fagen and Fagen, 2004). Here, however, captive juveniles are fed standard amounts, slightly
adjusted by individual consumption and at near ad libitum levels. Furthermore, each animal was
moved to a different area of the farm between the juvenile and adult stages, precluding the
possibility of the ambient environment having similar effects at both life stages. One untested
explanation is that general vigour or endurance is the common underlying factor. Anecdotally, I
have witnessed whole rows of juveniles “crashing” and going to sleep after a pre-feeding play
frenzy, when food failed to arrive when expected. Experienced farmers often suggest that males
become physically exhausted during the breeding season, and farmers may in fact curtail the
length of sexual encounters for this very reason. Among 2014 RBR breeder male subjects,
weighed before and after the breeding season, all but one lost weight, for an average of 12.5%
of their body mass over the course of the mating season. Rough-and-tumble play and
courtship/mating may both be energetically-demanding behaviours at which particularly
vigorous individuals excel. If this is the case, then highly playful juveniles and effective breeders
should, for example, show physiological evidence of increased vigour, such as larger leg muscle
or cardiac mass, and differential food provision should have parallel effects on both types of
behaviour.
In females, meanwhile, the only variable I tested as a potential mediator of the
relationship between rough-and-tumble play and latency to copulate was overall activity level.
As in males, this did not cancel out the significant effect of play. Of particular concern is the
possibility that the most playful females are masculinized and therefore less sexually receptive
overall, rather than being more discriminating and capable of fending off lower quality mates.
One obvious approach would be to test, as I did in males, whether the effect is mediated by
androgen production or anogenital distance – unfortunately both measures that I did not have
for my female subjects. Another approach, which would require a much larger number of female
subjects, each of them paired with several different males, would be to test whether the most
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playful females are slower to begin copulating with all males, or only with less preferred males.
Finally, if rough-and-tumble play is negatively correlated with the rate at which females choose
to visit males in a self-paced mating situation, this would suggest that they indeed have lower
libido.
In conclusion, the research detailed in this chapter reveals the first known correlations
between juvenile rough-and-tumble play and adult sexual behaviour in any species. Frequent
rough-and-tumble play is associated with longer copulations in males, potentially providing an
advantage in sperm competition, and with long latencies to begin copulating in females,
potentially allowing for enhanced pre-copulatory mate choice. This evidence is purely
correlational, however, and observations of socially isolated animals did not support the
hypothesis that rough-and-tumble play has a causal role in this relationship. Finally, further
research will be required to elucidate its underlying causes.
202
Chapter 6 – Conclusion
General project summary
After four years of active research, my work has produced the following main insights
into the play of young farmed American mink (Neovison vison). First, rough-and-tumble play is
moderately susceptible to litter effects, only somewhat stable over the juvenile period, and
motivationally distinct from object play. Second, contrary to expectations based on other species
(Chau et al., 2008), rough-and-tumble play frequency is not sexually dimorphic in very young
mink living with their littermates. Third, I showed for numerous experimental treatments,
involving the provision of environmental enrichments or of social partners expected to differ in
playfulness, that these did not affect the frequency of rough-and-tumble play. Fourth, contrary to
predictions of the hypothesis of play as training for the unexpected (Špinka et al., 2001), juvenile
rough-and-tumble play frequency was not correlated with adult fearful behaviour in response to
aversive stimuli associated with handling. Finally, partially consistent with the hypothesis that
rough-and-tumble play prepares mink for sexual behaviour (Pellis and Pellis, 2009), juvenile
rough-and-tumble play was, as predicted, associated with high copulation durations in males
and long latencies or low probabilities to begin copulating in females, though no other measured
aspects of sexual behaviour were affected. To my knowledge, this is the first demonstration of a
spontaneous association between rough-and-tumble play and later sexual behaviour in any
species; previous studies have instead shown that social isolation, which prevents rough-andtumble play, can additionally profoundly disrupt sexual behaviour, at least in males, in mink and
other species. Here, however, the apparently normal sexual behaviour of a small sample of
male and female social isolates did not support the hypothesis that rough-and-tumble play,
during the latter part of the juvenile period, has a crucial preparatory role in sexual development.
203
Unfortunately, there is much that I had hoped to learn but could not because of
methodological problems. First, experimental treatments that appeared to elevate rough-andtumble play frequency had opposite effects when replicated the following year. Given my
inability to test these using proper experimental designs that did not confound treatment with
location, it is uncertain whether rough-and-tumble play is affected by mixed-strain or same-sex
pair-housing, or by housing groups of three juveniles in extra-large cages. Second, because I
failed to identify experimental treatments that reliably and chronically elicit or depress roughand-tumble play, I could not experimentally test my hypotheses about long-term functional
benefits of rough-and-tumble play. Third, while I have quite clearly shown that rough-and-tumble
play is not correlated with later fearful behaviour in response to the threat of handling, in stick or
glove tests, my data are not so conclusive with respect to its lack of a relationship to
physiological activation in response to handling itself. Fourth, despite testing several potential
endocrine, morphological, and behavioural mediators, I could not discern what underlies the
relationship between juvenile rough-and-tumble play and male copulation duration. Finally, and
most importantly, my work has not provided satisfactory evidence in favour of any hypothesis
about the functions of rough-and-tumble play in mink, which had been my primary aim.
The research detailed in this thesis does provide valuable lessons about how I or
another researcher should tackle this problem in the future. First, while working on commercial
farms certainly has its advantages – inexpensive access to an immense pool of subjects, and
collaboration with caretakers possessing decades of accumulated knowledge and expertise –
experimental priorities can clash with management priorities. Here, this resulted in sub-optimal
experimental design, misleading results, and pursuit of a fruitless line of research. Next time, it
may be wise to begin by testing the potential of different experimental treatments to modulate
the frequency of play in more tightly controlled laboratory or experimental farm conditions. If
large numbers of subjects are then needed (e.g. to study effects on sexual behaviour, where
204
only a minority of juveniles will go on to breed), the researcher may then move on to the
commercial farm with a more well-founded understanding of the effect of various experimental
treatments on play. Second, longitudinal studies of seasonal breeders who only breed once
annually are incredibly risky, even with ideal experimental design. Having to wait for the next
cohort of juvenile mink between each experiment meant that I was unable to rapidly tweak my
methods. This does not leave much room for error, particularly in the context of a graduate
program meant to last only a few years. For future students intending to conduct a replicated
longitudinal study of this scope, it would be more appropriate to choose a study species that
does not breed seasonally and/or has a short generation time. The laboratory rat (Rattus
norvegicus), in particular, is just such a species with an already immense body of existing
rough-and-tumble play research to build on.
The state of play in ethology and psychology
At present, popular discourse is rife with claims about the developmental benefits of
play. To illustrate, at the time of this writing, a Google search for “play in children” turns up a first
page of results that unanimously herald play’s positive effects, ranging over social, cognitive,
emotional, and physical domains. Play, we learn, is “essential to development” (Ginsburg et al.,
2007), “one of the most important things you can do with your child” (Raising Children Network,
2015), “the vital activity that children use to learn about and interact with their world” (Boston
Children’s Museum, 2015), and “may even be the cornerstone of society”3 (Goldstein, 2012).
These optimistic assessments fit squarely into what Sutton-Smith (1997) called the “rhetoric of
play as progress”, which views play as foundational to development, and which has gained
prominence in industrialized or post-industrial Western societies (Cohen, 1993; Larson and
Verma, 1999). They also come as a reaction to what may be called the ‘domestication’ of
3
This last statement echoes Johan Huizinga's (1950, original 1938) fascinating Homo Ludens, a cultural
history that places play at the root of human endeavours including art, philosophy, law, and war.
205
children’s play: a marked reduction in the time available for discretionary activity, and in the
opportunities for unstructured and even risky play, over the last several decades in these
societies (Brussoni et al., 2012; Hofferth and Sandberg, 2001; Larson and Verma, 1999;
Staempfli, 2009). While these claims about play’s importance in development are employed in
service of a noble goal, and in my opinion are likely to be broadly correct, what these authors
and I still lack is solid evidence.
To this day, our collective knowledge about the functions and effects of play remains
unsatisfactory. Much of the available evidence is inadequate due to methodological flaws and to
an over-reliance on correlation. For example, Lillard and co-authors (2013) show in an
enlightening review that after careful consideration, the dozens of papers focusing on effects of
pretend play in children (Homo sapiens sapiens) fail to convincingly demonstrate causal links to
later social or cognitive outcomes. At best, they argue, the relationship between pretend play
and these outcomes is one of “equifinality” (Martin and Caro, 1985), meaning that play is only
one of several routes by which they may be achieved; alternatively, observed relationships may
be driven by unmeasured third variables or even altogether illusory, where methodological
rigour is wanting. While ethologists have the advantage of being able to use social deprivation,
which child psychologist Lynn Barnett (1990) points to as an absurd, unethical, and unattainable
gold standard for her field, I have argued in previous chapters that the concurrent effects of
social deprivation on non-play behaviour limit its usefulness. There is unfortunately still much
truth to Muller-Schwarze’s claim (1971, cited in Smith, 1978) that “the amount of time and paper
spent on speculations about motor play in immature animals is in inverse proportion to the
amount of facts available on the question”. Ethologist and experienced play researcher Lynda
Sharpe more recently summarized this state of affairs in a humorous Scientific American blog
post (2011), stating that “in quiet desperation, play researchers have come up with more than
206
two dozen possible benefits of play but, in spite of four decades of effort, they’ve found
conclusive evidence for none.”
What, then, can we say about the functions of play with any confidence? While we do
have fairly good evidence that play has acute effects on e.g. hormonal (Arelis, 2006; Horváth et
al., 2008) and neurobiological outcomes (Burgdorf et al., 2010; Vanderschuren, 2010), it is
unknown whether these have cumulative long-term consequences. While some studies have
identified longitudinal effects of housing rats with non-playful (adult or drugged) partners for
several weeks, providing better evidence than outright social deprivation experiments,
interpretation is difficult because the quality of social interaction provided by these stimulus
animals still differs in respects other than play (Bean and Lee, 1991; Bell et al., 2010). The best
currently available evidence that play has long-term benefits comes from two sources: a study
relating juvenile brown bear (Ursus arctos) play to yearling survival even after carefully
controlling for potential confounds like maternal care and food availability (Fagen and Fagen,
2004); and studies of partially isolated juvenile rats, who spend their brief daily periods of
socialization engaged largely in rough-and-tumble play to the exclusion of other social
behaviour (Einon et al., 1978; Pellis and Pellis, 2009), and who have then been shown to differ
from full isolates in terms of temperament (Einon et al., 1978) and male sexual behaviour (Hole
et al., 1986; Zimbardo, 1958).
In the present study, my primary methodological ambition was to study functional
hypotheses about rough-and-tumble play using a triangulation approach, in which multiple
experimental treatments affect the frequency of play in different ways throughout the juvenile
period, and convergent effects on adult outcomes are then tested for. This approach, building
on the work of Humphreys and Einon (1981), who used it to study acute effects of play on maze
learning in rats, demands stringent evidence in order to generate support for a hypothesis. The
likelihood that observed outcomes are due to confounding, non-play effects of any single
207
experimental treatment is dramatically reduced if multiple treatments generate similar results.
To my mind, this was to be the most novel methodological contribution of my doctoral work to
the study of long-term benefits and functional hypotheses about play. Sadly, the potential
strength and feasibility of this approach remain untested despite my efforts. I intend to try again.
208
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237
Appendix
Here, I present model outputs for each statistical model presented in Chapters 4 and 5.
The position of each model in the main text is indicated by page number and, where relevant,
table or figure number, along with a brief description of what is being tested, quoted from the
main text. Where multiple analyses are presented for the same page or table, they are shown in
the same order as in the text. Where multiple entries in the main text refer to the same
underlying model, this is indicated here by referring to the earliest such instance (e.g. “see
model output 1”). The test statistics presented here are not the same for each test, due to
differences in the output given by the various R statistical packages from which they were
obtained.
1. p. 156 “the probability of fearful responses differed between experiments”
Df Sum Sq Mean Sq
Experiment
4 203.181 50.795
as.factor(DateNum)
3 75.200 25.067
Sex
1 35.414 35.414
ObsType
1
8.303
8.303
Play
1
1.095
1.095
Experiment:Strain
3 69.764 23.255
Experiment:paste(Treatment, ExEuro) 11 41.484
3.771
Experiment:Play
4
4.359
1.090
F value
50.7951
25.0665
35.4144
8.3025
1.0950
23.2548
3.7712
1.0898
2. p. 157 “responding most fearfully on the first day of testing”
See model output 1.
3. p. 157 “fearful responding on Day 1 predicting fearful responding on Day 2”
Experiment
Sex
ObsType
FirstRepIgnoreMM == "F"
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
Df Sum Sq Mean Sq F value
1 3.5854 3.5854 3.5854
1 18.1425 18.1425 18.1425
1 3.2961 3.2961 3.2961
1 7.8494 7.8494 7.8494
1 8.2539 8.2539 8.2539
3 3.7907 1.2636 1.2636
4. p. 157 “and confidence similarly predicting confidence”
Experiment
Sex
ObsType
FirstRepIgnoreMM == "C"
Df Sum Sq Mean Sq F value
1 23.6581 23.6581 23.6581
1 16.0572 16.0572 16.0572
1 5.5296 5.5296 5.5296
1 5.1793 5.1793 5.1793
238
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
1
3
2.0739
0.3523
2.0739
0.1174
2.0739
0.1174
5. p. 158 “main effect of collection time […] on FCM concentration”
(Intercept)
Farm
Sex
Batch.Type.Glom
Farm:Batch.Type.Glom
numDF denDF F-value p-value
1
185 3489.439 <.0001
1
155
2.733 0.1003
1
155
21.037 <.0001
2
185
16.211 <.0001
2
185
21.990 <.0001
6. p. 158 “along with an interaction between collection time and farm”
See model output 5.
7. p. 158 “and a significant effect of sex”
See model output 5.
8. p. 158 “no effect of collection time at Millbank Fur Farm”
(Intercept)
Treatment
Sex
Batch.Type.Glom
Treatment:Batch.Type.Glom
numDF denDF
F-value p-value
1
90 2401.7339 <.0001
2
88
1.0987 0.3378
1
88
1.3783 0.2436
2
90
0.7162 0.4914
4
90
1.0714 0.3754
9. p. 158 “at RBR Fur Farm, in contrast, there were significant effects of both
treatment […] and sex”
(Intercept)
Treatment
Sex
Batch.Type.Glom
Treatment:Batch.Type.Glom
numDF denDF
F-value p-value
1
87 1701.0244 <.0001
2
62
6.3210 0.0032
1
62
30.7355 <.0001
2
87
37.2670 <.0001
4
87
0.4284 0.7877
10. p. 158 “collection time did have a significant effect on FCM, which surprisingly
decreased with increasing time since capture”
See model output 9.
11. p. 159 (Figure 4–1) “significant interaction between collection period and day”
(Intercept)
Treatment
Sex
Date.As.Factor
Batch.Number.As.Factor
Date.As.Factor:Batch.Number.As.Factor
numDF denDF F-value p-value
1
359 3329.600 <.0001
2
63
8.735 0.0004
1
63
45.601 <.0001
1
359
0.000 0.9865
3
359
87.888 <.0001
3
359
16.986 <.0001
239
12. p. 159 “did not predict higher FCM at 4-6 hours, relative to the 0-2 hour baseline”
Df Sum Sq Mean Sq F value Pr(>F)
log(ngg_adj) 1 3.539 3.5393 1.7360 0.1980
Sex
1 0.031 0.0312 0.0153 0.9024
Treatment
2 6.265 3.1324 1.5365 0.2321
Scream
1 0.704 0.7044 0.3455 0.5612
Residuals
29 59.122 2.0387
13. p. 159 “screaming when handled was not significantly correlated with the
probability of fearful behaviour toward the stick or glove”
Experiment
as.factor(DateNum)
Sex
as.factor(ScreamWhenHandled)
Experiment:Strain
Experiment:Treatment
Df Sum Sq Mean Sq F value
1 33.828 33.828 33.8284
1 7.848
7.848 7.8480
1 0.878
0.878 0.8778
1 2.713
2.713 2.7125
1 18.968 18.968 18.9678
3 5.881
1.960 1.9602
14. p. 160 “a lower probability of confident behaviour in this test”
Experiment
as.factor(DateNum)
Sex
as.factor(ScreamWhenHandled)
Experiment:Strain
Experiment:Treatment
Df Sum Sq Mean Sq F value
1 68.951 68.951 68.9511
1 0.655
0.655 0.6554
1 1.402
1.402 1.4019
1 3.779
3.779 3.7792
1 8.907
8.907 8.9072
3 6.293
2.098 2.0978
15. p. 160 “did not predict the probability of fearful responses in the glove test”
Experiment
as.factor(DateNum)
Sex
log(ngg_adj_02)
log(ngg_adj_46)
Experiment:Treatment
Df
1
3
1
1
1
2
Sum Sq
0.25928
1.17912
2.71157
0.13412
0.95386
2.37493
Mean Sq F value
0.25928 0.2593
0.39304 0.3930
2.71157 2.7116
0.13412 0.1341
0.95386 0.9539
1.18746 1.1875
16. p. 160 “tended to negatively predict confident responding”
Experiment
as.factor(DateNum)
Sex
log(ngg_adj_02)
log(ngg_adj_46)
Experiment:Treatment
Df
1
3
1
1
1
2
Sum Sq Mean Sq F value
0.0836 0.0836 0.0836
5.1293 1.7098 1.7098
2.4054 2.4054 2.4054
0.2657 0.2657 0.2657
3.8946 3.8946 3.8946
0.0000 0.0000 0.0000
17. p. 160 “did not predict the probability of responding fearfully to the stick or glove
[…] and had no interaction with experiment”
Experiment
as.factor(DateNum)
Sex
Df Sum Sq Mean Sq
4 203.181 50.795
3 75.200 25.067
1 35.414 35.414
240
F value
50.7951
25.0665
35.4144
ObsType
1
Play
1
Experiment:Strain
3
Experiment:paste(Treatment, ExEuro) 11
Experiment:Play
4
8.303
1.095
69.764
41.484
4.359
8.303 8.3025
1.095 1.0950
23.255 23.2548
3.771 3.7712
1.090 1.0898
18. p. 160 “higher among females than among males […] and differed between
strains”
See model output 17.
19. p. 160 “the main effect of treatment was also significant”
See model output 17.
20. p. 160 “R&T was no more predictive of confident than of fearful responses”
Df Sum Sq Mean Sq F value
Experiment
4 486.49 121.624 121.6235
as.factor(DateNum)
3 40.68 13.559 13.5589
Sex
1 39.79 39.787 39.7865
ObsType
1
0.26
0.261
0.2613
Play
1
0.40
0.397
0.3966
Experiment:Strain
3 34.21 11.402 11.4020
Experiment:paste(Treatment, ExEuro) 11 44.66
4.060
4.0597
Experiment:Play
4
0.57
0.142
0.1420
21. p. 161 “Rough-and-tumble play did not predict screaming when handled”
Farm
Split
Sex
Play
Farm:Strain
Farm:Treatment
Farm:Play
Df Sum Sq Mean Sq F value
1 0.4098 0.4098 0.4098
1 1.5945 1.5945 1.5945
1 16.5182 16.5182 16.5182
1 0.1916 0.1916 0.1916
1 20.0835 20.0835 20.0835
4 4.7606 1.1902 1.1902
1 0.4860 0.4860 0.4860
22. p. 161 “and there was no significant interaction of play with experiment”
See model output 21.
23. p. 161 “females were more likely to scream than males […] as were black mink
compared to pastels”
See model output 21.
241
24. p. 161 “Rough-and-tumble play did not predict FCM 4-6 hours after capture,
controlled for baseline FCM at 0-2 hours”
Df
log(ngg_adj)
1
Farm
1
Treatment
4
Sex
1
Play
1
Treatment:Play 5
Residuals
30
Sum Sq Mean Sq F value
2.194 2.1940 1.1478
20.454 20.4542 10.7007
11.366 2.8415 1.4866
0.991 0.9914 0.5187
0.004 0.0035 0.0018
4.373 0.8746 0.4576
57.344 1.9115
Pr(>F)
0.292549
0.002694 **
0.231076
0.476980
0.966101
0.804461
25. p. 161 “weak but significant negative relationship between time spent alert and
observed rough-and-tumble play in juveniles”
Df
Farm
1
Obs.Type
1
AlertAnalyze
1
Farm:Type
3
Farm:AlertAnalyze
1
Obs.Type:AlertAnalyze
1
Residuals
304
Sum Sq
0.020334
0.013337
0.001757
0.009705
0.002333
0.001965
0.102947
Mean Sq F value
Pr(>F)
0.0203343 60.0468 1.398e-13 ***
0.0133374 39.3851 1.197e-09 ***
0.0017570 5.1884 0.023432 *
0.0032350 9.5528 4.780e-06 ***
0.0023334 6.8906 0.009103 **
0.0019646 5.8015 0.016607 *
0.0003386
26. p. 162 “alert behaviour did not positively predict the probability of fearful
responses”
Experiment
as.factor(DateNum)
Sex
ObsType
Alert
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
Experiment:Alert
Df Sum Sq Mean Sq F value
1 2.9235 2.9235 2.9235
1 28.0137 28.0137 28.0137
1 2.7607 2.7607 2.7607
1 0.2241 0.2241 0.2241
1 0.5121 0.5121 0.5121
1 10.0406 10.0406 10.0406
2 3.4208 1.7104 1.7104
1 1.2770 1.2770 1.2770
27. p. 162 “it was negatively correlated with confident responses”
Experiment
as.factor(DateNum)
Sex
ObsType
Alert
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
Experiment:Alert
Df Sum Sq Mean Sq F value
1 18.990 18.990 18.9903
1 42.730 42.730 42.7302
1 9.707
9.707 9.7066
1 0.392
0.392 0.3922
1 4.979
4.979 4.9790
1 0.000
0.000 0.0000
2 3.318
1.659 1.6588
1 0.022
0.022 0.0221
28. p. 162 “alert behaviour then tended to be positively correlated with fearful
responding”
Experiment
as.factor(DateNum)
Sex
Alert
Df
1
1
1
1
Sum Sq Mean Sq F value
1.4843 1.4843 1.4843
0.3281 0.3281 0.3281
0.0811 0.0811 0.0811
3.7762 3.7762 3.7762
242
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
Experiment:Alert
1 7.2452
2 1.8077
1 1.9770
7.2452
0.9039
1.9770
7.2452
0.9039
1.9770
29. p. 162 “but no longer predicted confident responses”
Experiment
as.factor(DateNum)
Sex
Alert
Experiment:Strain
Experiment:paste(Treatment, ExEuro)
Experiment:Alert
Df
1
1
1
1
1
2
1
Sum Sq Mean Sq F value
3.6479 3.6479 3.6479
6.7753 6.7753 6.7753
6.3409 6.3409 6.3409
0.0510 0.0510 0.0510
0.0000 0.0000 0.0000
2.9090 1.4545 1.4545
2.5777 2.5777 2.5777
30. p. 189 (Table 5–2) “Latency to bite neck – RBR”
Fixed coefficients
FY.ObsKirk 2014 Close
FY.ObsKirk 2014 Far
F.Failed.This.RoundTRUE
F.Lit.BinTRUE
Date.Uni
J.SocPlay
F.SuccessRateCorFull
FY.ObsKirk 2014 Close:J.SocPlay
FY.ObsKirk 2014 Far:J.SocPlay
coef
-0.72698645
-0.32137151
-0.03085343
-0.05384392
-0.04565373
-0.30412405
1.32864241
17.37797262
5.94028199
exp(coef)
se(coef)
4.833634e-01 0.45133883
7.251538e-01 0.40254026
9.696177e-01 0.10145406
9.475800e-01 0.08981390
9.553727e-01 0.01160439
7.377693e-01 5.23824081
3.775914e+00 0.16786081
3.524988e+07 17.59031823
3.800421e+02 8.41168345
z
-1.61
-0.80
-0.30
-0.60
-3.93
-0.06
7.92
0.99
0.71
p
1.1e-01
4.2e-01
7.6e-01
5.5e-01
8.3e-05
9.5e-01
2.4e-15
3.2e-01
4.8e-01
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.14944819 0.02233476
31. p. 189 (Table 5–2) “Latency to bite neck – Millbank”
Fixed coefficients
coef
exp(coef)
F.Failed.This.RoundTRUE
-0.34339267
0.7093596
F.Lit.BinTRUE
-0.23620191
0.7896212
Date.Uni
-0.04538442
0.9556301
F.First.BinTRUE
0.87805633
2.4062183
J.SocPlay
2.46385523 11.7500234
J.SocPlay:FY.ObsTed 2014 Close 6.34533228 569.8267018
se(coef)
z
p
0.100108673 -3.43 6.0e-04
0.085095041 -2.78 5.5e-03
0.009149568 -4.96 7.0e-07
0.296370634 2.96 3.0e-03
3.212994218 0.77 4.4e-01
6.477669599 0.98 3.3e-01
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.12858937 0.01653523
32. p. 189 (Table 5–2) “Latency to copulate – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
z
FY.ObsKirk 2014 Close
0.31275110 1.367181e+00 0.76072976 0.41
FY.ObsKirk 2014 Far
0.29895844 1.348454e+00 0.74206209 0.40
F.Failed.This.RoundTRUE
0.01059486 1.010651e+00 0.11723976 0.09
F.Lit.BinTRUE
0.41240350 1.510444e+00 0.10085007 4.09
Date.Uni
-0.02276231 9.774948e-01 0.01342326 -1.70
J.SocPlay
-0.08117547 9.220319e-01 9.64017885 -0.01
F.SuccessRateCorFull
1.36895713 3.931249e+00 0.18746911 7.30
FY.ObsKirk 2014 Close:J.SocPlay -22.70703548 1.375497e-10 31.15531688 -0.73
FY.ObsKirk 2014 Far:J.SocPlay
-4.34253406 1.300353e-02 15.55807190 -0.28
Random effects
Group
Variable
Std Dev
Variance
243
p
6.8e-01
6.9e-01
9.3e-01
4.3e-05
9.0e-02
9.9e-01
2.8e-13
4.7e-01
7.8e-01
B.Year.ID Intercept 0.4939354 0.2439722
33. p. 189 (Table 5–2) “Latency to copulate – Millbank”
Fixed coefficients
F.Failed.This.RoundTRUE
F.Lit.BinTRUE
Date.Uni
F.First.BinTRUE
J.SocPlay
J.SocPlay:FY.ObsTed 2014 Close
coef
exp(coef)
-0.38763242
0.6786618
-0.03396616
0.9666042
-0.00572028
0.9942961
0.67588051
1.9657631
-0.45995681
0.6313109
5.28965708 198.2754217
se(coef)
0.11241578
0.09778165
0.01026381
0.32006818
4.98531312
9.46523728
z
-3.45
-0.35
-0.56
2.11
-0.09
0.56
p
0.00056
0.73000
0.58000
0.03500
0.93000
0.58000
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.3843287 0.1477086
34. p. 189 (Table 5–2) “Latency to copulate after biting neck – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
F.Failed.This.RoundTRUE
0.16917946 1.184333e+00 0.11928076 1.42 1.6e-01
F.Lit.BinTRUE
0.58889969 1.802005e+00 0.10366116 5.68 1.3e-08
Date.Uni
0.01486789 1.014979e+00 0.01340126 1.11 2.7e-01
J.SocPlay
-1.04490669 3.517246e-01 10.47660141 -0.10 9.2e-01
FY.ObsKirk 2014 Close
0.97277036 2.645263e+00 0.82188380 1.18 2.4e-01
FY.ObsKirk 2014 Far
0.25877012 1.295336e+00 0.80785749 0.32 7.5e-01
F.SuccessRateCorFull
0.46869562 1.597909e+00 0.18431054 2.54 1.1e-02
J.SocPlay:FY.ObsKirk 2014 Close -47.41498855 2.558186e-21 33.66818371 -1.41 1.6e-01
J.SocPlay:FY.ObsKirk 2014 Far
-2.21159010 1.095264e-01 16.93176846 -0.13 9.0e-01
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.5518906 0.3045832
35. p. 189 (Table 5–2) “Latency to copulate after biting neck – Millbank”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
F.Failed.This.RoundTRUE
-0.16796612 0.8453825 0.11073934 -1.52 1.3e-01
F.Lit.BinTRUE
0.27437049 1.3157022 0.10327126 2.66 7.9e-03
Date.Uni
0.05615868 1.0577655 0.01077077 5.21 1.8e-07
J.SocPlay
-0.18882041 0.8279352 5.25507391 -0.04 9.7e-01
J.SocPlay:FY.ObsTed 2014 Close -2.18504913 0.1124722 10.35069017 -0.21 8.3e-01
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.4173738 0.1742009
36. p. 189 (Table 5–2) “Copulation duration – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
F.Lit.BinTRUE
0.27343629 1.314474e+00 0.15703214
Date.Uni
-0.03933599 9.614276e-01 0.02079113
CopNum
0.10731053 1.113280e+00 0.12309534
F.SuccessRateCorFull
-0.75324740 4.708351e-01 0.26655580
J.SocPlay
-19.30154950 4.144227e-09 9.70626884
J.SocPlay:FY.ObsKirk 2014 Close -44.68081381 3.938847e-20 38.60446014
J.SocPlay:FY.ObsKirk 2014 Far
26.03354787 2.024073e+11 16.75837967
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.30807625 0.09491098
B.Year.ID
(Intercept) 0.31678743 0.10035427
244
z
1.74
-1.89
0.87
-2.83
-1.99
-1.16
1.55
p
0.0820
0.0580
0.3800
0.0047
0.0470
0.2500
0.1200
37. p. 189 (Table 5–2) “Copulation duration – Millbank”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06603279 9.361002e-01 0.01449277 -4.56 5.2e-06
CopNum
0.02630242 1.026651e+00 0.27589146 0.10 9.2e-01
F.First.BinTRUE
-0.19486955 8.229420e-01 0.62732038 -0.31 7.6e-01
J.SocPlay
-18.61804088 8.208949e-09 5.15117163 -3.61 3.0e-04
J.SocPlay:FY.ObsTed 2014 Close 26.57240623 3.469360e+11 11.12127903 2.39 1.7e-02
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.019712713 0.000388591
B.Year.ID
(Intercept) 0.252087532 0.063548124
38. p. 189 (Table 5–2) “Copulation duration – Millbank 2012”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06536670 9.367239e-01 0.01503516 -4.35 1.4e-05
CopNum
0.07607891 1.079048e+00 0.29355329 0.26 8.0e-01
J.SocPlay
-22.03918857 2.682267e-10 6.83539807 -3.22 1.3e-03
J.SocPlay:J.TreatmentAllTed Black STD Black
7.87914444 2.641612e+03 10.20467129 0.77 4.4e-01
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.0197373045 0.0003895612
B.Year.ID
(Intercept) 0.2439638437 0.0595183570
39. p. 189 (Table 5–2) “Copulation duration – Millbank 2014”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06371690
0.9382706 0.05417459 -1.18 0.24
CopNum
-0.16256115
0.8499641 0.81125098 -0.20 0.84
F.First.BinTRUE
-0.19967035
0.8190007 0.62425794 -0.32 0.75
J.SocPlay
9.10594641 9008.7032037 10.49150552 0.87 0.39
J.SocPlay:J.TreatmentAllTed Black STD Black MM 0.05347251
1.0549280 24.45152948 0.00 1.00
Random effects
Group
Variable Std Dev
Variance
B.Year.ID Intercept 0.099966324 0.009993266
40. p. 191 “play as a proportion of active time changed no other results but made this
trend significant”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06784579 0.9344045579 0.01517768 -4.47 7.8e-06
CopNum
0.11144874 1.1178964385 0.29469811 0.38 7.1e-01
J.SocPlay.Activity
-6.98608960 0.0009246552 2.83479228 -2.46 1.4e-02
log(BFCMng)
-0.08164669 0.9215975086 0.11652509 -0.70 4.8e-01
log(B.EPIng)
0.37017763 1.4479918043 0.18036962 2.05 4.0e-02
J.SocPlay.Activity:J.TreatmentAllTed Black STD Black 2.12712603 8.3907174736 3.93203924 0.54 5.9e-01
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.0196936174 0.0003878386
B.Year.ID
(Intercept) 0.2479780796 0.0614931280
245
41. p. 192 (Table 5–3) “Testosterone – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
F.Lit.BinTRUE
0.28011737 1.323285e+00 0.2243613 1.25 0.2100
Date.Uni
-0.02644965 9.738971e-01 0.0303592 -0.87 0.3800
CopNum
0.17002614 1.185336e+00 0.1749930 0.97 0.3300
F.SuccessRateCorFull
-1.10455624 3.313579e-01 0.3845806 -2.87 0.0041
J.SocPlay
-44.30096562 5.758841e-20 21.7939264 -2.03 0.0420
log(BFCMng)
0.54449368 1.723735e+00 0.2298358 2.37 0.0180
log(B.Tng)
-0.47818657 6.199065e-01 0.5452881 -0.88 0.3800
J.SocPlay:FY.ObsKirk 2014 Close
2.37295231 1.072902e+01 47.2218002 0.05 0.9600
J.SocPlay:FY.ObsKirk 2014 Far
46.09709889 1.046443e+20 28.0963455 1.64 0.1000
log(B.Tng):FY.ObsKirk 2014 Close -0.46760579 6.265005e-01 0.6686998 -0.70 0.4800
log(B.Tng):FY.ObsKirk 2014 Far
-0.00176051 9.982410e-01 0.6060530 0.00 1.0000
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.5063166 0.2563565
B.Year.ID
(Intercept) 0.3927242 0.1542323
42. p. 192 (Table 5–3) “Testosterone – Millbank”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06651791 9.356462e-01 0.01508595 -4.41 0.00001
CopNum
0.13505715 1.144602e+00 0.29581548 0.46 0.65000
J.SocPlay
-21.32191351 5.495543e-10 6.93613415 -3.07 0.00210
log(BFCMng)
-0.07320573 9.294096e-01 0.13700768 -0.53 0.59000
log(B.Tng)
0.32701041 1.386816e+00 0.23640616 1.38 0.17000
J.SocPlay:J.TreatmentAllTed Black STD Black
6.99455714 1.090681e+03 10.34124481 0.68 0.50000
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.0197557527 0.0003902898
B.Year.ID
(Intercept) 0.2441552144 0.0596117687
43. p. 192 (Table 5–3) “Epiandrostenone – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
F.Lit.BinTRUE
0.29077556 1.337464e+00 0.22404526 1.30 0.1900
Date.Uni
-0.02772736 9.726535e-01 0.03037994 -0.91 0.3600
CopNum
0.17645973 1.192986e+00 0.17527622 1.01 0.3100
F.SuccessRateCorFull
-1.09717470 3.338129e-01 0.38511361 -2.85 0.0044
J.SocPlay
-55.03320493 1.257137e-24 24.51055418 -2.25 0.0250
log(BFCMng)
0.43271722 1.541440e+00 0.19729021 2.19 0.0280
log(B.EPIng)
-0.44895224 6.382966e-01 0.35503930 -1.26 0.2100
J.SocPlay:FY.ObsKirk 2014 Close
44.18323496 1.543601e+19 55.47956077 0.80 0.4300
J.SocPlay:FY.ObsKirk 2014 Far
57.71935825 1.167344e+25 30.89193389 1.87 0.0620
log(B.EPIng):FY.ObsKirk 2014 Close -0.79309482 4.524424e-01 0.73151973 -1.08 0.2800
log(B.EPIng):FY.ObsKirk 2014 Far
0.34839587 1.416793e+00 0.45356584 0.77 0.4400
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.5043868 0.2544060
B.Year.ID
(Intercept) 0.4037517 0.1630155
44. p. 192 (Table 5–3) “Epiandrostenone – Millbank”
Fixed coefficients
Date.Uni
CopNum
J.SocPlay
coef
exp(coef)
-0.06761897 9.346165e-01
0.12406386 1.132088e+00
-20.34811156 1.455216e-09
246
se(coef)
z
p
0.01512478 -4.47 7.8e-06
0.29240326 0.42 6.7e-01
6.78838191 -3.00 2.7e-03
log(BFCMng)
log(B.EPIng)
J.SocPlay:J.TreatmentAllTed Black STD Black
-0.04877013 9.524000e-01 0.11446781 -0.43 6.7e-01
0.32931197 1.390011e+00 0.17771674 1.85 6.4e-02
5.61471348 2.744347e+02 10.08897850 0.56 5.8e-01
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.0197528665 0.0003901757
B.Year.ID
(Intercept) 0.2168260562 0.0470135387
45. p. 192 (Table 5–3) “AGD – RBR”
Fixed coefficients
coef
exp(coef)
se(coef)
F.Lit.BinTRUE
-0.003403599 9.966022e-01 0.269699820
Date.Uni
-0.068642316 9.336606e-01 0.037021198
CopNum
-0.399122381 6.709086e-01 0.237060943
F.SuccessRateCorFull
-0.661876180 5.158825e-01 0.442117562
J.SocPlay
-10.243187933 3.559918e-05 15.374048798
B.Mass.Post
0.001302497 1.001303e+00 0.001183774
B.AGD
0.054194438 1.055690e+00 0.033829097
J.SocPlay:FY.ObsKirk 2014 Far 53.334001935 1.454329e+23 24.490569076
z
-0.01
-1.85
-1.68
-1.50
-0.67
1.10
1.60
2.18
p
0.990
0.064
0.092
0.130
0.510
0.270
0.110
0.029
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.2935040020 0.0861445992
B.Year.ID
(Intercept) 0.0199211479 0.0003968521
46. p. 192 (Table 5–3) “Stereotypic behaviour – Millbank”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date.Uni
-0.06538999 9.367021e-01 0.01503595 -4.35 1.4e-05
CopNum
0.07667409 1.079690e+00 0.29350798 0.26 7.9e-01
J.SocPlay
-21.57371411 4.272234e-10 7.65858438 -2.82 4.8e-03
SB
4.80095671 1.216267e+02 35.74080241 0.13 8.9e-01
J.SocPlay:J.TreatmentAllTed Black STD Black
7.43522782 1.694644e+03 10.72167641 0.69 4.9e-01
Random effects
Group
Variable
Std Dev
Variance
B.Year.ID/Encounter (Intercept) 0.0197329355 0.0003893887
B.Year.ID
(Intercept) 0.2437431760 0.0594107358
47. p. 193 (Table 5–4) “Latency to bite neck – Juvenile”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
DateNum
1.791700e-07 1.0000002 1.916634e-07 0.93 3.5e-01
F.Failed.This.RoundTRUE -1.275270e+00 0.2793556 1.796273e-01 -7.10 1.3e-12
FemOldTRUE
-1.505156e-01 0.8602643 2.240410e-01 -0.67 5.0e-01
SingleTRUE
-1.618886e-01 0.8505359 1.730993e-01 -0.94 3.5e-01
Random effects
Group
Variable
Std Dev
Variance
SingleBlockPre/Male (Intercept) 0.15232697 0.02320351
SingleBlockPre
(Intercept) 0.39350709 0.15484783
48. p. 193 (Table 5–4) “Latency to bite neck – Adult”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
DateNum
5.891106e-08 1.0000001 1.672746e-07 0.35 7.2e-01
F.Failed.This.RoundTRUE -9.017735e-01 0.4058492 1.393786e-01 -6.47 9.8e-11
FemOldTRUE
-9.141868e-02 0.9126355 1.337864e-01 -0.68 4.9e-01
SingleTRUE
-2.470041e-01 0.7811375 2.198741e-01 -1.12 2.6e-01
247
Random effects
Group
Variable
Std Dev
Variance
SingleBlockPost/Male (Intercept) 0.30991598 0.09604791
SingleBlockPost
(Intercept) 0.50385898 0.25387387
49. p. 193 (Table 5–4) “Latency to copulate – Juvenile”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
DateNum
5.691719e-07 1.0000006 2.233244e-07 2.55 1.1e-02
F.Failed.This.RoundTRUE -1.284612e+00 0.2767581 1.967957e-01 -6.53 6.7e-11
FemOldTRUE
-2.522691e-01 0.7770356 2.369798e-01 -1.06 2.9e-01
SingleTRUE
-1.523186e-01 0.8587146 2.505038e-01 -0.61 5.4e-01
Random effects
Group Variable Std Dev
Variance
Male Intercept 0.3782191 0.1430497
50. p. 193 (Table 5–4) “Latency to copulate – Adult”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
DateNum
4.249288e-07 1.0000004 1.819553e-07 2.34 2.0e-02
F.Failed.This.RoundTRUE -1.004286e+00 0.3663062 1.571548e-01 -6.39 1.7e-10
FemOldTRUE
-3.558810e-01 0.7005560 1.537869e-01 -2.31 2.1e-02
SingleTRUE
8.364740e-02 1.0872455 3.807430e-01 0.22 8.3e-01
Random effects
Group Variable Std Dev
Variance
Male Intercept 0.6871203 0.4721343
51. p. 193 (Table 5–4) “Latency to copulate after biting neck – Juvenile”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
DateNum
4.158048e-07 1.0000004 2.345984e-07 1.77 0.076
F.Failed.This.RoundTRUE -3.719766e-01 0.6893703 2.118675e-01 -1.76 0.079
SingleTRUE
9.183972e-02 1.0961891 2.683816e-01 0.34 0.730
Random effects
Group Variable Std Dev
Variance
Male Intercept 0.3879472 0.1505030
52. p. 193 (Table 5–4) “Latency to copulate after biting neck – Adult”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
FemOldTRUE
-1.786868e-01 0.8363678 1.568154e-01 -1.14 0.2500
DateNum
5.602781e-07 1.0000006 1.830660e-07 3.06 0.0022
F.Failed.This.RoundTRUE -5.116948e-01 0.5994787 1.673786e-01 -3.06 0.0022
SingleTRUE
3.102928e-01 1.3638244 3.094449e-01 1.00 0.3200
Random effects
Group
Variable
Std Dev
Variance
SingleBlockPost/Male (Intercept) 0.5837626864 0.3407788740
SingleBlockPost
(Intercept) 0.0198652342 0.0003946275
248
53. p. 193 (Table 5–4) “Copulation duration – Juvenile”
Fixed coefficients
FemOldTRUE
CopNum
DateNum
F.Failed.This.RoundTRUE
SingleTRUE
coef
-4.937935e-01
-9.752773e-02
-1.113518e-06
-5.729551e-01
4.217445e-02
exp(coef)
0.6103068
0.9070772
0.9999989
0.5638567
1.0430764
se(coef)
4.353147e-01
3.808715e-01
3.742884e-07
3.762002e-01
5.194883e-01
z
-1.13
-0.26
-2.98
-1.52
0.08
p
0.2600
0.8000
0.0029
0.1300
0.9400
Random effects
Group
Variable
Std Dev
Variance
Male/Entry (Intercept) 0.0199970140 0.0003998806
Male
(Intercept) 0.8845421153 0.7824147537
54. p. 193 (Table 5–4) “Copulation duration – Adult”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
CopNum
6.776441e-01 1.9692329 2.771050e-01 2.45 0.014
DateNum
-4.109157e-07 0.9999996 3.523005e-07 -1.17 0.240
SingleTRUE -2.690328e-02 0.9734554 4.018576e-01 -0.07 0.950
Random effects
Group
Variable
Std Dev
Variance
Male/Entry (Intercept) 0.3987227 0.1589798
Male
(Intercept) 0.5137039 0.2638917
55. p. 194 (Table 5–5) “Latency to bite neck – Juvenile play”
Fixed coefficients
coef exp(coef)
se(coef)
z
paste(Colour, ObsTyp)Pastel Close -1.206551e+00 0.2992276 9.285919e-01 -1.30
paste(Colour, ObsTyp)Pastel Far
-3.274133e-01 0.7207857 5.113219e-01 -0.64
Date
9.422534e-05 1.0000942 1.253676e-04 0.75
StartPlay
-4.774416e-01 0.6203685 1.297445e+01 -0.04
p
0.19
0.52
0.45
0.97
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.019997074 0.000399883
56. p. 194 (Table 5–5) “Latency to bite neck – Single-housing”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
Date
-0.0001345732 0.9998654 0.0001060599 -1.27 0.20
SingleTypeSingleCTL -0.0528293509 0.9485419 0.3917791996 -0.13 0.89
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.25467005 0.06485683
57. p. 194 (Table 5–5) “Latency to copulate – Juvenile play”
Fixed coefficients
coef
exp(coef)
se(coef)
z
paste(Colour, ObsTyp)Pastel Close -4.199558e+00 1.500220e-02 1.353029e+00 -3.10
paste(Colour, ObsTyp)Pastel Far
1.425260e+00 4.158938e+00 6.532536e-01 2.18
Date
2.301183e-04 1.000230e+00 1.491049e-04 1.54
StartPlay
-4.293678e+01 2.253176e-19 1.638428e+01 -2.62
Random effects
249
p
0.0019
0.0290
0.1200
0.0088
Group Variable Std Dev
Variance
Female Intercept 0.0199849408 0.0003993979
58. p. 194 (Table 5–5) “Latency to copulate – Single-housing”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
Date
0.0003077576 1.0003078 0.0001084508 2.84 0.0045
SingleTypeSingleCTL -0.3599355162 0.6977213 0.4102619050 -0.88 0.3800
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.2887243 0.0833617
59. p. 194 (Table 5–5) “Latency to copulate after biting neck – Juvenile play”
Fixed coefficients
coef
exp(coef)
se(coef)
z
paste(Colour, ObsTyp)Pastel Close -3.940595e+00 1.943665e-02 1.370708e+00 -2.87
paste(Colour, ObsTyp)Pastel Far
2.513745e+00 1.235109e+01 8.673721e-01 2.90
Date
1.087426e-04 1.000109e+00 1.478744e-04 0.74
StartPlay
-5.582938e+01 5.670325e-25 1.850876e+01 -3.02
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.019993424 0.000399737
60. p. 194 (Table 5–5) “Latency to copulate after biting neck – Single-housing”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
Date
0.0003915872 1.0003917 0.0001198535 3.27 0.0011
SingleTypeSingleCTL -0.8467694644 0.4287979 0.5383177657 -1.57 0.1200
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.3361198 0.1129765
61. p. 194 (Table 5–5) “Copulation duration – Juvenile play”
Fixed coefficients
coef
exp(coef)
se(coef)
z
p
Date
-0.0006045899 9.993956e-01 0.000662222 -0.91 0.36
StartPlay 32.9849245411 2.114320e+14 34.382056009 0.96 0.34
Random effects
Group Variable Std Dev
Variance
Female Intercept 0.0199990591 0.0003999624
62. p. 194 (Table 5–5) “Copulation duration – Single-housing”
Fixed coefficients
coef exp(coef)
se(coef)
z
p
Date
-0.0004969497 0.9995032 0.0002427733 -2.05 0.041
SingleTypeSingleCTL 0.4113265459 1.5088180 0.6486916419 0.63 0.530
Random effects
Group
Variable
Std Dev
Variance
Female/Entry (Intercept) 0.0199939977 0.0003997599
Female
(Intercept) 0.0199963135 0.0003998526
250
p
0.0040
0.0038
0.4600
0.0026