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This is to certify that the
dissertation entitled

SOCIAL RECOGNITION IN NEONATAL PIGS AND
THE EFFECTS OF ACUTE STRESSORS ON IT

presented by

ADRIANA SILVEIRA DE SOUZA

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in DEPARTMENT OF ANIMAL
SCIENCE

 

 

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SOCIAL RECOGNITION IN NEONATAL PIGS AND THE
EFFECTS OF ACUTE STRESSORS ON IT

By

Adriana Silveira de Souza

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Animal Science

2006

ABSTRACT

SOCIAL RECOGNITION IN NEONATAL PIGS AND
THE EFFECTS OF ACUTE STRESSORS ON IT

By
Adriana Silveira de Souza

Social recognition (SR), used here to refer to the ability to discriminate a
previously encountered individual from a novel individual, is of fundamental
importance for a wide range of social behaviors and, in many species, is
sensitive to interference by acute stressors. In pigs, failure in SR may increase
aggression, yet the effects of acute stressors on SR of neonatal pigs have not
been investigated. This PhD thesis therefore presents protocols to assess short-
and long-term SR in neonatal pigs and the effect of acute stressors on it.

The ‘habituation-dishabituation’ paradigm was used in 11-day old female
piglets to assess short-term SR and the effect of acute increases in cortisol on it.
Although an intra-muscular hydrocortisone injection administered 15 minutes
prior to testing significantly increased salivary cortisol levels, it did not interfere
with short-term SR. This protocol involved considerable handling and fighting
among subject and stimuli piglets, which could interfere with the outcome of the
subsequent studies.

Therefore, a novel method to test SR abilities in pigs in which handling is
minimized and fighting during the familiarization phase is prevented was
developed and tested. This protocol proved to be suited for assessing SR in

neonatal pigs, since SR could also be assessed in both 11-day-old and 21-day-

old animals, and the latter showed SR for up to 24 hours after the last
familiarization prodecure.

The last study was based on the results of the previous three and
investigated whether weaning age (D11 or D21) and an acute stressor (15
minutes of social isolation) interfered with the ability of female piglets to
recognize familiar conspecifics. SR deficit was only observed in isolated D11
pigs, while non-isolated D11 and isolated and non-isolated D21 pigs did not show
this impairment. This was taken as an indication that acute social stress may
impair SR ability of D11 pigs, if so it may explain at least in part the increase in
aggression during the first day of post-mixing in D11 animals that was previously

reported by our lab.

I dedicate this dissertation to my god-father and god-mother Joao e Consténcia
they gave me great support throught my studies
although they left from this life before this work was concluded

they were always in my thoughts, heart and prayers

Eu dedico esta dissertacé’o aos meus padrinhos Joéo e Consténcia
eles me deram grande apoio durante os meus estudos
apesar deles terem partido desta Vida antes que esta obra fosse concluida

eles sempre estiveram nos meus pensamentos, coraca‘o e preces

ACKNOWLEDGEMENTS

“An education isn’t how much you have committed to memory, or even how
much you know. It’s being able to differenciate between what you know and what
you don’t.”

-Malcolm Forbes

A dissertation is more than only one among a number of requirements to
conquer a PhD title. I see it as a product of systematic evaluations of an
individual in his/her desire to find answers that can contribute to a better
understanding of the world (fundamental research) and/or to solve existing
problems (applied research).

My dissertation marks the end of a long and eventful journey and without
mentioning those who have contributed to it would leave it incomplete. I admit
that this list is just an attempt to acknowledge all those who have directly or
indirectly contributed to this work, and that in writing this I have probably left out
people who in one way or another did contribute to it. Please forgive me for that.

I would like to thank all those that were or still are part of the Animal
Behavior and Welfare Group. I was fortunate to be part of this research group for
a year as a Visiting Scholar, a joyful period of terrific teamwork that lead us to the
USDA/NRI grant awarded to Dr. Adroaldo J. Zanella. I thank him for giving me
the opportunity to continue my research as a PhD student. He was available
whenever I ran into trouble or had questions about my work. Very significant for

the completion of this dissertation were my committee members Dr. Robert J.

Tempelman, Dr. Michael Mendl, Dr. Catherine W. Ernst, and Dr. Fred C. Dyer,

pivotal forces in guiding me throughout my program. At Michigan State University
(MSU), I would like to thank the Animal Science Department, in particular the
chairperson Dr. Karen Plaut, Dr. Steve Bursian, and Jackie Christie. I also thank
the RCPD for the assistance I received during my comprehensive exams.

My doctoral project was funded by a USDA-NRI grant and the Michigan
Agricultural Experiment Station. However, I also acknowledge two other
fellowships I received from the Graduate School of MSU, the travel funding to
present part of my research at the 37th lSAE congress (Italy) and the Dissertation
Completion Fellowship which financially supported me during the process of
writing this dissertation. I also was fortunate to be awarded with another
fellowship for a collaborative research (during an internship) at the ADP group of
the Department of Animal Science at Wageningen University (Netherlands).

My life has totally changed since I started my doctoral studies and l was
privileged to also find support with family and old and new friends I made in many
places, speaking many languages. My deepest thanks go to my friends and
family, who do not speak better English than I did at the beginning of my PhD.
Therefore:

Eu agradeco aos meus pais (Joaquim e Alcélia) pelo amor que meu
forcas, pela simplicidade e sabedoria que me educaram para vida, pelo sacrificio
incondicional que me possibilitou sair em busca de novas experiéncias. As
minhas innas (Regina, Kétia, Roberta e Jane) pelo carinho e amizade que a
disténcia fisica tem tornado ainda mais forte. Eu agradeco também ao

companheirismo constante dos meus ‘velhos' amigos que estao no Brazil:

vi

especialmente Adriana Geaquinto, Rosileide, Adriana Franco, e aos membros do
grupo espirita da Casa da Esperanca.

I also want to sincerely and most gratefully thank my closest friends in the
USA: Charles, Rosana, Eraldo and Cleusa, Magali and Fernando, Betty and
Bruce, John and Angélica, Sandrinha, lsménia, and Miriana; in Canada: Flévia
and Dalmo; in Italy: Denise and lano; in Argentina: Vanesa. Even though my PhD
was entirely done at MSU, I also had critical input and support from the ADP
group at Wageningen University (WUR), in the Netherlands. In particular, I thank
Dr. Bas Kemp and Dr. Liesbeth Bolhuis, but the effort of too many people to
mention for practicing my presentation and discussing my results is also
acknowledged and deeply appreciated.

Among those who believed in my potential is Dr. Jurij Sobestiansky, my
supervisor during my Master’s at Vet school at the Federal University of Goias
who also became a very good friend. He has considerably influenced my
professional career and offered me assistance during my stay in Goiénia (Brazil).

I am grateful to Peter, Anke, and Jasper Jansen, who have provided a
loving family environment that afforded me the necessary tranquility and peace
since I have ‘formally’ entered their lives.

Finally, I thank my fiancé Jarno Jansen, for carefully reviewing this
manuscript, pointing out small and not-so-small mistakes, for his constant
encouragement and endless support as my true soul mate and best friend (even
though during the period of writing this dissertation it seemed as if my computer

was my closest friend).

vii

TABLE OF CONTENTS

List of tables ....................................................................................................... xi
List of figures ..................................................................................................... xii
Chapter 1 General introduction ......................................................................... 1
1. Pigs living in (semi-)natural conditions .......................................... 3
2. Domestication .............................................................................. 6
3. Pigs living in intensive husbandry systems .................................. 9
4. HPA-axis .................................................................................... 13
5. Stress in early stages of life ....................................................... 15
6. Social recognition ...................................................................... 1 7
7. Social recognition terminology and assessment ........................ 18
8. Aim and outline of the dissertation ............................................. 22
Chapter 2 The first examination of the role of cortisol in social recognition in
neonatal pigs ................................................................................. 24
Abstract .......................................................................................... 25
1. Introduction ................................................................................ 26
2. Material and methods ................................................................ 29
2.1 Animals and housing ........................................................... 29
2.2 Acclimatization: apparatus and experimental protocol ......... 29
2.3 Cortisol injection .................................................................. 30
2.4 Social recognition testing ..................................................... 31
2.5 Behavioral observations ...................................................... 32
2.6 Saliva sampling and cortisol analysis .................................. 32
2.7 Statistical analysis ............................................................... 33
3. Results ........................................................................................ 34
3.1 Salivary cortisol ................................................................... 34
3.2 Investigative behavior .......................................................... 35
4. Discussion ................................................................................... 37
4.1 The role of cortisol in short-term SR ......................................... 37
4.2 Habituation-dishabituation protocol ........................................... 39
5. Conclusions ................................................................................ 41

Chapter 3

A novel method for testing social recognition in young pigs

and the modulating effects of relocation ......................................... 43
Abstract ........................................................................................... 44
1. Introduction ............................................................................... 45
2. Material and methods ............................................................... 48

viii

Chapter 4

Chapter 5

2.1 Animals and housing ............................................................ 48

2.2 Habituation and familiarisation of piglets ............................. 49
2.3 Relocation ............................................................................ 51
2.4 Social recognition test ......................................................... 52
2.5 Behavioural observations .................................................... 53
2.6 Statistical analysis ............................................................... 54
3. Results ...................................................................................... 55
4. Discussion ................................................................................ 56
5. Conclusions .............................................................................. 60
Long-lasting social recognition in 3-week female pigs .................... 61
Abstract ........................................................................................... 62
1. Introduction ............................................................................... 63
2. Material and methods ................................................................ 65
2.1 Animals and apparatus ........................................................ 65
2.2 Habituation and familiarization protocol ................................ 66
2.3 Relocation ............................................................................ 68
2.4 Testing .................................................................................. 68
2.5 Behavioral observations during SR test ................................ 69
2.6 Statistical analysis ................................................................ 69
3. Results ....................................................................................... 70
4. Discussion ................................................................................ 71
5. Conclusions ............................................................................... 72
Social isolation elicits deficits in the ability of newly-weaned

pig to recognize familiar conspecifics .............................................. 74
Abstract ............................................................................................ 75
1. Introduction ................................................................................ 76
2. Material and methods ................................................................ 78
2.1 Weaning and isolation treatments ........................................ 79
2.2 Animals, housing, and weaning ............................................ 79
2.3 Familiarisation ...................................................................... 80
2.4 Social recognition test .......................................................... 81
2.5 Behavioural observations ..................................................... 82
2.6 Statistical analysis ................................................................ 83
3. Results ....................................................................................... 83
3.1 General behavioural activity ................................................. 83
3.2 Social investigation ............................................................... 84
4. Discussion ................................................................................. 87
5. Conclusions ............................................................................... 91

Chapter 6 General discussion .......................................................................... 93

1. Summary of the results ................................................................ 94
2. Stress susceptibility in early-weaned pigs .................................... 98
3. Concluding remarks ................................................................... 103
References ...................................................................................................... 104
CHAPTER 1 ................................................................................... 105
CHAPTER 2 ................................................................................... 116
CHAPTER 3 ................................................................................... 121
CHAPTER 4 ................................................................................... 126
CHAPTER 5 ................................................................................... 128
CHAPTER 5 ................................................................................... 132

Chapter 1

Chapter 3

Chapter 5

Chapter 6

LIST OF TABLES

General introduction .......................................................................... 1

Table 1- Distribution of weaning age by size of site in pig production
in the USA ........................................................................................ 12

A novel method for testing social recognition in young pigs
and the modulating effects of relocation .......................................... 43

Table 1- Familiarisation between litters and pairs of piglets ............ 51

Social isolation elicits deficits in the ability of newly-weaned
pig to recognize familiar conspecifics .............................................. 74

Table 1- Familiarisation between litters and pairs of piglets ........... 81

Table 2- Percentages of general behavioural activity (means :I:
S.E.M.) during familiar and unfamiliar exposures in the SR test ..... 84

General discussion .......................................................................... 93

xi

Chapter 2

Chapter 3

Chapter 4

LIST OF FIGURES

The first examination of the role of cortisol in social recognition in
neonatal pigs .................................................................................. 24

Figure 1- Design of the farrowing environment (white, Pen 1 and Pen
2) and the arenas (grey). During acclimatization of the piglets to the
arena, a solid barrier dividing the space in two halves prevented
mixing of litters and the door-ways were open so the animals could
freely access the arena. During the habituation-dishabituation test the
doors to access the arena were closed .......................................... 31

Figure 2- Salivary cortisol concentration (a) and amounts of social
investigative behavior (b) by treatment during the SR test in 11—day-
old female pigs. The grey line and bars indicate cortisol treatment
and the black line and bars indicate vehicle (saline). Hydrocortisone
and saline were injected 15 minutes before SR test started. Data are
presented as :t S.E.M. For significant differences see results ........ 36

A novel method for testing social recognition in young pigs and the
modulating effects of relocation ...................................................... 43

Figure 1-Side view (left) and top view (right, showing the netting
used for familiarisation) of the arenas ............................................. 49

Figure 2- Allocation of the farrowing pens and treatments before
(Pre-Test) and after (Test) relocation. Dark pens indicate NR-
treatment, grey pens indicate RC-treatment, and white pens indicate
RM-treatment. Dashed line indicates netting in arenas between pens.
Numbers indicate litters .................................................................. 52

Figure 3- Effects of familiarity and relocation of 12-day-old piglets on
time spent on social investigation and exploration of the arena during
the SR test. Familiarity is indicated by dark bars (familiar) and grey
bars (unfamiliar). NR, RM, and RC indicate relocation treatments.
Values represent adjusted means :I:S.E.M. Significant differences (P
< 0.01) within familiarity are specified with different letters ............. 56

Long lasting social recognition in neonatal female pigs .................. 61
Figure 1- Farrowing crates and testing arenas. Adjacent farrowing
crates shared the same arenas. During ‘familiarization’, both front

and rear arenas were used. During ‘testing’ only the rear arena, out
of view of the sow, was used .......................................................... 67

xii

Chapter 5

Figure 2- Effects delay period length and spatial learning
performance between familiarization and testing on duration of social
investigation. Dark bars indicate short-term (4h delay) and light bars
indicate long-term (24h delay) SR memory. Values represent
adjusted means i S.E.M. ................................................................ 71

Social isolation elicits deficits in the ability of newly-weaned pig to
recognize familiar conspecifics ....................................................... 74

Figure 1- Effects of social isolation and weaning age on the
percentage of time spent on social investigation during familiar and
unfamiliar exposures in the SR test. Dark bars indicate pre-test social
isolation, light bars indicate non-isolation. Values represent adjusted
means :I: S.E.M. Different letters indicate difference between
treatments (P < 0.001) .................................................................... 86

xiii

CHAPTER 1

GENERAL INTRODUCTION

The significance of the domestic pig for our society has considerably
increased since its domestication. There now is a high demand for pigs not only
for food supply, but also as an animal model for various types of research in
biomedicine and agriculture. Society has responded to this increased demand by
further optimizing pig production systems which considerably differ from the
natural environment in which they live, and the conditions under which they were
domesticated. In order to increase efficiency, farms have adopted drastic
changes in both the social and physical environment of pigs. There is growing
evidence that certain management practices have led to behavioral and
physiological alterations of the animals that indicate problems in coping with the
living conditions. The higher levels of aggression after mixing in pigs that were
weaned at a very young age that was reported by our lab (Yuan et al., 2004)
constituted the main motivation for starting this PhD research. The main purpose
of this introductory chapter therefore is to briefly summarize natural pig behavior
and the conditions under which pigs have been domesticated, focusing on social
structure, and relate these to the environments that modern production systems
have imposed on pigs. It follows by a description of data generated in our lab that
led to this PhD project stating how the present thesis primarily aims at looking at
some basic aspects of the raised hypotheses. Then, it presents a short
explanation of the Hypothalamic-Pituitary-Adrenocortical (HPA) axis and its
regulation as well as a brief on the neonatal stress sensitivity period. Information
on social recognition (SR) process is then introduced. At last, a short summary of

the aim and outline of this thesis is presented.

1. Pigs living in (semi-)natural conditions

Wild and domestic pigs are highly social, gregarious animals that use
olfactory, auditory, and visual signals to communicate with conspecifics (von
Klingholz and Meynhardt, 1979; Graves, 1984). Under free-ranging conditions,
pigs tend to form subgroups and maintain closer social bonds to certain
individuals than others, resulting in animals sleeping in separate nests (Stolba
and Wood-Gush, 1984). Non-member sows are usually not allowed to integrate
into a stable group (Stolba and Wood-Gush, 1989).

The basic social unit is commonly a maternal group of 2-6 sows, their
most recent litters, and pre-pubertal gilts (Graves, 1984). The maternal group is
characterized by long-lasting associations among members (Newberry and
Wood-Gush, 1986; Petersen et al., 1989; Stolba and Wood-Gush, 1989). Males
leave the social group when they reach 7-8 months of age (Fradrich, 1974).
When boars mature, they generally live in seclusion (Graves, 1984; Mauget,
1981), but temporarily reunite with maternal groups when the sows are in oestrus
(Graves, 1984).

In the wild, pigs have opportunity to freely interact with conspecifics and
naturally develop social behavioral patterns leading to the formation of
dominance hierarchy and group stability. Social behavior is extremely developed
in young pigs. Within hours, newborn piglets start forming social dominance
relationships among littermates that will lead to the formation of a stable social
hierarchy. Juveniles also maintain a clear hierarchy that is stable over time. In

stable social groups, sows are dominant to all other members and a linear

hierarchy is observed between sows (Mauget, 1981). In general, dominance is
determined by age and strength, and the social hierarchy is maintained through
the avoidance of fighting by subordinate animals rather than enforcement by the
dominants (Jensen and Wood-Gush, 1984), thus preventing unnecessary
distress. Only during the mating season the social hierarchy changes, when a
boar joins the group and assumes dominance (Schnebel and Griswold, 1983). At
this time, the group consists only of the breeding sows and boar. Non-breeding
females and pre-pubertal animals stay apart from the main group (Mauget, 1981;
Blasetti et al., 1988).

In maternal groups, newborn offspring integrate into the maternal group
over a period of around 7 weeks (Petersen et al., 1989). Pregnant sows separate
from their group approximately 10 days prior to parturition (Graves, 1984). This
separation seems to have evolved to give the mother and young enough time to
learn specific cues for mutual recognition, contributing to the formation of a social
bond between sow and litter and among littermates (Mauget, 1981). When
piglets reach 10-14 days of age they start following the sow and by doing so
gradually integrate into the maternal group (Jensen and Redbo, 1987; Stangel
and Jensen, 1991). From about one week post-partum up to weaning, non-
Iittermates slowly mingle. This process is usually non-aggressive, and social
interactions are often playful (Petersen et al., 1989). As the pigs grow older, non-
litterrnates are more often seen together on long excursions, without the
presence of the mother (Petersen et al., 1989). However, the social bond

between Iitterrnates remains strong (Stolba and Wood-Gush, 1981; Jensen,

1986; Newberry et al., 1988; Petersen et al., 1989). At 8 weeks of age, social
interactions between non-littermates are seen more frequently than between
littermates (Petersen et al., 1989). The amount of social interactions then
stabilizes and the animals are totally integrated into the maternal group (Petersen
etaL,1989)

Under natural conditions weaning is a gradual process. The length of the
lactation period is dependent on the nutritional state of the sow and the amount
of food available in the environment (Jensen and Recén, 1989). Usually, milk let-
down gradually decreases when piglets are 6 weeks-old (Newberry and Wood-
Gush, 1985, 1986) and Petersen (1994) reported a marked increase in solid food
intake around 5 and 8 weeks of age, indicating a transition from a complete
dependence on milk to nutritional independence from the sow. The nutritional
independence also contributes to the independence of piglets from the sow and a
broader integration within the maternal group (Jensen, 1986). Weaning hardly
ever includes aggressive behavior (Jensen, 1986). Instead, the sow makes
suckling more costly for the piglets by terminating nursing (Jensen and Recén,
1989). If weaning is defined as the time when nursing of the litter is entirely
ceased, then natural weaning is not complete until 12-19 weeks post-partum
(Stolba and Wood-Gush, 1989; Newberry and Wood-Gush, 1985; Jensen and

Recén, 1989; Petersen, 1994).

2. Domestication

Pigs were domesticated nearly 9,000 years ago and presumably derive
from the Eurasian wild boar (Sus scrofa) that is common in Europe, Asia, and
North Africa (CIutton-Brock, 1999). Due to profound phenotypic differences
between domestic pigs from those regions, Darwin recognized a possibility of
distinct origins (Darwin, 1868), although this suggestion was only later confirmed
by genetic studies showing clear differences between European and Asian
domestic pigs (Watanabe et al., 1986; Okumura et al., 1996; Larson et al., 2005).
Recently, by means of a more comprehensive molecular comparison using
mitochondrial DNA and nuclear genes of wild and domestic pigs from Europe
and Asia, the time of divergence of the two was estimated at around 500,000
years ago (Giuffra et al., 2000), much earlier than the time of domestication. It is
therefore suggested that instead of importing domesticated pigs around the
world, people from several distinct areas domesticated the existing wild animals
themselves for meat production. Among others, the pig its ability to procreate in
captivity and prolificy contributed to the domestication process and may explain
at least in part why pigs, rather than more local meat sources that may have
been more difficult to domesticate, were chosen for meat production in various
distinct areas.

One can assume that at the time of early domestication, pigs were kept
under fairly natural conditions, slaughtering young pigs when meat was needed,
but further causing little disturbance to the animals. Over the course of

domestication, however, animals with better genetically based behavioral

features for living in captivity were preferred (selected) and through selection,
domestication has had profound effects on the anatomy (e.g. higher meat
production) and physiology (e.g. diminished stress response) of the pig
(Mormede, 1995; Price, 1997; Désautés et al. 2002; O’Regan and Kitchener,
2005). On one hand, selection has enabled farmers to increase production
efficiency and meet the desires of the consumer. The development of genetic
maps in livestock has allowed the detection of genomic regions that influence
growth, body composition, meat quality, or reproduction (Bidanel et al., 2001;
Malek et al., 2001a,b), behavior and stress neuroendocrine responses (Fuji et al.,
1991; McGlone et al., 1998; Mormede et al., 2002). On the other hand, selection
for high production efficiency may also lead to a number of undesirable effects
(Breuer et al., 2005; for a review see Rauw et al., 1998). In pig production, highly
lean pigs have been reported to demonstrate leg problems due to a decrease in
leg strength (Webb et al., 1983; Sather, 1987) and also more excitable
temperaments (during handling: Grandin, 1994; increased anxiety in a open field
test: Shea-Moore, 1998; more aggressive: Busse and Shea-Moore, 1999). Thus
far, little scientific evidence have shown the effects of lean growth lines on
behavioral traits.

In spite of selection, the basic species-specific behavioral repertoire (e.g.
nest-building) of domestic pigs resembles that of their ancestors (Stolba and
Wood-Gush, 1984; Jensen, 1986; Spinka et al., 2000; Edwards, 2003). It seems
that the behavioral differences between wild and domestic pigs are more

quantitative rather than qualitative in character (Stolba and Wood-Gush, 1984;

Graves, 1984; Jensen, 1986), and can be better explained by differences in
response threshold (Price, 1999). The reduced responsiveness (i.e. sensitivity)
towards environmental changes has been pointed at as being the most
prominent difference, resulting from adaptation of living in a biologically ‘safe’
environment (Price, 1999). In an environment that is characterized by limited
opportunities for perceptual and locomotive stimulation, constant space intrusion
by humans, and limited opportunity to avoid confrontation with dominants, pigs
have adapted to be less reactive towards environmental changes (e.g. novel
objects and humans) than wild animals.

In the past decades, however, living conditions for domestic pigs have
changed drastically. Producers strive for higher production, economic benefits,
and labor efficiency, and this has led to the development of intensive husbandry
systems. These systems largely differ from the natural conditions in which wild
pigs evolved and have been domesticated. While wild pigs live in the forest, living
in small and stable social groups and spend most of their time foraging and
eating, in commercial husbandry systems, domestic pigs are now housed in
barren pens, with limited space allowance, and in highly unstable social groups,
since they are frequently mixed with unfamiliar pigs. The barren environment
offers little opportunity for exploration, and pigs spent most of the time lying
inactive.

In sum, the conditions in which pigs live in ‘nature’ are characterized by a
vast physical environment with abundant substrate for rooting and exploration.

The animals live in fairly stable and heterogeneous social groups. During the first

days of life the young stay in close contact with the closest family members and
are slowly introduced to the maternal group. This gradual social experience
seems to contribute to the normal development of social behavior which may
also involve social learning experience, perhaps preparing the individual for
social events. Weaning is also a gradual process in which the individual slowly
adapt to different sources of food and by increasing social interactions with other

members of the group the separation from the mother occurs naturally.

3. Pigs living in intensive husbandry systems

Social grouping in husbandry systems occurs in different social (e.g. group
size, composition, instability) and physical (e.g. restricted space, absence of
substrate) conditions from what animals usually encounter in more natural
environments. Although pigs are very social animals, high levels of aggression in
social groups are often seen in modern farms. lf living conditions are not offering
the opportunity for the animals to develop appropriate social communication and
behavior, this could interfere with group formation and stability.

The group structure in farm conditions, as in natural circumstances, is also
based upon dominance hierarchy (Meese and Ewbank, 1973). However, due to
the practice of regrouping (mixing) to obtain uniform cohorts, unfamiliar pigs that
are brought together often engage in vigorous fight that will end once the
dominance hierarchy is settled (Puppe and Tuchscherer, 1994). Different from
more natural enclosures, from the first weeks of life, confined piglets only have

social contact with littermates and sow. Furthermore, piglets are usually abruptly

mixed after weaning at 3-5 weeks post-partum, whereas under semi-natural
conditions piglets meet non-littermates for the first time after about 2 weeks.
Mixing of unfamiliar pigs disrupts group stability and increases aggression
(Algers et al., 1990; Fraser et al., 1998).

Pigs may benefit from early social experience that reduces aggressive
behavior (Weary et al., 1999). The first weeks after birth are crucial for
developing social skills, as indicated by increases in social interaction, such as
play and play-fight behavior (Petersen et al., 1989; Blackshaw et al., 1997). On
the other hand, if piglets are hampered in social skills at this age, they show
improper agonistic behavior at later ages (Schouten, 1986). The absence of
gradual social experience with other litters seeing in husbandry systems may
explain at least in part the high occurrence of aggressive behavior at regrouping.

One-week old pigs fight less during a second meeting, indicating that they
use the information of experiences acquired from earlier interactions (Jensen,
1994). However, it was also suggested that they may be less motivated to
continue fighting, because at this age the animal has only a very limited
experience in defending resources (teat). The shorter fighting time in young pigs
may therefore be a sign of a lesser interest in potential resources rather than
better assessment capacity.

In order to reduce the incidence of fights and injuries caused by mixing,
researchers have investigated the possibility of socializing litters prior to weaning.
Several methods involving socializing and/or mixing piglets during this period

have demonstranted positive results in minimizing aggression (Pitts et al., 2000;

10

Weary et al., 2002; D’Eath, 2005; Parratt et al., in press). A recent study by
D’Eath (2005) revealed that young pigs that were socialized (mixed with
unfamiliar litter) before weaning start fighting more quickly and form a stable
social hierarchy more rapidly than the controls pigs (kept in littermate groups).
However, contrary to what had been observed in free-ranging systems,
socialization of litters in confinement does not seem to be playful in nature and
can cause skin lesions primarily as a consequence of fights between unfamiliar
pigs (Wattanakul et al., 1997).

In commercial farms, weaning is a multifactorial stressor for the piglets as
it simultaneously involves abrupt separation from the sow, changes in diet, and
transfer to a novel environment (Varley et al., 1985; Dybkjar, 1992; Weary and
Fraser, 1997; Fraser et al., 1998). This is in contrast with (semi-)natural
conditions where weaning is a gradual process occurring at a later age (e.g.
Newberry and Wood-Gush, 1985). Aiming at enhancing productivity and
producing high health status breeding stock, the North American swine industry
has adopted practices in which the animals are weaned within a few days after
birth (e.g. Medicated Early Weaning: at 5-6 days of age; Segregated Early
Weaning: at 5-10 days of age). The latest report of the NAHMS (2001) revealed
that in the USA piglets are weaned at 19.3 days-old on average, with 63.9% of
the pigs being weaned between 16 and 20 days of age and 15% under 16 days-
old. However, these figures vary according to the size of the production sites (for

more detailed information see Table 1).

11

Studies have shown that weaning at early stages of life causes distress in
pigs, with prolonged vocalization, restlessness, and long-term behavioral
changes (Metz and Gonyou, 1990; Dybkjer, 1992; Hohenshell et al., 2000;
Patience et al., 1997; Weary et al., 1999; Worobec et al., 1999; Orgeur et al.,
2001), and enhanced cortisol concentration in plasma (Blecha et al., 1985,

Mason et al., 2003) and in urine (Hay et al., 2001).

Table 1- Distribution of weaning age by size of site in pig production in the USA

 

Size of Sites (Sow and Gilt inventory)

 

 

 

Small Medium Large

(<250) (250 - 499) (2 500)
Weaning Age (Days) % SE % SE % SE
< 16 2.3 1.0 8.8 2.4 25.5 4.6
16 - 20 11.2 1.7 65.3 4.0 67.0 4.4
21 — 27 30.1 2.7 20.7 3.3 6.3 1.3
28 - 34 22.3 2.4 3.3 1.0 0.6 0.3
2 35 34.1 2.9 1.9 0.8 0.6 0.4

 

 

 

 

Adapted from the National Animal Health Monitoring System of the USA (NAHMS,
2001).

A recent study conducted in our lab (Yuan et al., 2004) demonstrated that
when they were mixed at 9 weeks of age, piglets that were weaned between 9
and 12 days of age (EVV) fought longer and initiated more fights that they did not
subsequently win than piglets that were weaned between 21 and 23 days of age
(CW). The authors hypothesized that a possible impairment in social skills may
be at the heart of these findings, but also proposed that the stress of mixing may

have affected cognitive processes in EW pigs, since CW animals did not seem to

12

be affected. As deficits in discrimination of conspecifics may be a triggering point
for initiating aggression (Ewbank and Meese, 1971), studies on discrimination
and social recognition (SR) and the effects of stressors on it could improve our
understanding of at least some of the underlying mechanisms of aggression in
pig production. As mentioned previously, the first weeks after birth are crucial for
developing social skills of a pig seen by an augment in social interactions
(Petersen et al., 1989; Blackshaw et al., 1997), and if piglets are hampered in
social skills at this age, they show inappropriate agonistic behavior at later ages
(Schouten, 1986), sustaining Yuan et al.’s (2004) first hypothesis. Relatively little
is known, however, about the influence of acute stressors on SR abilities of
neonatal pigs, and therefore, the research in this thesis focused on the neonatal
period. In commercial pig production, several standard management practices
that neonatal pigs are subjected to such as castration and weaning, induce
biological changes that are indicative of stress (Hay et al., 2001; Mason et al.,

2003; Puppe et al., 2005).

4. HPA-axis

The hypothalamic-pituitary-adrenocortical (HPA) axis is assumed to play a
critical role in adaptation and homeostasis in times of stress. The end products of
the HPA axis activation are glucocorticoid (GC) hormones (e.g. corticosterone in
rats, cortisol in humans and pigs). The activation of the HPA axis occurs via the
neurosecretory neurons in the medial parvocellular zone of the paraventricular

hypothalamic nucleus (PVN). These neurons synthesize and realease

13

corticotropin-releasing hormone (CRH) and neuropeptides (e.g. arginine
vasopressin, AVP) into the pituitary. The corticotropes of the pituitary then
releases adrenocorticotropic hormone (ACTH) into the bloodstream (Whitnall,
1993). This mechanism is crucial for adaptation (Dallman et al., 1992) of the
individual with its environment, enabling a coping response. The adrenal gland
responds to the increase in ACTH by synthesizing and secreting GCs. By binding
to GO receptors within the brain, GCs also inhibit the further secretion of CRH
from the hypothalamus and ACTH from the pituitary (negative feedback).

During the stress response, GCs perform various functions. Most
importantly, they provide the body with the energy needed after the initial
activation of the sympathetic nervous system in response to an acute stressor.
GCs facilitate the release of glucose and fatty acids into the bloodstream
(providing energy for the muscles) and contribute to trigger a redistribution of
lymphocytes (preparing the immune system for a possible defense reaction)
(Miller et al., 1994; Anderson et al., 1999). Furthermore, they inhibit systems not
necessary for immediate survival (such as the reproductive and digestive
system).

Acute stress responses facilitate the readjustment of behavioral and
metabolic priorities and are usually adaptive, i.e. through a learning process they
allow an individual to respond more adequately in future similar stressful
situations (Moberg, 1985; McEwen, 2001). However, if the animals have
problems in coping with constant and severe stressors (i.e. chronic stress) a

sustained activation of the stress response systems will cause a variety of

14

behavioral and physiological problems, and increase the susceptibility to
diseases (e.g. Wiepkema and Koolhaas, 1993).

G05 affect not only bodily functions, but also brain processes (for a review
see de Kloet, 2000). GC receptors, the mineralocorticoid receptor (MR) and
glucocorticoid receptor (GR) can be found in various brain structures and their
occupation by GCs affect brain functioning, such as Ieaming and memory
(Fenoglio et al., 2005). MRs have high affinity for G05 and are believed to
modulate the circadian rhythm of GCs secretion, specially during trough. In
contrast, the GRs have lower affinity for GCs and are primarily responsible for
reactive negative feedback during circadian peak and following an acute stressor
(de Kloet et al., 2004, for a review). The severity of the effects of GCs on the
brain is dependent on the central concentrations and sensitive period of the

individual.

5. Stress in early stages of life

Stress of both physical and social/psychological origin can have profound
consequences for various neurophysiological, cognitive, and behavioral functions
(e.g. Washburn and Rumbaugh, 1991; de Kloet, 2000; Blanchard et al., 2001).
Stress sensitivity may change over the course of development of the individual,
however, and it has been suggested that stress during early developmental
periods may sensitize animals to the effects of stressors later in life (Graham et
al., 1999; Parker et al., 2004; Yuan et al., 2004; Sandstrom and Hart, 2005).

During early developmental periods, the brain experiences a “growth spurt” -

15

defined as the transient period of development when the brain is growing most
rapidly. This period may occur prenatally and/or postnatally, depending on the
species (guinea pig: Dickerson and Dobbing, 1967; rats: van den Hove et al.,
2006; sheep: Richardson and Hebert, 1978; primates: Leigh, 2004). Like in
humans, this developmental growth spurt extends from the late prenatal to early
postnatal period in pigs (Dickerson and Dobbing, 1967; Dobbing, 1974; Dobbing
and Sands, 1979).

During the first few weeks after birth, porcine brain maturation is
characterized by the continued proliferation of neuronal cells and development of
neural pathways (Dobbing, 1974; Brust et al., 2004) and during this period the
brain is sensitive to alterations (Weaver et al., 2000). Interestingly, during this
period, levels of circulating cortisol are usually low (Daniel et al., 1999; Carroll et
al., 1998). It is therefore possible that during this period, increases in circulating
cortisol may negatively affect porcine brain development. There is growing
evidence that stress during this sensitive period may result in long-term alteration
of emotional reactivity (Weiss et al., 2004), behavioral responses (Schouten,
1986; de Jonge et al., 1996), immune response (Tuchscherer et al., 2004; Kanitz
et al., 2004), in addition to dysregulation of the HPA axis (Weaver et al., 2000),
but studies examining the effects on brain development and functioning in pigs
are limited (Tuchscherer et al., 2004; Poletto et al., 2005; Schwerin et al., 2005).
Thus, this thesis is based on the hypothesis that, aside from possible deficits in

social competence in EW pigs, the increase in post-mixing aggression reported

16

by Yuan et al. (2004) is caused by impairments in SR due to an increased

susceptibility to the effects of acute stressors.

6. Social recognition

The ability of animals to identify the nature of conspecifics increases their
chances of survival. Animals can use this information for their own benefit and
develop context appropriate behavior minimizing social conflicts, optimizing
energy use through cooperation, defending against predators, and protecting
resources. Animals that chose to live in small groups, the formation and
maintenance of social relationships is an indication that they are able to
recognize other individuals.

Sows and litters can benefit from mutual recognition. From a sow’s
perspective, it ensures investment of the resources towards the proper offspring.
Sows seem to be able to recognize their piglets by using whole body odors within
a day post-partum (Horrell and Hodgson, 1992a) or using only vocalizations
(lllmann et al., 2002), and usually react aggressively towards alien pigs
(Wattanakul et al., 1997). As piglets can easily be separated from the sow when
she is foraging (Gundlach, 1968), auditory recognition (e.g. Maletinska and
Spinka, 2001) may help the sow to attend to distressing calls (i.e. presence of
predators, starvation, hypothermia) from her litter. From a litter’s perspective,
recognition enables them to approach the sow when in search of milk and
protection. Piglets are able to distinguish their own mother and also identify

several odor cues and/or auditory stimuli derived from her (Horrell and Hodgson,

17

1992b). In systems where sows and litters are kept in communal areas it is
probably advantageous for the piglets to discriminate their dam and stay close,
since other sows could recognize them as aliens and attack during attempts to

suckle (Newberry and Wood—Gush, 1985).

7. Social recognition terminology and assessment

Social discrimination is the individual’s perception of a difference between
conspecifics. This is a pre-requisite for SR as it allows individuals to establish
mental representations of social categories. SR however, is the individual’s
perception that the presented conspecific belongs to a known class of individuals
(e.g. species, familiarity, kinship, social hierarchy, sex, and age), whereas
individual recognition is the ability to identify others as being a particular and
unique individual (for reviews see Gheusi et al., 1994; Zayan and Vauchair,
1998). In animal behavioral studies, the term SR memory has been widely used
to indicate changes in behavioral responses towards a previously encountered
stimulus (e.g. Thor and Holloway, 1982).

Tests to assess SR ability are based on the animal’s natural tendency to
intensely investigate novel conspecifics. Most of the protocols to study SR are
primarily designed for rodents. Rodents are highly social animals (Barnett, 1958;
Lore and Flannelly, 1977) and usually engage in spontaneous investigatory
behavior as a way of learning the nature of conspecifics or their signatures (e.g.
urine). In the laboratory, it is possible to make use of spontaneous (e.g. in

preference tests, habituation-dishabituation test) or learned behavioral responses

18

(operant conditioning systems). Tests involving spontaneous responses measure
the response of the subject to a social stimulus, whereas tests involving learned
responses involve the association of a stimulus and a proper response using
rewards to motivate the subjects. The subjects can be tested either in its home
environment or not (e.g. in an apparatus). As in the present dissertation SR was
assessed using spontaneous response, learned behavioral methods will not be
further mentioned.

The advantage of using spontaneous behavior is that it does not require
high motivation of the subject nor numerous trials for learning, which could be
ideal for assessing SR in very young animals. The tests based on the
assumption that the subject in study has innate interest or is motivated enough to
develop a specific response in the presence of the stimulus or stimuli. In addition,
it better resembles what occurs in more natural conditions. The response of the
subject towards a social stimulus is usually assessed by measuring latency of
approaches towards the stimulus, duration or frequency of social investigation,
aggressive behavior, and others. Different responses towards different stimuli
indicate that social discrimination has occurred. In preference tests, no Ieaming
is required and social discrimination is demonstrated by comparing exploratory
behavior of the subject two or more stimuli (pigs: Kristensen et al., 2001; lambs:
Ligout and Porter, 2003; hens: Dawkins and Woodington, 1997; rats: Levy et al.,
2003). The main disadvantage of using preference tests is that negative results
cannot be interpreted as inability to discriminate. It is possible that although the

subject perceives the difference between stimuli it does not repond differently, for

19

example, because the subject has different motivation (aggression towards
strangers and curiosity tomards group-members). As an example, Hauser and
Huber-Eicher (2004) demonstrated that, in hens, variations in testing experience
can cause negative results in a preference test.

SR can also be assessed by using the habituation-dishabituation test.
Habituation is a form of learning that is defined as a progressive decrease in
responsiveness resulting from the repeated presentation of a particular stimulus
(Thompson and Spencer, 1966). It enables individuals to behave efficiently
(Hinde, 1970), once an animal becomes acquainted with a stimulus, less
investigation is needed. The research investigating the nature of SR using
habituation was originally described by Thor and Holloway who proposed a social
memory test using mature male laboratory rats as a model (Thor and Holloway,
1982). Juvenile male rats were used as stimuli to eliminate possible confounding
effects of aggression and sexual behavior during the testing exposures. The test
consisted of repeatedly presenting adult rats to the same juvenile resulting in a
decrease of social investigation, which is a reliable index of SR. The habituation
is considered to reflect the presence of a memory for the presented individual.
However, this decrease in social investigation after repeated exposures to the
same stimulus could also be interpreted as sensory adaptation or social fatigue,
which lead to another experiment where the subject was habituated to a stimulus
and subsequently exposed to either the same or to a novel stimulus. The results

demonstrated an increase in social investigation only towards the different

20

stimulus at levels comparable to the first encounter (Thor and Holloway, 1982),
indicating SR rather social fatigue.

A combination of habituation and dishabltuation procedures in one single
experiment was later presented (Dluzen and Kreutzberg, 1993; Winslow and
Camacho, 1995). Further adaptation of the habituation—dishabituation paradigm
is presented by Engelmann et al., (1995) in which during the testing phase the
previously exposed individual and a novel individual are presented
simultaneously. SR is assessed by comparing the differences between the time
spent investigating familiar and unfamiliar conspecifics. This adaptation of the
original SR test can be completed in two trials only and is suitable for testing
sexually mature males as well as females.

The habituation-dishabituation procedure and its variations have been
described as being easy to implement and offer the opportunity to test a large
number of animals in a short-period of time (Gheusi et al., 1994; Ferguson et al.,
2002), ideal for pharmacological studies. Winslow and Camacho found that
ovariectomized females can be used as an alternative for stimuli and can be
repeatedly used over months (anlow and Camacho, 1995). Studies have
reported SR in individually housed rodents to last no longer than 2 hours (Thor
and Holloway, 1982; Bluthe and Dantzer, 1990; Ferguson et al., 2001). Recently,
Kogan and colleagues demonstrated that individually housed mice were able to
only form short-term SR (with delay of 30 minutes), whereas group housed mice
formed a more robust memory that lasts for at least 7 days (Kogan et al., 2000).

The later study demonstrates that in rodents the habituation-dishabituation

21

procedure can be used in short- and long-term SR memory studies, however, the
housing condition should be taken into account in the experimental design, at

least in rodents.

8. Aim and outline of the dissertation

SR is essential for a broad range of social behaviors and is an element of
learning and memory that, in many species, is sensitive of interference by
stressors. It has been suggested that failure to remember a previously
encountered individual can increase aggression in social groups. This PhD
project aims at gaining insight in the modulating effects of acute stressors on SR
of neonatal pigs. For that purpose, the objective of the research was three-fold.
Firstly, to develop a protocol to assess SR that is suitable for using in non-
weaned piglets. Secondly, to examine whether the protocol designed could be
used to assess short- and long-term SR memory. Thirdly, to investigate whether
acute stressors interfere with SR ability of neonatal pigs.

Chapter 2 describes a study in which the ‘habituation-dishabituation’
paradigm is used to test SR in 11 day-old pigs. This protocol has been widely
used for assessing SR in weaned animals; therefore, an adaptation for use in
non-weaned pigs was necessary. This study also investigates whether acute
increases of cortisol impair short-term SR. Constraints associated with the
protocol such as distress of handling and fighting between animals lead to the

subsequent experiment.

22

Chapter 3 presents a different method to assess SR abilities in 12 day-old
pigs primarily aimed at minimizing problems associated with the previous
protocol. This method proved to be suited for use in non-weaned pigs and will the
used in the subsequent studies.

Chapter 4 reports an experiment in which short- and long-term SR
memory of 21-day-old pigs were assessed. For short-term memory the animals
were tested at 4h after the last familiarization procedure whereas for the long-
term memory the animals were tested 24 hours after familiarization.

Chapter 5 describes a study designed to investigate whether early
weaned (11 days of age) pigs are more susceptible than conventionally weaned
(21 days of age) animals to SR disturbance by an acute stressor (15 minutes of
social isolation). This information is fundamental in the context of the body of
evidence produced in our lab showing that these animals seem be more
susceptible to acute stressors.

Chapter 6 reports a summary of the major findings of chapters 2-5 and
further discusses the main findings. It subsequent presents the final conclusions

and proposes future studies.

23

CHAPTER 2

THE FIRST EXAMINATION OF THE ROLE OF CORTISOL

IN SOCIAL RECOGNITION IN NEONATAL PIGS

Adriana S. Souza, Robert J. Tempelman, Magali M. P. Moura,
and Adroaldo J. Zanella

Submitted

24

Abstract

The ability of pigs to acquire and retrieve information regarding previously
encountered conspecifics may be impaired if circulating cortisol levels are acutely
elevated. The main objective of this experiment was to investigate whether acute
increases of cortisol impair short-term social recognition (SR) in 11-day-old
female piglets. The habituation-dishabituation procedure, originally designed to
assess SR in adult rodents, was adapted for use in neonatal pigs. Twenty-four
female piglets were randomly assigned to an intramuscular injection of 1 mglkg
BW of hydrocortisone sodium succinate (SOLU-CORTEF) or vehicle (saline).
Salivary samples were collected 15 minutes before injection (baseline) and at 15,
35, and 55 minutes after the treatment. Fifteen minutes after injection, the female
subjects were exposed to a male piglet (stimulus) for four one-minute periods
separated by 10-minute intervals (habituation phase), after which a different
stimulus male was presented (dishabltuation phase). The results show that
female piglets decreased the amount of time spent investigating a repeatedly
presented male piglet (familiar) and increased investigation when exposed to a
novel male in the subsequent exposure (unfamiliar). Salivary cortisol levels in 11-
day-old piglets significantly increased after the cortisol injection. There was no
evidence that the cortisol treatment affected their ability to recognize a familiar
individual. The habituation-dishabituation method for assessing SR can be

further used in neonatal pigs.

25

1. Introduction

Glucocorticoids (GCs), mainly corticosterone in rodents and cortisol in
other mammals, are released in response to stressful situations. GCs are
Iipophillc and can freely cross the blood-brain barrier to act directly on the brain
(Dallman, 1993; Wolf, 2003), affecting areas involved in learning and memory
(for review, see Lupien and Lepage, 2001).

The modulating effects of circulating GCs on learning and memory depend
on a multitude of factors, including concentration in the body (Pavlides et al.,
1993), length of the exposure (acute or chronic, e.g. Shors, 2001; Fuchs et al.,
2001), moment of the exposure (i.e. during memory acquisition, consolidation, or
retrieval, e.g. de Quervain et al., 2000; Roozendaal, 2002, 2003; Elzinga et al.,
2005), time of the day (i.e. levels of basal GCs according to circadian rhythm,
Lupien et al., 2002ab), sex of the animal (Szuran et al., 2000; Bowman et al.,
2004; Shors et al., 2004), and age of the individual (Frisone et al., 2002; Driscoll
etaL,2005)

Circulating GCs affect cognition by binding with two GC receptor subtypes
that are present throughout the brain, but are specifically abundant in the
hippocampus, an area that plays an important role in Ieaming and memory (for
reviews, see McEwen and Sapolsky, 1995; de Kloet, 2000). The
mineralocorticoid receptor (MR or Type I) has a 10-fold higher affinity for GCs
than the glucocorticoid receptor (GR or Type II) and thus at low GC
concentrations primarily MRs are activated. In contrast, high levels of

endogenous or exogenous GCs saturate MRs and activate the GRs in the

26

hippocampus (Reul and de Kloet, 1985). MR activation in the hippocampus
enhances memory formation (Sandi and Rose, 1994, 1997) while GR activation
hampers memory formation (Wolkowitz et al., 1990). In line with these findings,
Pfaff and collaborators demonstrated that an acute exposure to high
concentrations of GCs (corticosterone injection of 1 mglkg BVV) decreases
hippocampal activity with a delay of approximately 30 minutes (Pfaff et al., 1971),
possibly caused by the activation of GRs (Joéls, 2001).

The hippocampus has been shown to play a role in social recognition (SR)
(Terranova et al., 1994; Maaswinkel et al., 1996; Kogan et al., 2000). In the
present study, SR is used to refer to the ability to discriminate a previously
encountered individual from a novel individual conspecific. Most of the
procedures to assess SR rely on spontaneous behavioral responses; the natural
tendency of animals (e.g. rodents, pigs) to intensely investigate novel
conspecifics. Therefore, decreases in the duration of social investigation after
successive exposures to the same individual can be used as an indication of SR
ability (Thor and Holloway, 1982).

The most common technique to test the ability of animals to recognize
(remember) pre-exposed (familiar) individuals is the habituation-dishabituation, in
which an animal is presented with the same conspecific over multiple exposures
and then presented with a novel stimulus conspecific (Dluzen and Kreutzberg,
1993; Winslow and Camacho, 1995). The decline in time spent on social
investigation between exposures from the first stimulus is inferred to reflect

habituation, indicating SR of the presented individual. After a novel and

27

unfamiliar stimulus animal is presented, dishabituation, indicated by an increase
in social investigation, should occur, serving to rule out the possibility of
habituation being a product of a generalized social fatigue rather than SR. The
habituation-dishabituation procedure has been described as being easy to
implement and offers the opportunity to test a wide range of animals in a short-
period of time (for a review, see Gheusi et al., 1994).

It has been demonstrated that pigs are capable of recognizing
conspecifics (Kristensen et al., 2001; McLeman et al., 2005; Souza et al., 2006).
Despite the vast literature investigating the impact of hormones released in
response to an acute stressor on learning and memory processes, little is known
about the effects of short-term increases of exogenous cortisol on SR of animals.
We have shown that SR in early-weaned pigs can be disrupted by an acute
social stressor (Souza and Zanella, submitted), but the role of GCs in this
disruption is still unclear. In the present study our goal therefore was to assess
the role of an acute increase in circulating cortisol levels on short-term SR of 11-
day—old non-weaned female piglets.

For that reason, we adapted the habituation-dishabituation SR paradigm
for use in non-weaned piglets. Based on preliminary studies (Zanella,
unpublished), we anticipated that a single injection of 1 mg/kg BW of
hydrocortisone (cortisol) sodium succinate prior to testing would significantly
increase salivary cortisol concentrations throughout the test time-period and that

SR would be impaired at these concentrations.

28

2. Material and methods

The study was carried out at the Swine Teaching and Research Center
(STRC) of Michigan State University (MSU), USA. All procedures used in this
study were reviewed and approved by the All-University Committee for Animal

Use and Care of MSU.

2.1 Animals and housing

Six unweaned litters (Yorkshire x Duroc) were used in this experiment.
Within 36 hours post farrowing, litters were standardized to 10 piglets by cross-
fostering (balanced for sex) and all animals were weighed following the standard
operating procedures of the STRC. Sows and litters were housed in standard
commercial farrowing crates (1.8 x 2.3 m) throughout the experiment.

The testing arena (91.4 x 71.1 x 60.9 cm) consisted of a metal bar crate
with two doors. The doors allowed two neighboring litters to access the arena at
the same time. A solid plastic barrier dividing the arena in two halves prevented
mixing of animals from neighboring litters (Figure 1).

Water was available ad libitum and sows were fed standard commercial
feed twice a day. Environmental temperature was controlled (200C 1 1) and

artificial lights were provided from 08:00 to 17:00 throughout the experiment.

2.2 Acclimatization: apparatus and experimental protocol
All litters were acclimatized to the testing arena and experimental protocol.

From day 4 until day 11 after birth, the doors to access the arena were

29

continuously opened allowing all litters to freely explore the testing arena. A solid
plastic barrier divided the arena in half, allowing two litters to be acclimatized at
the same time.

The piglets were tested at 11 days of age. In the four days prior to testing
(7-10 days of age), all piglets were acclimatized to the testing procedure by
holding each piglet for 10 seconds and subsequently placing them individually in
the arena for four 1-minute periods with 10-minute intervals. While the piglets
showed signs of distress (intense vocalization and attempts to escape) during the
first exposure to the arena, they were noticeably calmer on the last day,

indicating acclimatization to the experimental procedure.

2.3 Cortisol injection

A total of 24 female piglets (subjects) were selected (4/litter) and within
each litter, two piglets were randomly assigned to either vehicle (0.2 ml saline, n
= 12) or cortisol (hydrocortisone sodium succinate (SOLU-CORTEF; the Upjohn
Co., Kalamazoo, Mich.), 1 mglkg BW diluted in 0.2 ml of saline, n = 12)
treatment. All subjects were given an intramuscular injection behind the right ear
in the side of the neck 15 minutes prior to the SR test. After injection the subjects
returned to their farrowing crate and stayed with the sow and littermates until

testing. All animals were injected between 13:00 and 16:00 hours.

30

 

Habituation Test

 

Pen I Pen 2 Pen l Pen 2

 

 

 

 

 

 

 

   

 

 

 

Figure 1- Design of the farrowing environment (white, Pen 1 and Pen 2) and the
arenas (grey). During acclimatization of the piglets to the arena, a solid barrier
dividing the space in two halves prevented mixing of litters and the door-ways
were open so the animals could freely access the arena. During the habituation-

dishabituation test the doors to access the arena were closed.

2.4 Social recognition testing

The SR test was conducted in two phases. In phase 1 (habituation), the
subject was exposed to an unfamiliar castrated male (stimulus) for four
successive sessions of 60 seconds, each separated by a 10-minute interval,
during which both animals were placed back in their farrowing crate. In phase 2

(dishabltuation), the subject was exposed to a different stimulus male for 60

31

seconds. The selected stimulus animals were unfamiliar to the subject and were
20% lighter to minimize possible agonistic interactions. The subjects were tested
only once, at 11 days of age.

During testing, the doors by which the litters could voluntarily access or
exit the arena were closed. The test was always conducted in the subject’s home
environment (the arena attached to its farrowing crate). During each exposure,
subject and stimulus were placed on opposite sides of the arena at the same
time. After 30 seconds, the solid plastic barrier dividing the arena was removed
so the animals could freely interact. Each exposure lasted 60 seconds, but was

terminated if the animals were fighting for more than 15 seconds.

2.5 Behavioral observations

Behavior during the test was videotaped and subsequently observed using
“The Observer” behavioral recording software (Noldus Information Technology,
Wageningen, The Netherlands). Observations were done by an observer
unaware of the treatments. We measured the percentage of time that the subject
spent investigating the stimulus, defined as sniffing or touching (with the nose or
mouth) any part of the stimulus its head and/or body, or following within

approximately 10 cm.
2.6 Saliva sampling and cortisol analysis

Saliva was collected from the female subjects 15 minutes before injection

(baseline) and 15, 35, and 55 minutes after injection. For saliva collection, piglets

32

were allowed to chew on a cotton bud tied with dental floss. When thoroughly
moist, the samples were stored in a tube and kept on ice until centrifugation (5
minutes, 3,000 x G, 4°C). After centrifugation, the saliva was transferred to 1 ml
eppendorf containers and frozen at —20°C until further analysis.

Salivary cortisol concentrations were determined using the ACTIVE
Cortisol Enzyme Immunoassay (EIA) kit (Diagnostic Systems Laboratories, Inc.;
DSL-10-67100) modified for piglet salivary cortisol. According to the
manufacturer, cross reactivity of the assay for other components (cortisone, 11-
deoxycortisol, 21-deoxycortisol, 17o-hydroxycortisol) was 7.0, 5.7, 1.9, and 0.9%
respectively. Assay sensitivity was 0.072 pg/dL to a maximum concentration of
10pg/dL. Intra- and inter-assay coefficients of variance were 5.75% and 1.46%,

respectively.

2.7 Statistical analysis

The distribution of all variables was tested for normality and homogeneity
of variance, and appropriately transformed if not normally distributed.
Subsequently, the cortisol data was log-transformed. Data were analyzed using
SAS® 9.1 mixed model (PROC MIXED) software (SAS Institute inc., Cary, NC,
2003). The linear mixed model (LMM) included the effects of the treatments
(cortisol, saline), times for saliva collection (-T15, T15, T35, T55) or habituation
(habituation, dishabltuation) and their interaction. Furthermore, random litter
effects were specified as well as serial correlation between subsequent

measurements within piglet over time. Hence, a repeated measures analysis was

33

used to analyze effects of treatment on salivary cortisol levels and effects of
cortisol treatment on the amount of social investigation, correcting for repetition
effects of subject and litter. Key comparisons in the analysis for the social
investigation data were differences between the first and fourth exposures
(habituation phase) and between first and fifth exposures (habituation and
dishabituation).

A total of 30 salivary cortisol samples were omitted from the data set when
the subject had fought in a previous exposure or when cortisol levels were higher
than 10 pg/dl. All data are presented as means :I: S.E.M. Differences between the
experimental groups were considered to be significant at a Type I error rate of

5%.

3. Results
3.1 Salivary cortisol

Salivary cortisol concentrations before and after cortisol or saline injection
are presented in Figure 2a. Salivary cortisol levels were affected by treatment
and time (LMM, F3,37.1 = 2.45, P = 0.078). Post-hoc analysis revealed that
baseline salivary cortisol concentrations did not significantly differ between
treatment groups (P = 0.90). An increase in cortisol levels was observed only
after cortisol injection (LMM, Fug = 5.64, P < 0.05) and it changed over time
(LMM, F3374 = 5.38, P < 0.01). In the cortisol treated animals, salivary
concentrations increased over three-fold to a peak level 15 minutes after injection

(P < 0.001). Cortisol levels were still higher 35 minutes after injection compared

34

to the baseline levels in subjects injected with cortisol (P < 0.001) validating the
efficacy of the pharmacological treatment. At 55 minutes after injection of cortisol,
salivary concentrations declined but were still higher than baseline values (P =
0.073). Contrary to cortisol treatment, salivary cortisol levels in piglets injected
with saline did not significantly alter after 15, 35, and 55 minutes after injection (P

= 0.446, P = 0.152, and P = 0.106, respectively)

3.2 Investigative behavior

Social investigative behavior of subjects is presented in Figure 2b.
Repeated encounters to the same conspecific decreased social investigation in
cortisol and saline treatment groups across the four habituation sessions (LMM,
F4502 = 20.73, P < 0.0001). The subjects spent more time investigating the
stimulus pigs during the first than the second, third, and fourth habituation
exposures (P < 0.05, P < 0.0001, P < 0.0001, respectively). To rule out the
possibility that the above habituation was due to generalized social fatigue, the
last habituation (4"‘) exposure was compared with the dishabituation (unfamiliar
stimulus pig). Social investigation of the unfamiliar stimuli pigs was greater than
of the familiar one (DF = 58.2; t = -8.04; P = 0.0001). The amount of social
investigation during the dishabituation phase was comparable to that during the
first habituation subject (DF = 60.4; t= -0.87; P = 0.389). No effect of cortisol
administration was found on the amount of social investigation of the stimulus pig
during the habituation-dishabituation test (LMM, F1352 = 0.04, P = 0.849) or

interaction between treatment and exposures (LMM, F4502 = 0.55, P = 0.698).

35

 

 

 

(a)
3 a
“D
so
3
'3'.
E 4
8
E‘
.E‘
:75 2
o . .
t=—15 i=15 t=35 (=55
100
('1)

Social Investigation [%|
on
:

 

 

 

Hab l Hub 2 "ab 3 Hub 4 Dishab

Figure 2- Salivary cortisol concentration (a) and amounts of social investigative
behavior (b) by treatment during the SR test in 11-day-old female pigs. The grey
line and bars indicate cortisol treatment and the black line and bars indicate
vehicle (saline). Hydrocortisone and saline were injected 15 minutes before SR
test started. Data are presented as :t S.E.M. For significant differences see

results.

4. Discussion

While salivary cortisol concentrations of saline treated subjects did not
significantly alter, they considerably increased in those treated with cortisol.
However, no changes in habituation (SR) due to the cortisol treatment were
observed, as the duration of social investigation similarly declined during

habituation in both treatment groups.

4.1 The role of cortisol in short-term SR

It is well documented that exogenous and endogenous GCs not only
circulate in the body, but also enter in the brain (for reviews, see McEwen and
Sapolsky, 1995; Bremner 1999; de Kloet et al., 1999; Lupien and Lepage, 2001)
acting in regions related to learning and memory. In general, very high or very
low concentrations of GCs cause memory disruption, while moderate levels
enhance it (for reviews, see Sapolsky, 1999; Het et al., 2005). Our initial
hypothesis therefore was that administration of a high dose of exogenous cortisol
would lead to impairments in SR. Our results, however, indicate that although the
levels of circulating cortisol were increased during the habituation phase, the
ability of the subjects to acquire and recall information concerning previously
presented conspecifics was not significantly affected by the cortisol treatment.

Animal and human studies have reported conflicting results on the effects
of GC administration on learning and memory (for reviews, see Mendl, 1999; Het
et al., 2005). According to Mendl, methodological variations may underlie the

contradictory findings. It is likely that different routes of administration may

37

produce a different outcome and that pharmacological doses might not be
biologically relevant. In line with this suggestion, a noteworthy finding by Krugers
et al. (1997) suggests that while prolonged psychosocial stress caused spatial
learning deficits, artificial elevation of corticosterone to comparable levels did not.
It may therefore be that cortisol or corticosterone is not the only or even the
major mechanism by which stressors affect cognitive processes. During the
stress response, other hormones, such as catecholamines, are released at the
same time. There is evidence suggesting that these may interact with GCs in
affecting cognition (Roozendaal, 2000). Additionally, artificial manipulations of
single hormones are likely to result in feedback and cascade effects on other
hormones, thus questioning the precise mechanism of their interference with
cognitive processes (Mendl, 1999).

A review by Het et al., (2005), states that difference in methodology may
account for some of the conflicting results in studies on the effects of GCs on
cognition. In studies where cortisol was administered before learning, detrimental
effects on cognition were only found in those studies conducted in the morning
(high circulating cortisol), whereas those conducted in the afternoon (low
circulating cortisol) found enhancement or no effect (see also Maheu et al.,
2005). In our study, the subjects received a hydrocortisone injection in the
afternoon, which in that line may have obscured any potential effect of GCs on
SR. However, the effects of time of the day on cognition are likely caused by the
occurrence of a circadian rhythm in the levels of circulating cortisol. In humans,

these levels are higher in the morning (see Lupien et al., 2005 for a review),

38

leading to full occupation of MRs, and lower levels in the afternoon, resulting in a
lower occupation of MRs (de Kloet et al., 1998). This creates a ‘buffer’ of
unoccupied MRs to protect the brain from the detrimental effect of GR
occupation. Although it is possible that in our experiment this may have
accounted for our results, it is unlikely. In young piglets, as in human babies, a
circadian rhythm does not seem to be present (humans: de Weerth et al., 2003;
piglets: Evans et al., 1988; Kattesh et al., 1990; Klemcke and Pond, 1991; Ekkel
et al., 1996) and thus the time of injection should not have affected short-term
SR.

Unfortunately, we cannot determine with certainty whether the
ineffectiveness of exogenous GCs to affect cognition was due to methodological
constraints (e.g. injection effect, hydrocortisone dose) or whether exogenous
GCs do not alter short—term SR, as measured using the habituation-
dishabituation paradigm. Based on the results byKrugers et al. (1997), we
propose that exogenous GCs are not as potent as real-life stressors in affecting
SR in piglets, but stress the need for further research. For a better understanding
of the role of exogenous cortisol on short-term SR further studies on the dose

response curve are needed.

4.2 Habituation-dishabituation protocol
In the present study we have adapted a habituation-dishabituation
procedure previously used in adult mice (Dluzen and Kreutzberg, 1993; Winslow

and Camacho, 1995) to study the effects of GCs on SR in non-weaned piglets.

39

The protocol relies on the natural tendency of pigs to investigate novel and
unfamiliar conspecifics rather than familiar ones. As we anticipated, during
habituation, female 11-day-old pigs showed a decrease in social investigation
towards the same male stimulus pig, indicating recognition of the stimulus pig. To
rule out the possibility that this decrease was a result of social fatigue or
exhaustion instead of recognition of an encountered conspecific, we exposed the
subjects to a last exposure to a novel stimulus male. The presence of the novel
stimulus resulted in vigorous investigation, ruling out social exhaustion. The
habituation-dishabituation paradigm can therefore be used to test SR ability in
neonatal pigs.

In our study, we aimed to assess the effects of an acute exposure of
cortisol followed by increases in it on SR and thus needed to minimize stress
caused by the testing procedure itself. If testing conditions are stressful to the
subject, it may cause deficits in learning and memory functions. Indeed, Burman
and Mendl (2000) demonstrated that handling and moving rats to a different
environment may hamper SR, and it has been suggested that cognition in pigs is
also susceptible to disruption by acute stressors (spatial memory in pigs: Mendl
et al., 1997; social memory in weaned pigs: Souza and Zanella, submitted).
Therefore the subjects of the present study were tested in their home
environment and all animals were habituated to handling.

Interestingly, our results show that although the subject interacted with the
same stimulus pig for four trials, social investigation already significantly

decreased after the first exposure, indicating that a single 60-second exposure

40

was sufficient to acquire information concerning the identity of the stimulus. This
may be an important finding for future studies testing the effects of drugs with a
short half-life or studies requiring a minimum of handling (such as studies on the
effects of stressor in SR), since the number of learning trials can be reduced to

one.

5. Conclusions

In this experiment, a hydrocortisone injection and the resulting increase in
cortisol levels did not interfere with the ability of 11-day-old female piglets to
acquire and retrieve information about a male conspecific after repeated
exposures. It indicates that the injection of 1 mglkgBW of cortisol does not induce
impairments in acquisition and/or immediate recall of SR. However, future
studies are needed to clarify the role of cortisol on acquisition, consolidation, and
retrieval. This experiment does indicate that the habituation-dishabituation
paradigm can be adapted to investigate SR in non-weaned piglets. This
paradigm can be further used in very young pigs to address the effects of

stressors on SR.

Acknowledgments

This research was supported by USDA/NRI grant # 2001-35204-10810
(A.J.Z) and the Michigan Agricultural Experiment Station. We thank Cheryl Leece
and Christel Moons for helping with the data collection and personnel of the MSU

Swine Teaching and Research Center for assisting with animals care. We are

41

also grateful to Michael Mendl, Jarno Jansen, and Liesbeth Bolhuis for their

useful comments on an earlier version of this manuscript.

42

CHAPTER 3

A NOVEL METHOD FOR TESTING SOCIAL
RECOGNITION IN YOUNG PIGS AND THE MODULATING

EFFECTS OF RELOCATION

Souza, A.S.; Jansen, J.; Tempelman, R.J.; Mendl, M.; Zanella, A.J., 2006. A
novel method for testing social recognition in young pigs and the modulating

effects of relocation. Appl. Anim. Behav. Sci. 99: 77-87.

43

Abstract

Mixing litters of pigs often leads to short- or long-lasting aggression. As
social recognition (SR) is important for the development and maintenance of
stable social groups, a lack or disruption of SR could prolong agonistic
interactions and reduce welfare. Investigations of SR abilities are therefore of
considerable importance. However, experimental assessment of SR generally
involves moving subjects to unfamiliar environments, or excessive handling, or
aggression during the familiarisation period (in which subjects become
acquainted prior to testing their ability to recognise each other), which in turn may
change the outcome of the test. In this study, we tested a novel method for
assessing SR in young pigs aiming to minimise these problems. For the
familiarisation procedure, flexible netting was placed between farrowing pens
allowing physical contact between litters but preventing unwanted mixing and
fighting. We also investigated whether recognition of a familiar individual is
affected by relocation to a novel environment. Forty—eight piglets from 12 litters (4
piglets/litter) were tested in a split plot design with litters as the experimental
units for comparing two familiarity (familiar/unfamiliar) and three relocation (no
relocation/relocation to pens with the same spatial orientation/relocation to pens
with a different spatial orientation) treatments. Results indicated that piglets
exposed to unfamiliar animals spent more time on social investigation than those
exposed to familiar ones, suggesting that SR can be successfully tested using
this novel approach. The relocation treatment did not influence the amount of

time spent on social investigation, but did affect duration on exploration of the

44

arena. Piglets meeting familiar individuals were more susceptible to this effect

after relocation than piglets meeting unfamiliar conspecifics.

1. Introduction

Recognising familiar conspecifics enables pigs to form and maintain stable
social groups, thereby reducing social stress. In free-ranging systems, pigs tend
to form subgroups and maintain closer social bonds to certain individuals than
others, resulting in pigs sleeping in separate nests (Stolba and Wood-Gush,
1984). After forced mixing of different groups, the original bonds persist and are
distinguishable for over 190 days (Stolba and Wood-Gush, 1984). The low level
of aggression observed in free-ranging systems can be accounted for by the
availability of space and familiarity between animals (Jensen and Wood-Gush,
1984). In general, it is well documented that pigs usually react more aggressively
towards unfamiliar than familiar conspecifics (Arey and Franklin, 1995; Puppe,
1998; Turner et al., 2001). In intensive husbandry systems, social problems may
arise from disrupted bonding and restricted possibilities for group formation.
Fighting between pigs in stable social groups is rare, in contrast with groups of
unfamiliar pigs, which show a significantly higher incidence of aggression
(growing pigs: Ewbank and Meese, 1971, gilts: Spoolder et al., 1996, sows: Arey,
1999)

Many studies have investigated factors that affect and control aggression
among pigs such as familiarity and relatedness (Puppe, 1998; Stookey and

Gonyou, 1998), individual variation in aggressiveness (Erhard et al., 1997;

45

Bolhuis et al., 2005), visual cues (Ewbank et al., 1974; Friend et al., 1983), body
weight (Andersen et al., 2000), age (Jensen, 1994; Pitts et al., 2000), group size
(Turner et al., 2001; Morrison et al., 2003; Schmolke et al., 2004), pre-exposure
(Jensen and Yngvesson, 1998; D’Eath, 2005), familiarity with the environment
(Wattanakul et al., 1998), enrichment (Morgan et al., 1998) and space allowance
(Wregand et al., 1994; Turner et al., 2000), but while a potential role for social
recognition (SR) in moderating the levels of aggression observed has been
suggested (Yuan et al., 2004), it has not yet been thoroughly addressed.

Under commercial pig farming conditions, common husbandry practices
such as mixing of animals or even reintroduction of a previously familiar animal
contribute to the instability of social groups, leading to an increase in aggression
that may be partly explained by disruptions in SR memory (Puppe, 1998; Yuan et ,
al., 2004), particularly when the reintroduced animal is of lower rank in the social
hierarchy (Ewbank and Meese, 1971). If social memory is essential for
distinguishing familiar from unfamiliar individuals and thus for the maintenance of
stable social groups, then memory interference or impairments could increase
aggression leading to welfare problems (Mendl et al., 2001). However, there
have been limited studies on the role of social recognition memory - used here to
refer to the ability to discriminate a previously encountered individual from a
novel individual — in moderating aggression between pigs, and the effects of
changes in the environment on SR. lnforrnation on the effects of relocation on SR
is important as pigs are often moved to novel crates when weaned and mixed

(i.e. Graves et al., 1978).

46

A common method for assessing SR memory in rodents involves the
habituation - dishabituation technique, which consists of repeatedly exposing the
same subject to a stimulus animal (Thor and Holloway, 1982) and then, in a test
phase, exposing the same subject to either the same animal, or an unfamiliar
animal (Dluzen and Kreutzberg, 1993; Winslow and Camacho, 1995). Social
recognition is usually measured by a decrease (habituation) in social
investigation during successive exposures to the same conspecific, but an
increase (dishabituation) in social investigation when the unfamiliar animal is
presented. However, behavioural responses of subjects during SR tests are not
always easy to interpret. Agonistic interactions between subject and stimulus
animal can occur during the habituation period (e.g. hamsters: Johnston and
Jernigan, 1994, pigs: Souza and Zanella, in preparation), which in turn can
influence behaviour in subsequent encounters, interfering with the outcome of
the test. Also, considerable handling and moving of animals between different
environments during the repeated exposures of the habituation period can
distress and change the experience of individual animals. It has been
demonstrated that when rats are handled or placed in a different cage, these
procedures may cause interference with social memory (Burman and Mendl,
2000) which may be dependent in part on contextual cues from the environment
(Burman and Mendl, 1999).

In the current experiment, our main goal was therefore to test a novel
protocol for assessing SR memory in young piglets that minimised handling and

reduced the occurrence of aggression during familiarisation (habituation). The

47

familiarisation occurred in arenas situated between neighbouring crates that were
separated by flexible netting allowing interaction between litters but preventing
full physical contact and mixing, hence minimising handling, and aggression. By
using the netting we prevented some of the problems demonstrated in previous
studies where agonistic interactions between piglets and aggression of sows
towards foreign pigs were observed after mixing (i.e. Weary et al., 1999; D’Eath,
2005). Our second goal was to investigate whether recognition of familiar
individuals is affected by relocation to a novel but similar farrowing pen to clarify

potential effects of relocation on SR.

2. Materials and methods

This study was carried out at the Swine Teaching and Research Center
(STRC) at Michigan State University (MSU), USA. All procedures used in this
study were reviewed and approved by the All-University Committee for Animal

Use and Care of MSU.

2.1. Animals and housing

A total of 12 unweaned litters (Yorkshire x Duroc) were used in this study.
Within 36 h post-farrowing, piglets were cross-fostered so that litters totalled 10-
11 piglets (balanced for sex) following the standard operating procedures of the
STRC. The animals had ad libitum access to water and sows were fed according
to National Research Council (NRC, 1998). Sows and litters were housed in

standard farrowing pens (1.8 m x 2.3 m) with slatted floors throughout the

48

experiment. Two similar oval arenas (1 m x 1.5 m) were located between the
farrowing pens (Figure 1), enabling piglets to access the arena without handling
when the doors to the arena were opened. The temperature of the room was
controlled (20 :t 1°C) and artificial light was provided from 08:00 to 18:00 h

throughout the experiment.

 

Figure 1- Side view (left) and top view (right, showing the netting used for

familiarisation) of the arenas.

2.2 Habituation and familiarisation of piglets

All piglets were habituated to human presence and contact from birth until
12 days of age by the researcher holding each piglet in their home pen for 30 5
every 2 days. This procedure aimed to habituate piglets to handling needed for
the test protocol, where animals would be carried and placed in an arena. Piglets
were also habituated to the layout of the arenas from 08:00 to 18:00 h, every 2

days, by opening the doors between the farrowing pen and the arena. Litters

49

from neighbouring pens were habituated to the arenas on alternate days and the
arenas were cleaned daily to prevent odour transfer. We considered all litters to
be unfamiliar with each other prior to the familiarisation procedure.

The familiarisation procedure consisted of two familiarisation phases:
exposures between litters (Fam1) and exposures between pairs of siblings
(Fam2, see Table 1). Both familiarisation phases were carried out in the arenas.
Flexible netting (Figure 1) was used to enable body contact between litters but
prevent unwanted mixing and fighting between non-littermates. Fam1 occurred 1
day before the test, and consisted of opening the doors on both sides of the
arenas from 08:00 to 12:00 h and from 14:00 to 18:00 h, so that litters from.
adjacent pens could freely interact through the netting. Doors between the
arenas and the farrowing pens were left open during Fam1. For Fam2, a total of
48 unweaned piglets were randomly selected from the 12 litters (26 females and
22 males, 4 per litter). During Fam2, occurring on the day of testing, pairs of
littermate piglets from adjacent pens were placed in the arena for two 10-min
periods separated by a 10-min interval. Pigs were familiarised in pairs with their
siblings to prevent stress resulting from social isolation (e.g. Kanitz et al., 2004)
during the familiarisation procedure. During Fam2, the doors to the farrowing
pens were closed to prevent the animals from returning to their home pens and
to habituate them to being placed in a closed arena. After these two

familiarisation phases, the piglets were considered to be familiar.

50

Table 1- Familiarisation between litters and pairs of piglets

 

 

Familiarisation Animals in the Doors to Netting Exposure time
arena access the
arena
Between litters Up to 11 piglets Opened — Present 2 x 4 h
(Fami1) from each litter free access (2-h interval)
Between pairs Only 2 piglets Closed — Present 2 x 10 min
(Fami2) from each litter controlled (10-min interval)
access

 

2.3 Relocation

After Fam2, the piglets were randomly assigned by litter to one of the
three relocation treatments (see Figure 2): no relocation (NR, n = 12 piglets from
3 litters), relocation maintaining the arena orientation relative to the farrowing pen
(RM, n = 20 piglets from 5 litters) or relocation changing the arena orientation
relative to the farrowing pen (RC, n = 16 piglets from 4 litters). Relocation
consisted of moving sows and litters to novel but similar farrowing pens located
in the same room as their original pen. Relocation was completed within 40 min.
The relocation protocol was arranged so that litters to be tested together would
share the same arena (see Figure 2). Immediately after Fam2, litters and sows
subjected to the NR treatment remained in their home pens (acting as the control
for the relocation treatment). Litters and sows subjected to the RM treatment
were moved to novel farrowing pens located on the same side of the arena
relative to their original pens, while litters and sows subjected to the RC

treatment were moved to novel farrowing pens on the opposite side of the arena.

51

Allocation of the animals per treatment group is presented in Figure 2. A
balanced design comparison of the relocation treatments with the unfamiliar
treatment was not possible due to a constraint in size of the room, given that
pairs of unfamiliar piglets to be tested together could not be previously housed in

adjacent pens.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

j Pre-Test(0-11d)] | Pre-Test(0-11d) ]
08 09 12
[Unfamiliar Test (12d)]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2- Allocation of the farrowing pens and treatments before (Pre-Test) and
after (Test) relocation. Dark pens indicate NR-treatment, grey pens indicate RC-
treatment, and white pens indicate RM-treatment. Dashed line indicates netting

in arenas between pens. Numbers indicate litters.

2.4 Social recognition test
Summarising the treatment distribution, the subjects were allocated to one
of two familiarisation treatments (familiar or unfamiliar (n = 24, for each

treatment)) and one of three relocation treatments (NR, RM, and RC). The dyads

52

were formed by either females and males or male and female (balanced for the
total amount of dyads). The piglets were tested at 12 :I: 1 days of age. The SR
test began approximately 4 h after Fam2. Only the rear arena, out of view of the
sow, was used for the SR test to minimise the influence of the sows on piglets’
behaviour. Prior to testing, the netting was removed from the arena so that
piglets could freely interact.

‘ The SR test consisted of placing dyads of familiar or unfamiliar piglets in
the arena for a 5—min period per dyad. Dyads of familiar animals were formed by
assigning two piglets from different litters that had the opportunity to interact
during both phases of the familiarisation procedure. Conversely, unfamiliar dyads
were formed by pairing piglets that had never had the opportunity to interact

during the familiarisation procedures. Each animal was tested only once.

2.5 Behavioural observations

We investigated whether during the familiarisation between litters (Fam1)
the animals would visit the arena and interact with non-littermates by themselves.
During the total of two 4-h periods (= 480 min) the events in the arena were
videotaped and later analysed using instantaneous scan sampling performed at
5-min intervals. The amount of time the piglets spent in the arena, with and
without the future test pig (for the familiar group), are presented in percentages.
Fam2 was not analysed in this way because the test pairs of the familiar group

were always placed together in the arena for the two 10-min periods (= 20 min).

53

The behaviour of individual piglets in the arena during the SR test was
also videotaped and subsequently recorded using “The Observer 3.1”
behavioural recording software (Noldus Information Technology, Wageningen,
The Netherlands). The following behaviours were scored in a continuous focal
sampling paradigm: social investigation, defined as investigating the conspecific
(sniffing or touching (with the nose or mouth) any part of the other piglet’s head
and/or body, or following the other piglet within 10 cm) and exploring the arena

(rooting or licking the floor, doors or walls of the arena while sniffing).

2.6 Statistical analysis

The distribution of all variables was tested for normality and homogeneity
of variance, and appropriately transformed if not normally distributed. After
square-root transformations, all variables reasonably met the normality and
homogeneity criteria. The data were analysed using the mixed model analysis of
variance procedure (PROC MIXED) of SAS 8.2 (SAS Institute Inc., Cary, NC,
2001) to test the effects of, familiarity (familiar or unfamiliar), relocation (NR or RM
or RC), sex and test mate sex as well as all possible two-way interactions
between these four factors. Due to the split plot structure of our experimental
design, the experimental unit for relocation effects was defined by sow whereas
the experimental unit for familiarity was defined by both sow and dyad, thereby
necessitating the use of Satterthwaite’s approximation for determining degrees of
freedom (Sattherwaite, 1946) as invoked in PROC MIXED. The data are

presented as adjusted least-squares means :t S.E.M. with degrees of freedom

54

determined using Satterthwaite’s approximation. Differences between the
experimental groups were considered to be significant if P s 0.05. Sex, test mate
sex and their interaction (P > 0.10) did not significantly influence any of the

response variables and will not be mentioned in Section 3.

3. Results

During Fam1 (480 min), the test piglets spent 46.5% (223.2 min) of the
time in the arena. In 7.81% (37.49 min) of the total time, a piglet was in the arena
in presence of the future assigned ‘familiar’ test piglet. All the tested piglets
meeting familiar animals thus met their future test mates at least once during the
first familiarisation.

During the SR test, unfamiliar piglets spent significantly morertime on
social investigation (26.59 :t 3.5) than familiar piglets (11.55 i 3.21) (GLMM, F195
= 9.96; P < 0.01). Familiarity did not affect the amount of time spent on
exploration of the arena (GLMM, F1405 = 0; P = 0.96). The total percentage of
investigation and exploration between pairs of familiar and unfamiliar piglets by
relocation treatments is presented in Figure 3. There were no significant effects
of relocation on the duration of social investigation (GLMM, F2358 = 2.14; P =
0.17). Although relocation did not significantly affect the amount of time spent on
exploration of the arena (GLMM, F2103 = 2.21; P = 0.15), the interaction between
familiarity and relocation was significant (GLMM, F2915 = 4.50; P < 0.05). Familiar
piglets that were relocated to a farrowing pen with changed arena orientation

(RC) spent more time exploring the arena than familiar piglets relocated to a

55

farrowing pen that maintained the arena orientation (RM) (P < 0.01), see Figure

3.

0!
O

 

N 0) b
O O 0

Duration of Investigation (%)
"c3

 

 

Duration of Exploration (%)

 

 

 

NR RM RC

Figure 3- Effects of familiarity and relocation of 12~day-old piglets on time spent
on social investigation and exploration of the arena during the SR test. Familiarity
is indicated by dark bars (familiar) and grey bars (unfamiliar). NR, RM, and RC

indicate relocation treatments. Values represent adjusted means iS.E.M.

56

Significant differences (P < 0.01) within familiarity are specified with different

letters.

4. Discussion

In SR studies, most of current protocols use a habituation method to
familiarise animals, in which a subject is exposed to the same conspecific or
odour, repeatedly (e.g. in rats: Thor and Holloway, 1982, in pigs: Souza and
Zanella, 2001, using pigs urine odour: Mendl et al., 2002). It is not always easy to
carry out systematic evaluations of the behavioural responses of the tested
subjects. The most common problems using stimulus animals are related to
considerable handling of the animals and aggression that occurs during the
familiarisation procedure (e.g. in pigs: Souza and Zanella, in preparation;
hamsters: Johnston and Jernigan, 1994) and these effects should to be taken
into account during the interpretation of results. Challenges posed to the animals
during the learning process can cause an apparent failure in recognition and this
effect might be explained as a side effect of the experimental technique itself
(e.g. repeated handling), rather than the specific treatment (Burman and Mendl,
2000). We therefore designed a novel paradigm for familiarisation using netting
barriers which successfully prevented fighting between non-littermate piglets
during this period. There was only one pair of non-littermates that engaged in
aggressive interaction during familiarisation, but it did not last more than 4 s

because both animals went back to their home pen without being injured.

57

Furthermore, the animals were familiarised and tested in arenas located next to
the farrowing pens which seemed to reduce the stress of moving animals to a
different environment during the test or other possible confounding factors, such
as isolation (e.g. Kanitz et al., 2004). This method for familiarisation appeared to
reduce some of the previously mentioned challenges commonly seen during the
habituation phase.

Ewbank et al. (1974) suggested that distinction between a previously
encountered individual and an unfamiliar one might be improved by processing
the information through more than one sensory system. In our study, we
increased the chances of piglets to become familiar with individual conspecifics
by allowing them to use their olfactory, visual, auditory, and tactile cues during
the familiarisation period. By subjecting the piglets to Fam1, we increased their
chances of gaining information about non-littermates. The piglets spent almost
half of the total 480 min in the arena and more than 30 min with their future test
animal. We tested whether this novel familiarisation protocol would successfully
familiarise non-littermate piglets by analysing differences in social investigative
behaviour between dyads of familiar and unfamiliar 12—day-old piglets during
subsequent testing. Unfamiliar piglets spent significantly more time investigating
each other than familiar animals, a finding supported by previous results
(Kristensen et al., 2001; Souza and Zanella, 2001), suggesting that familiar
animals remembered each other from the preceding familiarisation period and

indicating successful familiarisation.

58

Relocation did not affect social investigation, indicating that, in pigs, minor
changes in a novel environment do not interfere with social memory, in contrast
with findings in rats by Burman and Mendl (2000). However, relocation did affect
the overall time the animals spent on the measured behaviours during testing,
most notably in familiar dyads. Familiar animals relocated with changes in the
arena orientation (RC) spent significantly more time exploring the environment
than those relocated with no change in arena orientation (RM). For RC animals,
the position of the testing arena had changed relative to the home pen and this
difference might have motivated the animals to explore the ‘new’ surroundings
rather than a familiar conspecific more than in RM animals where orientation of
the novel arena was the same. Novelty arouses a motivation to explore, likely an
adaptive response necessary to gather information (VVood-Gush and
Vestergaard, 1991; Wemelsfelder and Birke, 1997). No effects of relocation were
found in unfamiliar dyads, suggesting that investigation of an unfamiliar has
priority over exploration of a new environment. Conclusive remarks cannot be
made, but it is possible that animals prioritise information gathering, e.g. if an
animal is familiar, investigation of the environment becomes a priority. Further
investigation on this subject is needed to fully understand the role of the
environmental context on social memory and clarify the processes in which

animals prioritise their information processing.

59

5. Conclusions

We described a novel method to test social recognition in pigs which
avoids fighting and resulting distress during the familiarisation (habituation)
phase. This familiarisation methodology resulted in clear differences in time
spent on social investigation between familiar and unfamiliar dyads during the
testing phase, up to 4 h after familiarisation. Relocation of the animals between
the familiarisation and testing phases did not interfere in the outcome of the SR
test in terms of time spent on social investigation, but had effects on time spent in

exploration of the arena.

Acknowledgments

This research was supported by USDA/NRI grant # 2001-35204-10810
and the Michigan Agricultural Experiment Station. We thank Sandra Simone
Rigotti for helping with the data collection and personnel of the MSU Swine

Teaching and Research Center for assisting with animals care.

60

CHAPTER 4

LONG LASTING SOCIAL RECOGNITION IN 3 WEEK-OLD

FEMALE PIGS

Adriana S. Souza, Jarno Jansen, Janice M. Siegford,
and Adroaldo J. Zanella

In preparation for publication

61

Abstract

While the establishment of social recognition (SR) memory in rodents has
been extensively explored, studies that systematically investigate SR in pigs,
especially at very young ages, remain sparse. As in rodents, a decrease in
spontaneous social investigation is observed when a pig re-encounters a familiar
conspecific, and this phenomenon can be used as a valid measure of SR ability.
We have recently designed a protocol to test SR in non-weaned pigs and
showed that 12-day-old pigs were able to remember members of a familiar litter
for at least 4 hours after the last familiarization exposure. In the present study,
we aimed at investigating whether the same protocol can be used to assess SR
in 21-day-old female pigs up to 24 hours. The results demonstrated that the
animals were able to recognize familiar conspecifics at least 24 hours later, as
similar amounts in the duration of social investigation were found in piglets
meeting familiar conspecific at 4 and 24 hours after being familiarized. We
therefore concluded that the used SR protocol allows investigation of a more

robust SR memory in 21-day-old female pigs.

62

1. Introduction

The basis of social relationships in mammals involves at least the ability to
discriminate between classes of individuals, such as dominant/subordinate,
group-member/non-member, or familiar-unfamiliar. An animal’s ability to
discriminate between familiar and unfamiliar conspecifics has been demonstrated
in a number of species (a.o. pigs: Kristensen et al., 2001; rats: Engelmann et al.,
1995; ground-squirrels: Mateo and Johnston, 2000).

In pigs, discrimination probably relies on familiarity gained over a period of
mutual association rather than genetic relatedness, as pigs are similarly
aggressive towards unfamiliar kin and unfamiliar non-kin (Puppe, 1998; Stookey
and Gonyou, 1998). Familiarity, and recognition thereof, may therefore be a key
factor in attaining and maintaining stability within a social group, and failure in
distinguishing between conspecifics may result in outbreaks of fighting (Ewbank
and Meese, 1971; Puppe, 1998). There is evidence that pigs can retain
information about certain conspecifics for long-periods of time. As an example,
Ewbank and Meese (1971) have shown that top-ranking fattening pigs could be
safely returned to their original group even after 25 days of isolation. However,
while indirect assessment of SR based on the amount of fighting may indicate
SR of high-ranking animals, it does not provide insight in SR of low-ranking
animals, since fighting will occur with both low-ranking and unfamiliar animals.
Depending on the social rank of individuals, dominant or subordinate, in a social
encounter an appropriate behavioral response is usually expected. During

interactions between the same individuals in a stable social group, the behavioral

63

responses tend to be very consistent over time. This consistency may indicate
reflect a stability of social relationship which requires that individuals are able to
recognize or at least discriminate members of its group. However, this ability may
be disturbed, for example, as a result of incorporation of unfamiliar conspecifics,
illness or temporary removal.

In an experimental setting, SR may be assessed using the individual’s
natural ‘interest’ to intensely investigate novel conspecifics. Investigation is
important for acquisition of information and increases familiarity or at least
reduces novelty of the stimulus (T odrank et al., 1999), since a lesser amount of
investigation is often observed after repeated exposure of a (social) stimulus
(Thor and Holloway, 1982; Engelmann et al., 1995; Souza and Zanella, 2001).

Recently, we showed that 11-day-old piglets were able to distinguish
between familiar and unfamiliar conspecifics 4 hours after the last exposure time
(Souza et al., 2006). McLeman et al. (2005) have provided evidence that 6-week-
old pigs are able to discriminate conspecifics sharing similar characteristics 24
hours after the last training session. The required amount of handling and
training in McLeman’s et al. (2005) experiment, however, may not be ideal for
use in very young pigs. Therefore, the present study aimed at testing a protocol
to investigate SR in 21-day-old pigs using a method specifically designed for
neonatal animals (for details see Souza et al., 2006). In the present study, we
investigated a more robust SR of 21-old female pigs using two delay periods, 4

and 24 hours.

2. Materials and methods

This study was carried out at the Swine Teaching and Research Center
(STRC) at Michigan State University (MSU), USA. All procedures used in this
study were reviewed and approved by the MSU All-University Committee for

Animal Use and Care.

2.1 Animals and apparatus

Eight litters of piglets were used in a total of four trials. Within 36 hours of
farrowing, piglets were cross-fostered following the standard operating
procedures of the STRC so that each experimental litter contained 8 female
piglets. Sows and litters were housed in standard farrowing crates (1.8 x 2.3 m)
with slatted floors. The animals had ad libitum access to water and sows were
fed according to National Research Council (NRC, 1998). The temperature of the
room was controlled (20°C :I: 1) and artificial light was provided from 08:00 to
18:00 throughout the experiment.

The piglets in this study had been previously used in a spatial learning
task. The spatial learning task consisted of an adapted form of the Morris Water
Maze (MWM: for details see Laughlin and Zanella, 2003). The animals performed
the MWM task at 13 and 14 days of age and were classified for the spatial
experiment as ‘good performers’ or ‘poor performers’ (n = 24 per group: GP,
average latency time s 50 seconds; PP, average latency 2 70 seconds; Laughlin
and Zanella, 2003). The remaining littermates classified as ‘intermediate

performers’ were kept with the sow and eliminated from this study.

65

2.2 Habituation and familiarization protocols

Piglets were habituated to human presence and contact from birth until 12
days of age by the researcher holding each piglet in their home pen for 30
seconds every other day. This procedure aimed at accustoming piglets to the
handling needed for SR testing, where animals would be carried and placed in
the testing arena located between farrowing crates. Piglets were habituated to
the testing arenas from 08:00 to 18:00, every other day by opening the doors to
the arenas (located in between two neighboring farrowing crates; see Figure 1).
Neighboring litters were habituated to the arenas on alternate days and the
arenas were cleaned daily to minimize odor transfer between litters. All litters
were assumed to be unfamiliar to each other prior to the familiarization
procedure.

The familiarization procedure consisted of two phases: exposures
between whole litters (Fam1) and exposures between groups of 3 siblings
(Fam2). Six piglets/litter, balanced for spatial memory task performance, were
used (n = 48). Both familiarization phases were carried out in identical oval
arenas (1 m x 1.5 m) located between the farrowing crates (Figure 1). Flexible
netting divided each arena in half to enable body contact between litters while
preventing mixing and fighting between non-littermates.

Fam1 occurred one day before SR testing, and consisted of opening the
doors on each side of an arena from 08:00 to 12:00 and from 14:00 to 18:00, so
that two litters from adjacent crates could interact freely through the netting. Each

litter was exposed just once to Fam1. During Fam2, which was performed the

66

day after Fam1, three littermates from adjacent farrowing crates were placed in
the arena for two 10-minute periods separated by a 10-minute interval. As social
isolation can be highly stressful for pigs (Kanitz et al., 2004), the animals were
familiarized in groups with their siblings during the familiarization procedure.
During Fam2, the doors to the farrowing crates were closed to prevent the
animals from returning to their pens and to accustom them to being placed in a
closed arena. After Fam2, neighboring litters that had opportunity to social

interact in the arena were considered to be familiar.

F arrowing Crate Arena Farrowing C rate

 

Rear

 

——————-——.—q
——————-—u————.-

 

 

 

 

r
I
I
l
l
I
l
I
I
I
I
A

From

 

Figure 1- Farrowing crates and testing arenas. Adjacent farrowing crates shared
the same arenas. During ‘familiarization', both front and rear arenas were used.

During ‘testing’ only the rear arena, out of view of the sow, was used.

2.3 Relocation

After Fam2, all litters and sows were relocated to novel farrowing crates,
so that the animals assigned to be tested together (familiar or unfamiliar) were
placed in crates adjacent to the testing arenas. We moved the litters prior to
testing to minimize disturbance of the animals during testing. Relocation
consisted of moving sows and litters to novel but similar farrowing crates,
maintaining the orientation or the arena and located in the same room (for details
see Souza et al., 2006). Relocation of all animals was completed within 20

minutes.

2.4 Testing

Piglets were tested in dyads at the age of 21 :t 3 days. Each dyad was
made up of piglets from two different litters. Half the dyads comprised piglets that
had been exposed to each other during Fam1 and Fam2 (familiar) and half
comprised piglets that had not (unfamiliar). GP piglets were only tested with GP
piglets and PP piglets were tested only with PP piglets. For the SR test, only the
rear arena, out of view of the sow, was used for testing, in order to minimize the
influence of the sow on piglet behavior. Prior to testing, the doors to access the
arena were closed and the netting was removed, so that piglets could freely
interact, but could not return to their home crate. Dyads of piglets were placed in
the arena for a 3-minute period. Twenty-four animals (6 ‘familiar’ dyads and 6

‘unfamiliar’ dyads) were assigned to be tested after a delay period of 4 hours

68

after Fam2 (T4) whereas the remaining 24 were assigned to be tested 24 hours

after Fam2 (T24).

2.5 Behavioral observations during SR test

The behavior of individual piglets in the arena during the SR test was
videotaped and analyzed using “The Observer 3.1” behavioral recording software
(Noldus Information Technology, Wageningen, The Netherlands). Duration
(expressed as the percentage of the total testing exposure) of social
investigation, defined as sniffing or touching (with the nose or mouth) any part of
the conspecific’s head and/or body, or following within approximately 10 cm, was

scored by continuous focal sampling recording.

2.6 Statistical analysis

The distribution of all variables met the normality and homogeneity criteria.
The data were analyzed using the mixed model analysis of variance procedure
(PROC MIXED) in SAS 8.2 (SAS Institute inc., Cary, NC, 2001) to test for effects
of familiarity (familiar or unfamiliar), performance (GP, PP), and delay period (T4
or T24), as well as all interactions. All effects were randomized for trial within
litter and for pair. To establish equivalence between means of T4 and T24 a
Bioequivalence testing was used. The data are presented as adjusted means 1:
S.E.M. Differences between the experimental groups were considered to be

significant if P s 0.05.

69

3. Results

The duration of social investigation was affected by familiarity (GLMM;
F1,37= 15.20; P < 0.001); after the familiarization procedure unfamiliar pigs spent
significantly more time investigating the testing mate (26.35 :I: 1.91) than familiar
animals (14.18 i 1.28). No effect of delay period (T4, T24) was found for the
duration of social investigation (GLMM; F1,37= 2.6; P > 0.1). Pigs tested 4 hours
and 24 hours after being exposed to the familiarization procedure spent similar
amounts of time on social investigation (18.45 t 1.40 and 21.89 1 2.21,
respectively). The duration of social investigation was not significantly affected by
performance in the Morris-Water Maze test (GLMM; F1,37= 1.11; P > 0.1).

There was no interaction between familiarity, performance, and delay
period on the duration of social investigation (Figure 2; GLMM; F1,37 = 0.01; P >
0.1). The results on two-way interactions showed no effect between familiarity
and performance on the Morris-Water Maze test (GLMM; F1,37 = 1.06; P > 0.1),
between performance and delay period (GLMM; F1,37 = 0.12; P > 0.1), neither
between familiarity and delay period (GLMM; me = 15.20; P > 0.05). The testing
power of the interaction between familiarity and delay period falls into the 90%
rate; therefore bioequivalence testing was applied for comparisons between
familiar groups tested at T4 and T24. The results showed that the mean values of
T4 and T24 (14.57 i 1.58 and 13.78 :I: 2.05, respectively) are similar (t = 0.23, P
= 0.8). Also, comparisons between mean values of unfamiliar groups tested at T4
and T24 (22.33 :I: 2.04 and 30.74 :t 3.11) showed that they are not similar (t = -

2.48, P = 0.01).

70

4. Discussion

The present study demonstrated that 21 day-old female pigs are able to
remember members of a familiar litter (of the same sex, age, and non-related) for
up to 24 hours. The amount of social investigation did not differ between pigs
tested after a delay period of 4 hours and those tested after 24 hours, indicating
that this testing protocol can be used to assess a relatively long-term memory in

non-weaned 21 day-old pigs.

 

l
I 100 —

80-

Social investigation (%)

 

 

 

 

 

 

Figure 2- Effects delay period length and spatial learning performance between
familiarization and testing on duration of social investigation. Dark bars indicate
short-term (4h delay) and light bars indicate long-term (24h delay) SR memory.

Values represent adjusted means :t S.E.M.

71

The familiarization protocol provides a relatively long exposure of the
animals to each other (approximately 81/2 hours) with minimal distress as fighting
was prevented by the initial presence of mesh netting, all of which may favor a
more robust memory for members of familiar litters. The relevance of these
findings lays in the possibility to clarify the role of pharmacological compounds
and acute and chronic stressors on memory in young pigs. Such work may
potentially lead to a valid model for SR memory in humans (e.g. by using
neonatal pigs as a model more invasive interferences can be used), but can also
provide a method for investigating the underlying causes of prolonged
aggression in group-housed pigs. The SR familiarization and testing
methodology presented here is a good method for conducting such much needed

research.

5. Conclusion

The present study demonstrated that the protocol (familiarization and
testing procedure) used to assess the ability of neonatal pigs to discriminate
members of a familiar litter can be used to assess both short- and long-term
memory. Furthermore, this method offers the possibility for further investigation
of both short— and long-term memory in a single trial, and with minimal

disturbance to the animals.

Acknowledgements

72

This research was supported by the USDAINRI grant # 2001-35204-
10810 awarded to Adroaldo J. Zanella) and Michigan Agricultural Experiment
Station. We thank personnel of the MSU Swine Teaching and Research Center
for assisting with animal care. We are also grateful to Dr. Michael Mendl for his

useful comments on an earlier version of this manuscript.

73

CHAPTER 5

SOCIAL ISOLATION ELICITS DEFICITS IN THE ABILITY
OF NEWLY-WEANED PIGS TO RECOGNIZE FAMILIAR

FEMALE CONSPECIFICS

Adriana S. Souza and Adroaldo J. Zanella

Submitted

74

Abstract

In pigs, weaning and social isolation can be highly stressful and may
impair social recognition (SR). Using a modification of a previously presented
familiarisation protocol, newly-weaned pigs were familiarised within litters. The
goal of this study was to assess the ability of the piglets to recall the information
about familiar conspecifics and examined how this was affected by weaning age
and/or social isolation. A total of forty-eight female piglets were weaned at day 11
(D11, n = 24) or at day 22 (D22, n = 24) of age. To examine whether social
isolation impaired SR, 24 pigs were isolated for 15 minutes prior to the SR test,
while the control group (n = 24) remained with their littermates in nursery pens.
Immediately after social isolation pigs were exposed to a familiar (n = 24) or an
unfamiliar (n = 24) conspecific for a 3 minute-period. The animals were tested
only once, two days after weaning, i.e at 13 days of age for D11 piglets and 24
days of age for D22 piglets. The results indicated that during familiar interactions,
isolated D11 pigs investigated more than non-isolated 011 and 022 pigs,
whereas during unfamiliar interactions no differences between the treatment
groups were found. Interestingly, the amount of social investigation displayed by
familiar isolated D11 pigs was similar to that displayed by unfamiliar animals.
Taken together, this indicates that SR is disrupted by 15 minutes of social
isolation in D11, but not in 022 pigs. Possible mechanisms for this disruption are

discussed.

75

1. Introduction

In feral and free-ranging conditions pigs are weaned gradually. Weaning
begins when the sow leaves the farrowing nest for foraging, but the complete
cessation of suckling is normally not seen before 4 months of age, when the
young pigs continue to live in social contact with the sow and her social group
(Newberry and Wood-Gush, 1985; Jensen, 1986; Jensen and Stangel, 1992;
Petersen, 1994). In commercial husbandry conditions, piglets are abruptly
weaned at much younger ages. Under these circumstances, weaning is a
multifactorial stressor for the piglets as it simultaneously involves separation from
the sow, changes in diet, and relocation to a different environment (Varley, 1985;
Dybkjaer, 1992; Weary and Fraser, 1997). Studies have shown that weaning
causes distress in pigs, with prolonged vocalisation, restlessness, and long-term
behavioural changes (Week 4: Dybkjaer, 1992; Week 2 x Week 4: Weary et al.,
1999; Day 6 x Non Weaned : Orgeur et al., 2001; Week 3 x Week 4: Colson et
al., 2006), and enhanced cortisol concentration in plasma (Week 2: Blecha et al.,
1985; Week 3 X Week 5: Mason et al., 2003) and in urine (Day 6: Hay et al.,
2001). These studies show that biological changes are associated with weaning
these animals abruptly and at much early stages of life than would occur in a
more natural environment, which may in turn compromise pig’s welfare. The
latest report of the NAHMS (2001) revealed that, in the USA, 63.9% of the pigs
are weaned between 16 and 20 days of age and 15% are weaned before 16

days of age.

76

Social isolation is also stressful for a pig (Herskin and Jensen, 2000; Ruis
et al., 2001; Kanitz et al., 2004) and may occur during routine management
practices (e.g. tail docking, castration, iron injection, veterinary inspection) or
experimental purposes (e.g. metabolism chamber, Moeser et al., 2002). Still, the
effects of social isolation for young pigs from the mother and/or siblings are
poorly understood.

It is well established that glucocorticoid (GC) hormones, secreted by the
adrenal cortex after a stressful event, influence cognitive performance (for a
review see Roozendaal, 2002). A recent study conducted in our lab investigated
the effects of weaning age and social isolation on a spatial learning task.
Laughlin and Zanella (2003) demonstrated that the ability to reach a hidden
platform in a Morris Water Maze was hampered if female pigs were weaned at
D12 and tested at D14 or D23 of age, but only if they were socially isolated
immediately before testing, whereas performance of female pigs weaned at D21
and tested at D23 was not affected by social isolation. No differences in
performance were found between non-isolated animals and the animals did not
differ in post-isolation salivary GC levels (Laughlin and Zanella, 2002). This
finding may indicate a possible cognitive impairment in animals weaned at D12
pigs during heightened HPA axis activity caused by social isolation.

Social recognition, here defined as the process by which animals
discriminate familiar conspecifics, is of fundamental importance for a wide range
of social behaviours and is a component of learning and memory. In rodents, SR

is sensitive to disruption by acute stress (Burman and Mendl, 2000; Kogan et al.,

77

2000). In this study, we therefore aim at providing an insight on the effects of a
real life acute stressor on the learning ability of newly weaned female pigs. For
that purpose, we investigated whether the ability of D11 and D22 piglets to
recognise conspecifics is susceptible to disruption by a single acute stressor (15
minutes of social isolation).

To that end we used a SR protocol developed by Souza et al. (2006),
which is based on the social discrimination paradigm, using pigs’ natural
tendency to investigate novelty (novel environment: Stolba and Wood-Gush,
1980; de Jong et al., 2000; novel conspecifics: Souza and Zanella, 2001). In this
protocol, piglets are familiarised with neighbouring animals through flexible
netting, which has been shown to prevent mixing and fighting between unfamiliar
pigs during familiarisation (for details see Souza et al., 2006). A lack of
differences in the duration of social investigation between familiar and unfamiliar
encounters indicates impairments in recognising familiar individuals (in rats:

Heinrichs, 2003).

2. Material and methods

This study was carried out at the Swine Teaching and Research Center
(STRC) and the Intensive Metabolism Unit (IMU) at Michigan State University. All
procedures used in this study were reviewed and approved by the All-University

Committee for Animal Use and Care of MSU.

78

2.1 Weaning and isolation treatments

Three factors were studied, weaning age with 2 levels: Day 11 (D11) and
Day 22 (D22), isolation with 2 levels: Isolated and Non-Isolated and familiarity
with 2 levels: Familiar, Unfamiliar.. A total of 8 treatments were used in this

factorial design with 6 replicates per treatment group.

2.2 Animals, housing, and weaning

Eight litters (Yorkshire x Duroc) were selected for this study. VIfithin 36
hours post-farrowing the piglets were cross-fostered so litters were standardised
to 10 or 11 females and the males were fostered to non-experimental litters,
following the standard operating procedures of the STRC. During the suckling
period, sows and their piglets were housed in standard farrowing crates (1.8 x
2.3 m) with access to food and water.

Nine female piglets were selected per litter and balanced for two weaning
ages (4-5 piglets/ litter). Half the litter was weaned at 11 days of age (D11) and
the other half was weaned at 22 days of age (D22). Weaning was carried out by
taking the selected piglets from their farrowing crates and driving them to
experimental facility — Intensive Metabolism Unit (IMU) (distance: 1.6 km, time: 5
minutes) where they remained with littermates in nursery pens (1.5 m2, 4-5
animals/ pen). A heating pad, a nipple drinker and a 5-space feeder were
provided in each pen. Circular arenas (diameter, 1.5 m) were located between
the nursery pens enabling pigs from adjacent pens to access the common area

when the doors to the arena were opened. Flexible netting dividing each arena

79

into two halves prevented mixing and agonistic interactions between non-
penmates (for details see Souza et al., 2006). All animals had ad libitum access
to water and were fed according to National Research Council (NRC, 1998).
Artificial lights were provided from 06:00 to 23:00 hour. Room temperature was

controlled (27°C :I: 2°C).

2.3 Familiarisation

The day after weaning, the pigs were subjected to the familiarisation
procedure, which was conducted in two phases: exposure between non-
penmates (fam1, see Table 1) and exposure between pairs of penmates to pairs
of non-penmates (fam2). Fam1 occurred on the day after weaning, when the pigs .
were habituated to the neighbouring pigs by allowing them to freely access the
testing arena for two consecutive days from 08:00 - 12:00 and 14:00 - 18:00.
Fam2 was carried out on the subsequent day (testing day), when pairs of pigs
from adjacent pens (e.g. 2 pigs of pen-A with 2 pigs of pen-B) were placed in the
arena twice for 10 minutes. Both familiarisation phases occurred in the arena
with a netting barrier. After the familiarisation procedure pigs from adjacent pens

that had previously interacted were considered familiar.

80

Table 1- Familiarisation between litters and pairs of piglets

 

 

 

Familiarisation Animals in Doors to the Netting Exposure time
the arena arena
Between Up to 5 Open - free Present 2 x 4 hours
litters (Fam1) piglets from access (2 hour interval)
each litter
Between pairs Only 2 piglets Close - Present 2 x 10 minutes
(Fam2) from each controlled (10 minute
litter access interval)

 

2.4 Social recognition test

A total of 48 female pigs were selected for the SR test. As part of the
testing protocol, immediately after fam2 all litters were relocated (within approx.
10 minutes) to novel nursery pens. The novel pens were similar to their home
pens and were located in the same room. After relocation the pigs assigned to be
tested together were housed in adjacent pens and shared the same arena. The
spatial orientation of the novel pens, position of the arena, feeder, and drinker did
not change. The SR test began 4 hours after fam2.

Prior to testing, the 24 female pigs selected for the isolation treatment
were individually taken from their nursery pens placed in a weighing cart and
subsequently transported to another room located in the same experimental
building. Here every pig was kept alone in the weighing cart for 15 minutes. After
isolation each pig was immediately subjected to the SR test. The non-isolated
pigs were left undisturbed in their home pens during this time and only placed in

the arena for the testing. The SR test consisted of a single 3-minute exposure of

81

pigs in the arena without the netting, which allowed the animals to have full body
contact. The testing exposure was done by exposing pairs of non-penmates (i.e.
from different litters). For familiar groups, the pair was made by two pigs that
were familiarised together, whereas in the unfamiliar groups the pigs were
completely unfamiliar to each other. The pigs were tested two days after
weaning; 011 animals were tested at 13 days of age and 022 pigs at 24 days of

age. Every pig was tested only once.

2.5 Behavioural observations

Social behaviour of individual pigs in the arena during the SR test was
video recorded and subsequently analysed using “The Observer 3.1” behavioural
recording software (Noldus Information Technology, Wageningen, The
Netherlands). The duration of social investigation was the only behaviour used
for SR memory assessment. Social investigation was defined as sniffing or
touching (with the nose or mouth) any part of the other piglet’s head and/or body,
or following the other piglet within approximately 10 cm.

The total amount of general behavioural activity was recorded to detect
possible non-specific effects of social isolation, such as changes in activity
patterns that could alter social investigative behaviour and was calculated by
subtracting the duration of general inactivity from the total duration of the
exposure and in the results is presented as a percentage. Inactivity was defined

as lying on side or belly without visibly sniffing the arena or the other pig.

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2.6 Statistical analysis

The distribution of social investigative behaviour met the criteria for
normality and homogeneity. Data were analysed using a mixed model analysis of
variance procedure (PROC MIXED) in SAS® 9.1 (SAS Institute inc., Cary, NC,
2003) to test for effects of familiarity, weaning age, and social isolation and all
possible interactions between these three factors. All factors were tested against
the random effect of litter within familiarity and of pair. Post-hoc analyses were
done using Tukey adjustment. Data were collected from individual pigs and are
presented as means :I: S.E.M. Differences between the experimental groups
were considered to be significant if P s 0.05.

Due to a lack of normality and homogeneity of variance of general
behavioural activity, the difference between groups (isolated D11, non-isolated
D11, isolated 022, and non-isolated 022) was analyzed using a two-tailed

Wilcoxon Two Sample Test.

3. Results
3.1 General behavioural activity

Pos-hoc comparisons between treatment groups were performed and
showed that they were not significantly different from each other. Isolated 011
pigs did not significantly differ in amounts of general behavioural activity when
compared with non-isolated 011 (P = 0.31), isolated 022 (P = 0.31), and non-

isolated 022 (P = 0.98) pigs. No differences were found in general behavioral

83

atictivity when comparing non-isolated 011 with isolated 022 pigs (P = 0.86) and

isolated 022 with non-isolated 022 (P > 0.31).

Table 2 - Percentages of general behavioural activity (means :I: S.E.M.) during

familiar and unfamiliar exposures in the SR test.

 

Familiar Unfamiliar

 

Weaning at 011 Isolated 100.00 :I: 0.00 (n = 6) 100.00 1 0.00 (n = 6)

 

Non-Isolated 93.55 :I: 5.50 (n = 6) 98.53 :I: 1.47 (n = 6)

 

Isolated 95.48 :I: 4.52 (n = 6) 82.06 :I: 11.64 (n = 6)
Weaning at 022

 

Non-Isolated 100.00 :t 0.00 (n = 6) 100.00 :I: 0.00 (n = 6)

 

3.2 Social investigation

The percentage of social investigation in familiar and unfamiliar female pig
dyads during the SR test is presented in Figure 1. There was a significant effect
of familiarity (GLMM; F15 = 9.30, P < 0.05) with familiar dyads spending less time
on social investigation (16.70 :I: 2.86 %) than unfamiliar ones (29.09 :I: 3.71 %). A
significant effect of isolation (GLMM; F134 = 7.20, P = 0.01) showed that socially
isolated animals had in average higher duration of social investigation (28.35 :I:
3.59) than non-isolated (17.45 i 3.14). There was no significant effect of weaning
age (GLMM; F134 = 0.93, P > 0.1) on social investigation time.

An interaction effect between weaning age and social isolation was found
(GLMM; F134 = 8.25, P < 0.01), such that 011 pigs that were socially isolated
spent a significantly larger percentage of time on social investigative behaviour

than non-isolated 011 (P < 0.001), isolated 022 (P = 0.1), and non-isolated 022

84

animals (P = 0.01). There was no interaction between isolation and familiarity
factors (GLMM; F134 = 0.55, P > 0.1). A tendency towards significance for an
interaction between weaning and familiarity was found (GLMM; F134 = 3.50, P <
0.07), where familiar groups weaned at 011 (22.46 :t 4.75) spent more time on
social investigation then those weaned at 022 (10.95 :I: 2.39).

There was no evidence of a tree-way interaction between the factors
isolation familiarity and weaning (GLMM; F134 = 0.73, P > 0.1). Interestingly, the
mean values of amount of social investigation between familiar animals that were
weaned at 011 and social isolated (36.98 i 3.29) and unfamiliar animals from the
same weaning and isolation treatment group (35.29 :I: 4.64) seemed comparable.

The bioequivalence test showed that the means are indeed equal (P = 0.8).

85

 

Unfamiliar

N OJ A
O O 0

Social Investigation (%)

_\
O

 

 

50 _ .
Familiar
40
30

20

Social Investigation (%)

 

 

   

EW CW

Figure 1- Effects of social isolation and weaning age on the percentage of time
spent on social investigation during familiar and unfamiliar exposures in the SR
test. Dark bars indicate pre-test social isolation, light bars indicate non-isolation.
Values represent adjusted means i S.E.M. Different letters indicate difference

between treatments (P < 0.001).

4. Discussion

In this study we investigated whether 011 and 022 piglets, after being
socially isolated, differed in their ability to recognise familiar conspecifics, as
indicated by differences in their behavioural response during the SR test. We
have previously demonstrated that non-weaned piglets were able to distinguish
between familiar and unfamiliar conspecifics and that unfamiliar piglets spent
significantly more time on social investigative behaviour than familiar piglets
(Souza and Zanella, 2001; Souza et al., 2006). In our study the familiarisation
procedure occurred during the two days post-weaning. Recent reports have
shown that after weaning, piglets manifest abrupt changes in social and feeding
behaviours, signs that are frequently associated with stress (Henskin and
Jensen, 2000; Orgeur et al., 2001). Prior to our experiment we could not rule out
the possibility that weaning-induced stress would hamper social learning ability or
motivation to explore during the familiarisation period, which in turn could
eliminate possible differences in social investigation between familiar and
unfamiliar dyads. Although we did not design this experiment to investigate this
hypothesis, the outcome of the test showed that SR learning ability during the
two-day period following weaning was not impaired, in contrast with the
disrupting effect of social isolation in 011 pigs only. Our results indicated that
there is a significant difference in social investigation between familiar and
unfamiliar conspecifics, where unfamiliar dyads spent more time on social

investigation than familiar ones.

87

Our results demonstrated that SR memory of 022 pigs was not impaired 4
hours after the familiarisation procedure and that 15 minutes of social isolation
prior to testing did not significantly affect the SR retrieval per se. Isolated D11
pigs however showed possible deficits in recognising familiar conspecifics, as
indicated by a significant increase in the percentage of time spent on social
invesfigafion.

It could be argued that after social isolation the animal was more
motivated to socially investigate (see Marin et al., 2001), irrespective of whether
the subject recognized an individual or not. If so, we would expect to find an
increase in social investigation in animals weaned at 011 and 022 groups.
However, as noted only 011 animals were affected by social isolation, and only
when exposed to a familiar conspecific. Unfortunately, no conclusive remarks
can be made but it is clear that the weaning treatment had a different impact on
the social investigation time of familiar dyads which was age dependent. The
increase in social investigation may also be a result of altered general
behavioural activity caused by the social isolation rather than a deficit in
recognising familiar conspecifics. Rearing rats in social isolation post weaning
has been shown to significantly increase general locomotor activity (Heidbreder
et al., 2000; Paulus et al., 2000), whereas repeated social isolation of piglets
during the first days of life caused a decrease in their behavioural activity (Kanitz
et al., 2004). However, social isolation had no effect on general behavioural

activity regardless of weaning age, in contrast with findings by Kanitz et al.

88

(2004) and disproving the argument that the decrease in social investigation was
a side-effect of general behavioural inactivity.

Interestingly, the amount of social investigation of isolated D11 piglets was
comparable to the amounts found when unfamiliar animals were tested. These
results strengthen the hypothesis that SR in 011 pigs, but not in 022 pigs, is
susceptible to interference by social isolation prior to testing and that the
difference in social investigation found in the present study is likely to reflect
impairments in short-term SR. From now on we will explore the possibility that
weaning at 011 elicits deficits on the ability of neonatal pigs to recognize familiar
conspecifics.

Post-learning administration of GCs produces a dose-dependent effect on
memory consolidation and retrieval. Acute administration of low doses of GCs
post-learning enhances memory consolidation (Pugh et al., 1997; Roozendaal,
1999), but performance is impaired if the subjects are tested under high
circulating GC levels (Kirschbaum et al., 1996; Diamond et al., 1999; Kim and
Diamond, 2002). High GC levels shortly before testing impair retrieval of
previously learned information (de Quervain et al., 1998). In our study, although
the acute stressor was applied after the familiarisation (i.e. after acquisition and
retention), it did not significantly interfere with SR memory retrieval in 022 pigs,
but it negatively affected the ability of 011 pigs to recognise familiar conspecifics.
It is unlikely that the impairment in SR was caused by differences in the increase
of GC levels; i.e. EW leads to a higher sensitivity of the HPA-axis and an

increased glucocorticoid response to social isolation, since Laughlin and Zanella

89

(2002) found no differences between EW and CW pigs in HPA-reactivity using a
similar stress paradigm. To investigate possible mechanisms through which
weaning and/or acute social isolation may affect gene expression in the brain of
female piglets an experiment was conducted in our lab by Poletto et al. (2005).
Their results indicated that early weaning suppressed mRNA expression of
stress-related hippocampal genes, while no changes were observed in
conventionally-weaned piglets. It was suggested that the decrease in GR mRNA
in those younger animals is an indication of difficulties in reacting to increases of
GC levels. These deficits in SR memory are therefore possibly caused by a
higher sensitivity of EW animals to an increase in GC levels induced by the acute
social stressor.

Dean and Mattews (1999), studying the immature brain of guinea-pigs,
found that specifically during the brain growth spurt that occurs at 48-52 days of
gestation, increases in GCs resulted in significant changes in mRNA expression
of GC receptors in the hippocampus of females. These alterations may influence
hippocampal function, which could affect cognition under stress conditions.
Whether these changes are transitory or permanent is still unknown. In pigs, the
brain is still in development during the first few weeks after birth (Brust et al.,
2004). Pond et al. (2000) studied normal growth patterns of the pig brain and
found that the developmental peak occurs at two weeks of age. This period of
rapid growth is associated with glial cell multiplication, dendritic growth, and
synaptic connectivity (Dobbing, 1974). Interestingly, these first two weeks also

coincide with a time when the circulating cortisol levels are naturally low (Carroll

90

et al., 1998; Daniel et al., 1999). It Is therefore possible that by weaning pigs as
early as 11 days of age the animals are exposed to weaning-induced GC
elevations at a time when the brain is in a critical period of development, which,
in line with the findings in guinea pigs, may alter GC receptor levels,

subsequently increasing the animal’ 5 sensitivity to an acute stressor.

5. Conclusions

Weaning at 011 and social isolation do not impair SR per se, but socially
isolated D11 pigs show impairments in the ability to discriminate familiar from
unfamiliar conspecifics as indicated by increases in social investigation of familiar
animals to amounts comparable to those found in unfamiliar dyads. The SR
deficits after social isolation may indicate possible changes in sensitivity to stress
(hormones) in 011 pigs and this altered sensitivity to acute stress may be caused
by the age at which the developing brain is exposed to increases in stress
hormones. Further research is necessary to investigate possible mechanisms
through which 011 may affect both brain normal development and stress
sensitivity. From a welfare point of view, it should be further investigated whether
the increased stress sensitivity is long lasting or if it will compromise pig welfare

throughout life.

Acknowledgments

This research was supported by USDAINRI grant # 2001-35204-10810

(A.J.Z) and the Michigan Agricultural Experiment Station. The authors wish to

91

thank Jarno Jansen and Patricia E. Almeida for helping with the data collection
and personnel of the MSU Swine Teaching and Research Center for assisting
with animals care. We are also grateful to Michael Mendl, Jarno Jansen, and
Liesbeth Bolhuis for their useful comments on an earlier version of this

manuscript.

92

CHAPTER 6

GENERAL DISCUSSION

93

The primary goal of this thesis was to investigate the effects of acute
stressors on social recognition (SR) in neonatal pigs in order to determine
whether impairments in the SR process could be responsible for the increased
post-mixing aggression in early-weaned pigs that was previously reported by our
laboratory (Yuan et al., 2004). Because the animals were tested at a very young
age (starting at 11 days of age), experimental approaches based on a
spontaneous behavioral response, i.e. the amount of social investigation, were
chosen, since this does not require extensive training of the animals.

To achieve the main goal, two protocols for assessing SR were tested
(chapter 2 and 3). The novel SR paradigm proposed in chapter 3 proved to be
appropriate for use in neonatal pigs and was therefore further used to investigate
possible interference of an acute stressor on SR of early-weaned (EW) and
conventionally-weaned (CW) piglets. If weaning at a very young age has a
negative impact on social recognition abilities, it may also lead to deficits in the
formation and maintenance of stability in social groups.

In this chapter, the results are briefly described and compared to other
results generated in our lab. Subsequently, based on these results, interesting

future studies are presented and concluding remarks are drawn.

1. Summary of the results
In the first study (chapter 2), the ‘habituation-dishabituation’ paradigm was
used to assess SR in neonatal pigs. As this method was primarily designed for

adult rodents, special attention was given to the acclimatization of the animals to

94

the apparatus and procedures so that the method would inflict minimal
disturbance of the animals. The goal was to examine whether acute increases of
cortisol impair short-term SR in 11-day old pigs. The results showed that while an
intra-muscular hydrocortisone injection administered 15 minutes prior to testing
significantly increased salivary cortisol levels, it did not interfere with the ability of
the animals to acquire and retrieve information about a male stimulus pig during
successive exposures. Although the results indicated that this paradigm can be
further used in pigs (as the subjects were successfully habituated to the
stimulus), the protocol caused considerable distress to the experimental animals
that could possibly compromise the outcome of the main study focusing on
interference of acute stress on SR. If conditions of testing impose stress or fear
to the animals it may lead to disregulation of behavioral and cognitive functions
and misleading results, aside from the ethical implications for the animals (pigs:
Mendl et al., 1997; chicks: Marin et al., 2001; cattle: Grandin et al., 1994;
rodents: Kogan et al., 2000). In addition, exogenous GCs did not affect SR as
tested in this paradigm. We therefore decided that in future studies, a more
natural stressor, such as social isolation, should be used. This was, however, not
further investigated, since Laughlin and Zanella (2003) had already shown that
15 minutes of social isolation increased salivary cortisol to levels hampering
spatial memory in early weaned piglets.

In the second study (chapter 3), a novel protocol to test SR in pigs was
presented. The major accomplishments of this study were that fighting during the

familiarization (habituation) phase was prevented, thus reducing distress of the

95

animals, and that this familiarization procedure resulted in clear differences in
time spent on social investigation between familiar and unfamiliar pairs during the
testing phase, indicating that neonatal pigs were able to retrieve information
about familiar conspeclfics up to 4 hours after the familiarization. Additionally,
relocation of the animals to similar farrowing crates, between the familiarization
and testing phase did not affect SR test in terms of time spent on social
investigation. This test therefore proved to be more appropriate for assessing the
effects of an acute stressor on SR abilities in neonatal pigs that was the aim of
this thesis.

Using the familiarization procedure described in the previous study,
another study was designed to assess whether the novel protocol could be used
to assess a more robust SR In 20-day-old female pigs (chapter 4). In this
experiment, the subjects were ‘testwed at 4 and 24 hours after the last
familiarization procedure. The results revealed that the familiarization protocol
enabled the animals to successfully remember familiar conspecifics for at least
24 hours after familiarization. This finding is very interesting, since it will allow
future studies to further elucidate memory processes in young pigs by using
pharmacological agents or various disturbances at different stages of the
memory process. In addition, it will also allow for more in depth testing of a
relation between familiarization and later aggression, since animals can
potentially be familiarized at a very young age and mixed at much later ages (e.g.

D’Eath et al., 2005).

96

Chapter 5 then presented a study investigating whether weaning age (EW
at 11d and CW at 21d) and/or acute social isolation (15 minutes) interfered with
the ability of female piglets to remember familiar conspecifics. During interactions
with familiar animals, isolated EW pigs investigated the conspecific more than
non-isolated EW and all CW pigs. Interestingly, the amount of social investigation
displayed by familiar isolated EW pigs was similar to that displayed by unfamiliar
animals. There was no sign of differences in general locomotion between
treatment groups. These findings therefore provide strong evidence that acute
social isolation impaired SR ability of EW pigs, providing a second potential
cause of the increase in post-mixing aggression in EW pigs reported by Yuan et
al(2004)

The results of all studies have been described and discussed in previous
chapters, and at the end of this thesis, I will only aim to integrate these findings
with the results of other studies done in our lab. I have worked with other
members of the group towards providing a clearer picture of the early weaning
process and its effects on young pigs, but do all the results fit or do they
contradict? By discussing these findings, I intend to clarify the results of the initial
USDAINRI grant (# 2001-35204-10810), form new ideas on underlying
mechanisms and propose hypotheses and future studies aimed at testing these

ideas.

97

2. Stress susceptibility in early-weaned pigs

As previously mentioned in chapter 1, the increasing intensification of
husbandry systems has massively changed the natural course of the weaning
process for a piglet. In commercial farms, artificial weaning of piglets involves
simultaneous separation from both the nutrient supply and social contact with the
sow, and it occurs at much younger ages and much more abrupt than in more
natural environments.

Modern pig production systems have developed early weaning systems in
which pigs are weaned before 21 days of age. It is, therefore, important to
integrate the scientific knowledge on the effects of early weaning on different
biological functions (neurophysiological, behavioral, and cognitive aspects) of the
animal, as was done in our lab. By combining these results, the impact of early
weaning for the animal will become clearer, and general hypotheses on the
underlying mechanisms, as well as some future studies to test these hypotheses,
can be provided.

The research project aimed at investigating how early weaning, as a
model for early stress, affected post-mixing aggression, cognitive functions
(social and spatial learning), and brain processes (gene expression). The pioneer
study was conducted by Yuan et al. (2004), who found that EW pigs fought
longer and initiated more fights that they did not subsequently win than CW
animals, when they were regrouped at 9 weeks of age. Initially, this was
presented as a welfare concern, since pigs at this age are able to inflict serious

injuries during fighting, but with the development of a porcine brain library (Nobis

98

et al., 2003), more central processes could be studied that would be important for
both animal welfare and fundamental knowledge on central stress physiology.

Based on Yuan et al.’s (2004) hypothesis, we aimed at investigating
whether early weaning would alter stress sensitivity in pigs and how this would
reflect in both behavior/cognition and brain physiology. If EW animals were more
susceptible to the harmful effects of an acute stressor, this could be tested with
cognitive tasks. To this end, the Morris Water Maze (MWM) and the habituation-
dishabituation paradigms were adapted for use in neonatal pigs. A study
conducted by Laughlin and Zanella (2003) indicated that social isolation of EW
pigs impaired their performance in the MWM task at 12 and 23 days of age.
Interestingly, this impairment was only observed in EW pigs that were socially
isolated 15 minutes prior to testing, whereas performance was not affected in
non-isolated EW animals or isolated CW pigs. Furthermore, no differences in the
cortisol response to isolation were found, indicating that the differences could not
be explained by alterations in HPA-axis functioning, but may instead be the result
of changes in central sensitivity to GCs. Since the MWM task is a known
hippocampus-dependent learning task, the hippocampus was indicated as a
likely candidate for these changes in central sensitivity.

As was presented in this thesis, the results of the SR are in agreement
with those of the MWM task. Again, since the hippocampus seems to be involved
in SR processes, more support was obtained for a role of the hippocampus in the
adverse effects of early weaning on pig behavior. Furthermore, these results

provided direct support for the hypothesis that the increase in post-mixing fights

99

reported by Yuan et al. (2004) was possibly caused by SR deficits in acutely
stressed EW animals. More specifically, difficulties in recognizing or at least
distinguishing stronger opponents seemed to support their finding that EW
animals initiated more fights that they ended up not winning.

Thus, now two cognitive tasks in which the hippocampus might play a role
indicated that early weaning in combination with acute stress hampered
performance. To investigate possible mechanisms through which weaning and/or
acute social isolation may affect gene expression in the brain of female piglets an
experiment was conducted by Poletto et al. (2005). Their results indicated that
early weaning suppressed mRNA expression of stress-related hippocampal
genes, while no changes were observed in conventionally-weaned piglets. It was
suggested that the decrease in GR mRNA'in early weaned animals is an
indication of difficulties in reacting to increases of GC levels. This hypothesis was
further explored in our lab in an in situ hybridization study that explored cortisol
receptor mRNA in the hippocampus. The hippocampus expresses both MR5 and
GRs (Veldhuis et al., 1982). Hippocampal MRs bind GCs with a 10-fold higher
affinity than GRs (Reul and De Kloet, 1985). As a result, MRs are extensively
occupied under basal resting conditions, while substantial GR occupation occurs
in circadian peak and following stress (de Kloet et al., 1993). A theory of balance
of these receptors was developed by de Kloet, which states that an imbalance in
MR- and GR-mediated effects would alter individual-specific vulnerability to
stress (de Kloet, 1991). In other words, the ratio of MR:GR in the hippocampus

may determine sensitivity to GCs. By subjecting hippocampal slices of early-

100

weaned and conventionally-weaned piglets to in situ hybridization for GR and MR
mRNA, the MR:GR ratio could be calculated. Unfortunately, methodological
difficulties have so far hindered this experiment, but the question may in the
future be addressed by immunocytochemistry.

Altogether, the experiments on the effects of early weaning on post-mixing
aggression, spatial learning, SR, and brain gene expression indicated a possible
higher susceptibility of EW pigs to acute stressors (i.e. mixing, social isolation),
possibly due to a shift in receptor ratio that alters the optimal GC concentrations
for cognitive functioning (as theorized on in the inverted-U-shape models).

Future directions for further exploring the early weaning process seem to
be directed in two ways. Firstly, while we have provided a mechanism that might
explain why 50-day-old early weaned pigs initiate more fights that they end up
not winning, we have not actually tested whether the effect of an acute stressor
on the cognitive ability of pigs is actually altered at this age. It would therefore be
of interest to further adapt the social recognition protocol for use in 50-day-old
animals and test early- and conventionally weaned animals at this age. By
familiarizing the animals at a younger age, it may even be investigated whether
an acute stressor affects both a relatively short (4h) and long (24h) memory, or
whether only short-term memory is affected.

Secondly, while we have hypothesized a neurophysiological mechanism
responsible for the cognitive impairments in early-weaned pigs, and have
provided a basis for the disruption of this mechanism, we have not actually tested

it. The study carried out by Poletto et al. (2005) suggests that early weaning may

101

cause changes in the brain, but it did not provide a specific location, let alone a
biological mechanism. These are issues that should be addressed before any
concluding remarks on the effects of early weaning on the brain can be made.

Although the development of a novel SR protocol was only a step towards
answering a research question, the results offer very interesting possibilities. In
an applied setting, it would be interesting to see whether the familiarization
protocol as presented in this thesis would be able to reduce post-mixing
aggression at later ages. This is a study that has already been carried out in our
lab (Souza, Siegford, Jansen and Zanella, in preparation), but unfortunately
could not be presented in this thesis.

In a more fundamental setting, the SR test allows for testing the effects of
natural and pharmacological disturbance of various stages of memory formation
and allows further testing of the importance of different sensory cues for memory
formation. By administering drugs prior to familiarization, the effect of that drug
on acquisition can be tested, while isolating the animal immediately afier
familiarization allows investigating the role of stress on memory consolidation.
Furthermore, by replacing the flexible netting with a plastic see-through plate or
an opaque plastic barrier with small holes, we could test whether visual or
olfactory cues are more important for SR in pigs. Small adaptations in the SR
testing protocol may further clarify SR abilities of young piglets; since the
familiarization was done between litters, and testing between individuals, it
cannot be concluded that the animals recognized one another based on

individual cue or on cues shared by its litter litter. If during testing, the subject

102

would be exposed to an unfamiliar animal from a familiar litter (by, for instance,

only subjecting half the litter to Fam1), this question could be answered.

3. Concluding remarks

In conclusion, this thesis indicates that:

1. Short-term SR of neonatal pigs can be assessed using the habituation-
dishabituation technique.

2. A relatively short— and long-term SR of neonatal pigs can be assessed with
minimal disturbance by using a newly-developed protocol.

3. Early and conventional weaning do not impair the ability of pigs to
recognize familiar conspecifics per se, nor does acute social isolation by itself
impair the ability of newly weaned pigs to recognize conspecifics.

4. Acute social isolation may hamper the ability of female EW pigs to retrieve

information about familiar conspecifics, but does not affect SR in CW pigs.

103

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104

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