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SEGREGATED EARLY WEANING STRESS HAS ACUTE AND LONG-
LASTING EFFECTS ON GROWING PIGS

BY

Yan Yuan

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1999

 

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ABSTRACT

SEGREGATED EARLY WEANING STRESS HAS ACUTE AND LONG-LASTING
EFFECTS ON GROWING PIGS

BY

Yan Yuan

The North American swine industry is rapidly adopting a
segregated early weaning (SEW) system with little
information about potential welfare implications. In this
study, the welfare of SEW pigs was examined by studying the
development of abnormal behavior, agonistic interactions,
and cortisol concentrations post-weaning. Two trials were
conducted to investigate the short— and long—term effects
of SEW. Urinary cortisol stayed high in SEW pigs up to
three day post—weaning (P S 0.0339). Other responses
included high vocalization rate, an increase in activity
levels and the exhibition of belly-nosing. The long—term
effects were evidenced by higher level of belly-nosing (P S
0.0142), higher proportion of fights with no clear outcome
(P = 0.0430), and longer duration of fights (P = 0.0075) in
SEW pigs than in conventionally weaned pigs. In summary,
the behavioral and physiological measures indicate that the
welfare of SEW piglets may be poorer than conventionally

weaned piglets.

To the love and support of my parents —— Yuan, Xiaoping and
Yao, Jinmei. To the love, friendship and help of my husband
—— Qian, Jin

iii

ACKNOWLEGMENTS

I deeply appreciate my major advisor, Dr. Adroaldo
José Zanella, who has guided me through the Master program.
I am indebted to of my guidance committee members Dr.
Jeanne Burton, Dr. Mathew Doumit, and Dr. Cheryl Sisk, who
have also provided valuable help in my research. Special
thanks go to Dr. Jeanne Burton, who has helped me
throughout the thesis writing process.

All members of Dr. Zanella’s lab have made significant
contribution to my thesis research and thesis writing. They
are Dr. Miyuki Tauchi, Miss Debbie Charles, Mrs. Amy
Shelle, Miss Elissette Rivera, Miss Jennifer D'Agostino,
Mr. Joreci Federizzi, Dr. Telmo Oleas, Dr. Madonna
Benjamin, Mrs. Heidi Doza, and Mr. Domenic Perri.

Finally, I would like to thank Dr. Robert Tempelman
for statistical consultation, and the Rackham Foundation

for partial financial support.

iv

TABLE OF CONTENTS

 

 

 

 

 

LIST OF TABLES .......................................... Vii
LIST OF FIGURES ........................................ viii
CHAPTER 1 INTRODUCTION .................................... 1
CHAPTER 2 LITERATURE REVIEW ............................... 6
2.1 Segregated Early Weaning System ...................... 6
2.2 Stress Response ...................................... 8
2.2.1 Stress and Stressor ................................. 8
2.2.2 Behavioral Response to Stress ...................... 10
2.2.3 Physiological Response to Stress ................... 12
Autonomic Nervous System and Neuroendocrine
Systems ............................................. 12
Hypothalamus- Pituitarv- Adrenal Axis ................. 14
2.2.4 Cortisol as an Physiological Indicator of
Stress .............................................. 16
Plasma Cortisol ..................................... 16
Salivary Cortisol ................................... 18
Urinary Cortisol .................................... 21
2.3 Segregated Early Weaning Stress ..................... 23
2.3.1 weaning Stress ..................................... 23
2.3.2 Transportation Stress .............................. 25
2.4 Summary ............................................. 26
CHAPTER 3 HYPOTHESES AND OBJECTIVES ...................... 28

CHAPTER 4 EXPERIMENT l: IMMEDIATE RESPONSE OF NEONATAL

 

PIGS TO SEGREGATED EARLY WEANING ......................... 31
4.1 Introduction ........................................ 31
4.2 Material and Methods ................................ 32
4.2.1 Animals and Management ............................. 32
4.2.2 Behavioral Observations ............................ 34
4.2.3 Urine Sampling ..................................... 34
4.2.4 Urine Cortisol and Creatinine Analysis ............. 36
4.2.5 Statistical Analysis ............................... 37

Statistical MOdel ................................... 39
4.3 Results ............................................. 40
4.4 Discussion .......................................... 44
4.5 Conclusion .......................................... 49

CHAPTER 5 EXPERIMENT 2: LONG-TERM EFFECTS OF SEGREGATED

 

EARLY WEANING STRESS ON PIGS ............................. 50
5.1 Introduction ........................................ 50
5.2 Material and Methods ................................ 51
5.2.1 Animals and Management ............................. 51
5.2.2 Behavioral Observations ............................ 54
5.2.3 Saliva Sampling .................................... 55
5.2.4 Salivary Cortisol Analysis ......................... 56
5.2.5 Statistical Analysis ............................... 56
Statistical MOdel ................................... 57
5.3 Results ............................................. 58
5.4 Discussion .......................................... 66
5.5 Conclusion .......................................... 72
CHAPTER 6 GENERAL DISCUSSION ............................. 73
6.1 Behavior ............................................. 73
6.1.1 Belly-nosing ....................................... 73
6.1.2 Aggression ......................................... 75
6.2 Responsiveness of Hypothalamic—Pituitary—Adrenal
Axis ................................................. 78
6.3 Behavior and Hormones ................................ 80
6.4 Animal Welfare and Segregated Early Weaning .......... 81
APPENDIX ................................................. 85
REFERENCES ............................................... 89

W

LI ST OF TABLES

Table 1. Description of behavior categories in piglets..

Table 2. Mean Wilcoxon rank sum score (iSEM) in SEW
piglets and control piglets (not weaned) for frequency
of grunting, climbing, and duration of moving

immediately after and in the first day post weaning .....

Table 3. Mean Wilcoxon rank sum score (iSEM) in SEW
piglets and control piglets (not weaned) for frequency
of belly-nosing, duration of rooting, and duration and

frequency of fighting in days 1, 2 and 3 post weaning...

Table 4. Summary of basic statistics on behavioral and

physiological data in Experiment 1 ......................

Table 5. Summary of basic statistics on behavioral and

cortisol data in Experiment 2 ...........................

Table 6. Summary of body weight of SEW and CW piglets

in Experiment 2 .........................................

vii

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LIST OF FIGURES

Figure 1. Mean weight of SEW and control piglets before
and a week after weaning treatment ....................... 40

Figure 2. Average percentage of time spent lying from

0830 to 1530 h in SEW piglets over three days post

weaning, with reference to control piglets that stayed
with the sows ............................................ 43

Figure 3. Mean urinary cortisolzcreatinine ratio in
SEW and control piglets during pre— and post-weaning
period ................................................... 43

Figure 4. Size and design of a nursery pen ............... 53

Figure 5. Average body weight of SEW and CW piglets at
weeks 5, 6 and 8 of age .................................. 59

Figure 6. Average frequency of (a) belly-nosing, (b)
manipulating, (c) fighting, and (d) bar biting from

0900 to 1800 h in SEW and CW piglets at weeks 5, 6 and

7 of age ................................................. 60

Figure 7. Mean salivary cortisol concentration before
and after a 20-minute transportation in SEW and CW pigs
at 9 weeks of age ........................................ 63

Figure 8. The distribution of clear and unclear outcome
of fights in three days after regrouping when SEW pigs
were mixed with CW pigs at 9 weeks of age ................ 63

Figure 9. The distribution of clear and unclear outcome
of fights in three types of encounters when SEW pigs
were mixed with CW pigs at 9 weeks of age ................ 64

Figure 10. Average duration of fights when outcomes
were not clear in days 1, 2 and 3 after the mixing of
SEW and CW pigs at 9 weeks of age ........................ 64

Figure 11. Behavioral sequences of fighting from the

first bite to the first sign of submission for young
pigs. Body—turning was always preceded by ear bites ...... 77

viii

Chapter 1 INTRODUCTION

From 1988 to 1997, the number of swine farms in the US
has decreased from 326,600 to 138,690. However, swine farms
that produced 50,000 or more pigs annually increased their
market share from 7% to 37% during this time (1998 Pork
Industry Study by National Pork Producer Council). This
could be largely attributed to the implementation of multi-
production sites by large swine farms in order to manage
the larger number of animals per farm. For small swine
farms, survival strategies in today’s competitive market
have included formation of small cooperative operations and
participation in production contracts with larger
companies. Regardless of the strategy that small swine
farms implement, each individual farm has a special
function. For example, some farms primarily deal with
breeding herds and nursing piglets until they are weaned
and sold. Others raise the weaned piglets until they reach
a weight of 25 kg and/or raise the animals until they reach
market weight. These cooperative alliances or production
contracts are very similar to a multi-site operation of a
single large farm.

The existing multifunctional cooperative setting makes

it easy for the swine industry to adopt the segregated

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early weaning (SEW) system. The SEW system is a multi-site
production system in which piglets are weaned at a very
early age, usually between 10 to 19 days after birth, and
moved to a separate site for the initial growth period.
This system reduces the pig’s exposure to pathogens
compared to conventional weaning practices and has been
rapidly adopted by the North American swine industry
(Harris, 1988; Dritz et al., 1996). In a conventional
weaning system, piglets are weaned between 21 to 28 days of
age to an adjacent nursery room or building. The nursery
site has piglets moving in and out on a regular basis,
which makes it difficult to maintain a clean site and
prevent pathogens from passing from one group of pigs to
another.

Despite the potential benefits of preventing
infectious disease which results from the improved
cleanliness of the nursery housing in SEW, the welfare
implications of SEW have been ignored. Welfare is defined
as the state of an individual in regard to its attempts to
cope with its environment (Broom and Johnson, 1993). Since
stress implies difficulty in coping, the presence of stress
is regarded as a threat to the welfare of an animal.

Weaning (Hinde, 1966; Wurbel and Stauffacher, 1997) and

 

 

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transportation (Parrott and Misson, 1989; Baldock et al.,
1990) can be stressful to animals.

Stress has universal and multi-organ effects. Stress,
especially chronic stress, can result in sub—optimal immune
function, a delay in puberty, and a reduction in brain
hippocampal volume among many other malfunctions (Munck et
al., 1984; De Jonge et al., 1996; Bremner et al., 1995).
With a suppressed immune system, animals are not able to
properly respond to vaccines and disease challenges. The
onset of puberty is an important indicator of reproductive
fitness, and its delay could be an indicator of poor
welfare. The atrophy of the hippocampus is associated with
behavioral and memory problems (Bremner et al., 1995).

Of all these potential detrimental effects of stress
during the precocious weaning age, development of abnormal
behavior is the most consistent and long-term problem in a
variety of species, including mink (Mason, 1994), rats

(Wfirbel and Stauffacher, 1997), and pigs (Gonyou et al.,
1998L.“Abnormal” is defined as being statistically rare or

different from a chosen population which is living free or
in naturalistic conditions in captivity (Fraser and Broom,
1990). The occurrence of behavior abnormalities has been
used as an indicator of poor welfare (e.g. Broom and

Johnson, 1993). It concerns the public when they witness

the exhibition of abnormal behavior. The pacing behavior of
zoo animals, such as the red panda, tiger and polar bear,
is an example (Poulsen et al., 1996; Lyons et al., 1997).

Currently, the swine industry is harshly criticized on
environmental issues such as manure management. As a
result, pig producers have spent substantial amounts of
money to comply with new laws on manure management. If the
pork industry is not prepared, SEW may become an easy
target for criticism by welfare groups. An immediate
concern in the pork industry is that some abnormal
behaviors, such as belly-nosingl, and ear and tail biting
may decrease the carcass value (Hunter et al., 1999). In
the future, pork producers may have to change their
management practices in order to deal with the SEW welfare
concerns.

This research was conducted to examine the welfare of
SEW piglets. Behavior and physiological measures in
neonatal piglets can provide information on the short- and
long-term effects of the SEW stress. The hypothesis is that
neonatal piglets will respond behaviorally and

physiologically to SEW stress. This stressful experience

 

1 Belly-nosing is defined as a rhythmic up~and-down movement
with the snout on the belly or soft tissue between the hind
or forelegs of another pig (Dybkjer, 1992).

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will have long lasting effects as measured by increased
aggression among piglets, increased behavioral
abnormalities, impaired ability to establish social
hierarchy, and different physiological responses to an
acute stressor at an older age when compared to
conventional weaned pigs.

To my knowledge, this work is the first of its kind in
swine which integrates the behavioral and physiological
measures, and the results may be valuable to the pork
producers who are adopting or considering to adopt the SEW
system. In addition, the results may provide insight on how
neonatal stress may influence behavior and physiology in

animals.

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Chapter 2 LITERATURE REVIEW

2.1 SEGREGATED EARLY WEANING SYSTEM

The segregated early weaning (SEW) concept was
developed from the early work of Alexander et a1. (1980).
In their study, sows and their litters were medicated, the
piglets were weaned at about five days of age and moved to
a strictly isolated site. At the end of the trial, herds
raised by this method achieved a high health status and
gained more weight than conventionally raised herds. Recent
research showed that these health benefits can be
maintained when sows are not isolated or medicated, and
weaning occurs at 10 to 19 days (Harris, 1988; Dritz et
al., 1996). The success of SEW was not widely acknowledged
until recently. Reasons for the delayed acceptance of SEW
included understanding the nutritional needs of the
piglets. Diets had to be developed for immature digestive
system of neonatal piglets.

The objective of SEW is to control diseases by
reducing vertical and horizontal microbial contamination.
Vertical contamination refers to the spread of bacterial
and/or viral infections from sows to piglets. One way to
reduce the risk of spreading infectious diseases is to move

piglets away from the sow at an early age to a separated

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site. Horizontal contamination is defined as the spread of
infection from one group of pigs to another. This can be
controlled by using the all-in-all-out settings, where pigs
are placed in and removed together from rooms, whole
buildings, or complete sites (Alexander et al., 1980; Dritz
et al., 1996). SEW is a combination of the following two
practices: neonatal weaning to a separated site, and all-
in—all—out management.

Since neonatal weaning and transportation are
integrated in practical SEW systems, these two factors were
not separated in my research. As a result, there are at
least four stressors associated with SEW in this work:
maternal deprivation; nutritional deprivation; exposure to
an unfamiliar environment; and transportation. Among them,
maternal deprivation, nutritional deprivation and exposure
to an unfamiliar environment that accompany weaning are
referred to together as “weaning stress".

Few studies have examined the behavior of piglets
weaned at 2 weeks of age or younger. The occurrence of
abnormal behaviors, such as belly-nosing, increases as
weaning age decreases (Metz and Gonyou, 1990; Gonyou et
al. , 1998) .

In addition, some evidence suggests that the sow may

not be ready for weaning at an early stage of lactation.

After piglets were removed at an early age, sows were more
active, vocalized at a much higher rate, and returned
repeatedly to the piglets resting area (Pajor et al.,
1996). They also appeared to encounter some reproductive
problems such as a delay in return to estrus and a
reduction in subsequent litter size (Koketsu and Dial,
1997).

Due to the large size of SEW herds, there is a concern
that infectious diseases can spread very rapidly and affect

more animals than in smaller conventional herds.

2.2 STRESS RESPONSE
2.2.1 Stress and Stressor

Physiologists consider stress to be a biological
phenomenon. Psychologists argue that stress is a
psychosocial pressure being consciously sensed by an
individual. In many cases, stress has been defined as
either a behavioral or physiological response. In general,
stress is considered to be the physical and psychological
state of animals in response to environmental,
physiological and psychological stimuli. The definition
proposed by Broom and Johnson (1993), however, can best
describe the stress associated with weaning and

transportation of piglet. In their definition, stress is a

physical, environmental and/or psychological effect on an
individual that overtaxes its control systems and reduces
its fitness or appears likely to do so. From this
viewpoint, stress is induced by noxious environmental
stimuli and its occurrence implies a failure to cope. Since
the individual’s welfare is poor when it finds coping
difficult, stress invariably indicates poor welfare, but
welfare can be poor without stress (Broom and Johnson,
1993). On the other hand, the success of coping behavior
can be measured by its effectiveness in reducing
physiological measures of stress or by its effectiveness in
reducing the effect of aversive stimuli and thus restoring
fitness (Wechsler, 1995).

Stress responses vary among individuals and depend on
the following elements: species; the genetic make up; early
life experience; type and severity of the stressor; the
ability to cope with the situation; and experience
involving the same type of stressor (Plomin et al., 1994;
Steinhausen, 1994; Plotsky and Meaney, 1993; Levine, 1985).

Stressors can be physical, environmental, or
psychological in nature. Physical stressors include tail—
docking, castration and physical restraint. Environmental
stressors include extreme temperatures, infections.

Psychological stressors include maternal separation and

social isolation in social animals. Though the stressors
are different, they activate three major biological
systems: behavior, autonomic nervous system, and

neuroendocrine systems (Moberg, 1985).

2.2.2 Behavioral Response to Stress

vwmnian animal faces challenges, the most apparent
change is in its behavior. Acute stressors can induce fear—
related, anxiety—related, or frustration-related behaviors.
Fear responses in pigs include trembling body, moderately
loud vocalization, unnatural huddling together, climbing on
other pigs, and increased frequency of defecating and
urinating (Brundige, 1998).

The long-term behavioral consequences of stress in
animals include the occurrence of destructive, stereotypic,
redirected, aggressive, inactive, and escape behavior
(Fraser and Broom, 1990). Destructive behaviors are those
that cause damage to other animals, such as feather pecking
in poultry or tail biting in pigs. Stereotypic behaviors
are rhythmic, repetitive, and invariant behaviors without
apparent function or goal (Mason, 1991). Examples are head—
rolling in caged mink and sham chewing in tethered sows.
Redirected behaviors are normal behaviors either in

abnormal frequency or directed to a substitute object.

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Examples are increased drinking and massaging of penmates
in pigs kept in crowded environments (Dybkjer, 1992).
Aggressiveness is related to the environment in which the
animals are raised. De Jonge et al. (1996) showed that
piglets reared in farrowing crates displayed more
aggressive behaviors than piglets reared in outdoor pasture
with half—open crates. Inactive behaviors include
inactivity, apathy and unresponsiveness. Tethered sows
responded much less than group housed sows to environment
stimuli (Broom, 1987).

Destructive, stereotypic, redirected and inactive
behavior are considered abnormal. In the introduction, I
have introduced the definition of “abnormal”, which is
“being statistically rare or different from a chosen
population which is living free or in naturalistic
conditions in captivity” (Fraser and Broom, 1990).
Behavior abnormalities are the most focused issue in the
area of applied animal ethology and animal welfare. These
behaviors are widely spread in confined animals and can be
induced by aversive situation such as unpredictable and
uncontrollable draught (Broom, 1991; Scheepens et al.,
1991). It is suggested that the development of abnormal
behavior may result from the failure to cope with normal

behavior (Wechsler, 1995). Some studies indicate that

11

abnormal behaviors are associated with a reduction in
physiological measures of stress and can thus be regarded
as successful coping behavior (Wiepkema et al., 1987,
Mittleman et al., 1991). However, the relationship between
abnormal behavior and physiological measures of stress is
not unequivocal. For example, Terlouw et al. (1991)
reported no correlation between the exhibition of post-
feeding stereotypy and plasma cortisol levels in sows.
Furthermore, in a pain-sensitivity test, the sows with high
levels of stereotypy had shorter tail-flick latencies than
sows with low levels of stereotypy. This result suggests
that the performance of stereotypic behavior does not
narcotize the animal. Although the results of different
studies on abnormal behavior and physiological measurement
are not consistent, most researchers agree that abnormal
behavior indicates difficulties in coping, and therefore is
an important indicator of welfare (e.g. Wiepkema, 1983;
Fraser and Broom, 1990; Barnett and Hemsworth, 1990;

Wechsler, 1995).

2.2.3 Physiological Response to Stress
Autonomic nervous system and neuroendocrine systems
Besides the behavioral changes, biochemical changes

also take place when an animal responds to stress. The

12

autonomic nervous system (ANS) and neuroendocrine systems
are major pathways that are activated to regulate these
processes in stress responses.

The ANS has three central components, hypothalamus,
brain stem, and spinal cord. The peripheral ANS is
functionally composed of the sympathetic and
parasympathetic systems. During a stressful situation, the
sympathetic activity is enhanced leading to an increased
heart and respiratory rate, shunting of blood supply from
the viscera to the muscles and the brain, and margination
of lymphocytes. These prepare the body with the
physiological measures needed to handle an emergency
situation. The sympathetic activity also stimulates the
neuroendocrine system, which releases catecholamines
(norepinephrine and epinephrine), and further strengthens
the sympathetic activity (Rhoades and Pflanzer, 1996).
However, it is difficult to measure hormonal responses
associated with sympathetic activity because the
concentration of plasma hormones returns to basal levels
very quickly. Thus, alternative indicators of stress need
to be identified.

The hypothalamic—pituitary—adrenal (HPA) system, the
hypothalamic-pituitary-gonadal (HPG) system, and

catecholaminergic system are of most interest in stress

13

 

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research (Levine, 1985). The catecholamine responds to
conditions that require attention and vigilance. The HPG
system is mainly responsive to situations involving social
behavior, such as dominance. The HPA response occurs in
situations involving novelty and uncertainties, where the
animal perceives that it can not control its surroundings

(Levine, 1985).

Hypothalamus-pituitarv-adrenal axis:

Historically, the HPA axis has received the most
attention in stress research. This can be credited largely
to Hans Selye, a pioneer and authority in the modern stress
research, who interpreted stress as the nonspecific
response of the body to any demand made upon it (Selye,
1956). The HPA axis is activated during a variety of
situations such as injury, social interaction, and physical
restraint (Klemcke and Pond, 1991; Becker et al., 1985).
Therefore, Selye advocated that the activation of HPA axis
is a component of a nonspecific, stereotyped reaction to
stress (Selye, 1973).

Once a stimulus is perceived to be threatening by the
central nervous system (CNS), the hypothalamus of the brain
releases corticotropin releasing hormone (CRH, also known

as CRF). CRH reaches the pituitary gland via the

14

hypophyseal stalk and triggers adrenocorticotropic hormone
(ACTH) secretion from the anterior pituitary into the blood
(Vale, 1981). When circulating ACTH reaches the adrenal
gland, it stimulates the synthesis and release of adrenal
glucocorticoids, cortisol and corticosterone.
Glucocorticoids are the final effectors of the HPA
axis and participate in the control of whole body
homeostasis (Stratakis and Chrousos, 1995). These steroid
hormones promote the transformation of non-sugars into
sugars, glycogen storage, and protein and lipids
decomposition (Rhoades and Pflanzer, 1996). Through these
processes, the amount of glucose available to the CNS,
heart and skeletal muscles is increased, which enables the
animal to handle potential threatening situations. Thus,
the activation of HPA axis and the elevation of
glucocorticoid concentrations during a stressful event are
normal and necessary functions of the body. When in high
concentration, glucocorticoids also play a key role in
terminating the stress response by exerting negative
feedback involving ACTH and CRF at the level of pituitary
and hypothalamus (Munck et al., 1984; De Kloet, 1991). In
addition to their effects on carbohydrate metabolism and
neuropeptide regulation, glucocorticoids suppress

inflammation, delay the growth of new tissue, inhibit

15

 

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neutrophil migration and production of antibodies by B
lymphocytes, inhibit cytokine production and activity, and
lower lymphocyte counts (Munck et al., 1984; Burton and
Kehrli, 1995, 1996). In general, glucocorticoids suppress
normal defense mechanisms.

Chronic release of glucocorticoids results in
decreased growth rate, suppressed immune—inflammatory
reactions, suppressed reproductive function in both sexes,
and progressive neuronal cell loss in the hippocampus
(Stratakis and Chrousos, 1995; Sapolsky et al., 1985). In
summary, the HPA axis profoundly influences growth, immune
function, reproduction, and the thyroid axis (i.e.
metabolism), which are important parameters of fitness

(Stratakis and Chrousos, 1995).

2.2.4 Cortisol as Physiological Indicator of Stress
Glucocorticoids have frequently been used as
indicators of HPA axis activity. Cortisol is the
predominant glucocorticoid produced in dogs, cats, pigs,
sheep, and horses (reviewed in Stephens, 1980). Cortisol is
a small, hydrophobic molecule produced by the adrenal
cortex of the adrenal gland, and is detectable in all body
fluids including plasma, saliva, and urine. In piglets, the

urinary cortisol half—life is long enough (6.0 i 0.6 hour)

16

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s

 

 

 

 

 

 

 

 

 

so that it can be used as an indicator of stress (Kraan et

al., 1986).

Plasma cortisol

Most of the circulating cortisol is bound to plasma
proteins. In humans, approximately 80-90% of circulating
cortisol is bound to cortisol-binding protein (CBG), 5—10%
is loosely bound to albumin, and about 4.0% is unbound
(Brody, 1994). In pigs, approximately 9% of cortisol is
unbound (Cook et al., 1996). Unbound cortisol is the
physiologically active form.

Plasma cortisol concentration provides a reliable tool
for the assessment of HPA axis activity and estimates the
difficulty for animals to cope with short-term problems.
Mice being exposed to three increasingly unfamiliar
environments respond correspondingly with elevations in the
plasma glucocorticoids level (Hennessy and Levine, 1978).
The severity of surgical procedures used in sheep (Shutt et
al., 1987) also resulted in increased levels of plasma
glucocorticoids.

In pigs, plasma cortisol also increases during
stressful events. These events can be maternal separation,

social isolation, electric shock, physical restraint,

17

thermal extremes, and transportation (Klemcke et al., 1991;
Becker et al., 1985; Hicks et al., 1998).

When blood samples are collected via venipuncture,
pigs experience pain and additional stress due to
restraint. Therefore blood sampling may artifactually
influence the HPA axis response to stress. Consequently,
plasma cortisol as a stress marker gives ambiguous results
(Bassett and Hinks, 1969; Worsaae and Schmidt, 1980;
Moberg, 1987). A noninvasive technique that does not induce
an appreciable stress on the animals could eliminate this
ambiguity. Most research has focused on whether salivary
and urinary cortisol values could reflect plasma cortisol
concentration. In my first experiment, urinary cortisol was
used since I could not collect enough (about 500 pl) saliva

from 10-day old piglets.

Salivary cortisol

Many small organic compounds such as urea, glucose,
steroids, and lipids are present in saliva, usually much
less than their plasma concentration. The reliability of
using saliva to determine the fraction of unbound low
molecular weight hormones, such as cortisol, has been well

established for humans (Walker, 1989).

18

Most of the saliva is produced by three major pairs of
salivary glands (parotid, submandibular, and sublingual).
The numerous small buccal glands, which line the mouth,
secrete saliva continuously under local control. Secretion
by the major glands is mediated by both sympathetic and
parasympathetic pathways in response to physical, chemical
and psychological stimuli. In humans, the relative
proportion contributed by each of the major glands under
resting conditions is typically, submandibular 69%, parotid
26%, sublingual 5% (reviewed in Vining and McGinley, 1986).

Vining and McGinley (1986) proposed several routes
that may transport a hormone to saliva: 1) passive
intracellular diffusion is for high lipid-soluble molecules
like corisol and takes place in the lipid-rich cell
membranes of acinar cells or into the cells lining the
gland; 2) ultrafiltration, which brings the lipid-insoluble
compounds into saliva; and 3) active secretion or
transport, an active, energy-consuming process by which
many electrolytes and some proteins may be secreted to
saliva.

Most of the cortisol present in saliva appears to
enter intracellularly by diffusing through the cells of the
salivary glands. Saliva flow rates can range from less than

0.05 ml/min (during sleeping) to 10 ml/min (when stimulated

19

u
17-!"
u.¢~

1

 

 

 

‘ FL

 

 

 

by acid lemon drops). Fortunately, saliva cortisol levels
are not affected by the flow rate because the diffusion
rate is high enough to maintain a concentration equilibrium
between saliva and plasma (Hubert and De Jong—Meyer, 1989;
Cooper et al., 1989). There is a linear correlation between
unbound cortisol concentration in plasma and saliva.
Correlation coefficients between 0.89 to 0.97 have been
reported (reviewed in Vining and McGinley, 1986). Vining et
al. (1983) also found the immediate (approximately 1 min)
appearance of cortisol in saliva following an intravenous
injection of cortisol. In summary, saliva cortisol
concentration accurately reflects the current level of
unbound cortisol in plasma.

The measurement of cortisol in saliva offers several
advantages over the measurement of the hormone in blood: 1)
saliva is easily collected and plentiful in supply (Cooper
et al., 1989; Parrott and Misson, 1989; Cook et al., 1996);
2) the stress of venipuncture has been shown to increase
cortisol levels whereas the non—invasive saliva sampling
has not (Kirschbaum and Hellhammer, 1989); and 3) there is
no CBG present in saliva. The presence of CBS often
complicates the interpretation of plasma cortisol levels
because many factors may alter the level of CBG in the

blood or affect the binding of cortisol to them.

20

In pigs, Cook et a1. (1996) compared the efficacy of
salivary cortisol to serum cortisol for assessment of the
HPA response to exogenous ACTH stimulation. These authors
found the levels of cortisol in saliva are approximately 9%
of the total plasma cortisol. This result is consistent
with observation of Parrot et al. (1989). It is well
established that salivary cortisol significantly correlates
with plasma cortisol in pigs (r > 0.7)(Cook et al., 1996;

Schonreiter et al., 1999).

Urinary cortisol

Urinary cortisol is also a widely accepted index of
circulating cortisol in humans (Beisel et al., 1964;
Brooks, 1979). Circulating unbound cortisol distributes
along concentration gradients. Unbound plasma cortisol
continuously enters the urine pool via glomerular
filtration of blood by the kidneys (Beisel et al., 1964;
Brooks, 1979). This urine pool accumulates plasma cortisol
over several hours. Cortisol concentrations in voided urine
represent the average level of unbound cortisol circulating
between urinations. Therefore urinary cortisol levels can
dampen the temporal variability inherent in instantaneous
plasma and saliva values (Miller et al., 1991; Crockett et

al., 1993).

21

 

 

- . .t w. E .C U . . ,
C C C .C c 1 O a .2 O T E O a 1 . .. C

S a e C C C .F.

 

a: X . . A
a S a .n

1-;9‘

 

 

 

 

Twenty—four hour urine cortisol is used to evaluate
adrenal responses to stressors in varieties of species,
such as primates (Crockett et al., 1993), sheep (Berman et
al., 1980), and mink (Madej et al., 1992). To collect 24-
hour urine samples, animals need to be individually caged.
Social isolation is a potent stressor, which increases the
activity of HPA axis in social animals such as pigs (Ruis
et al., 1997). Thus, this sampling method interferes with
the endocrine response it is intended to measure.

To avoid the artifactual effects and the inconvenience
of standard 24—hour continuous sampling, the
cortisol:creatinine ratio in a single urine sample (UCCR)
has been evaluated. The diurnal fluctuation of UCCR is a
substitute for the total urinary cortisol secretion in 24
hours in the diagnosis of cortisol excess and deficiency in
human (Contreras et al., 1986). A positive linear
relationship (r=0.5 to 0.7) between UCCR and plasma
cortisol concentrations has been reported in dogs (Jones et
al., 1990) and bighorn sheep (Miller et al., 1991).

In the state of elevated plasma cortisol concentration
there is an increased percentage of unbound plasma
cortisol, an elevated renal clearance for unbound cortisol,

and an increased glomerular filtration rate (Jones et al.,

22

 

S . . . I .2 3 r h S C . c F; .
l .3 2 2 .1 C S 35 t .l "K mg C1 Mac n.

e

.5 E
\V

.p «l‘

l!

 

 

 

 

1990). This may explain why the rise in urinary cortisol is

disproportional to that of the plasma cortisol.

2.3 SEGREGATED EARLY WEANING STRESS
2.3.1 weaning Stress

In a commercial pig production system, weaning
includes an abrupt separation of the suckling piglets from
the sow when the piglets are still wholly nourished by the
sow’s milk. Under semi—natural environments, weaning is a
gradual process where stronger piglets wean earlier than
the weaker pigs in the litter. The natural weaning process
is completed between 12 to 17 weeks of age (Newberry and
Wood—Gush, 1985; Jensen, 1986; Stolba and Wood-Gush, 1989).

Premature separation from the mother has long been
acknowledged as a cause of stress in many species, which is
often evidenced by prolonged vocalization, restless
activity, HPA axis activation and long—term behavioral and
physiological changes (Fraser, 1978; Worsaae and Schmidt,
1980; Blackshaw, 1981; Klemcke and Pond, 1991; Plotsky and
Meaney, 1993).

Some studies have examined the behavioral effects of
weaning and weaning age in animals. Mason (1994) found that
early weaning in mink increased the performance of tail-

biting. Wfirbel and Stauffacher (1997) reported that

23

precociously weaned mice had high levels of stereotypes in
adulthood. Fraser (1976) compared the behavior development
of suckling and weaned piglets (at three weeks of age)
during the first six weeks after birth. He noticed that
newly weaned animals had high levels of general activity
and aggression. Belly—nosing occurred after weaning and
appeared to be stimulated by the sound of the neighboring
sow nursing its young. This behavior was never observed in
suckling piglets (Fraser, 1976). As piglets’ weaning age
decreases, more calls/min and higher frequency were
recorded post-weaning (Weary et al., 1997). In fact, in
many European countries, weaning piglets younger than three
weeks of age is considered inhumane and therefore is
illegal (The Welfare of Livestock Regulations, 1994).

Long term physiological effects of maternal care on
neonates have been examined in rodents. In rats, maternal
care during early postnatal age has been demonstrated to
have a persistent effect in the offspring’s response to
stress throughout their life. As adults, offspring who
received more maternal care had reduced behavioral
fearfulness in response to novelty, and reduced response of
HPA axis to acute stress (Caldji et al., 1998; Liu et al.,
1997). In addition, Plotsky and Meaney (1993) found that as

adults, rats’ offspring who were subjected to 3-hour/day

24

h.
.a.
.W

Y

2.3

 

 

 

 

“.‘\(

 

maternal separation during 2—14 days of age had a high
level of plasma corticosterone (predominant glucocorticoid
in rodents) in response to restraint stress when compared
with undisturbed offspring. Wurbel and Stauffacher (1997)
also reported that precociously weaned mice had elevated

plasma corticosterone levels post-weaning.

2.3.2 Transportation Stress

A number of reports (Dow, 1976; van Putten and Elshof,
1978; Stephens, 1980) indicate that transport can have a
detrimental effect on pigs. Economic loss due to
transportation was estimated at more than 43 million
dollars in 1994 in the United States (Pork Chain Quality
Audit, 1994). The loss resulted from sudden death of pigs,
excess bruise of the carcass, and pale, soft, and exudative
(PSE) meat. Many environmental conditions contribute to the
transportation stress, such as vibration, noise, changes in
speed, mixing with unfamiliar animals, and deprivation of
food and water (Stephens and Perry, 1990). Stephens et al.
(1985) found that pigs can make an operant response to
terminate the vibration of their pen and do not habituate
to vibration and noise. But the noise alone does not

motivate the animals to turn the apparatus off.

25

11¢

Y'I“

.i.

I11.lu|:

 

 

at

L.»
r a

is
a»

,\\
.nu

A number of researchers have examined the
physiological response to transportation in pigs. Elevated
plasma cortisol, reduced body weight, increased numbers of
blood neutrophils, and decreased numbers of lymphocytes are
reported after a four—hour transportation (McGlone et al.,
1993). The transportation of sheep leads to an increase in
vocalization, heart rate, and serum and salivary cortisol
levels (Fell et al., 1985; Baldock et al., 1990). In
horses, transportation leads to immune suppression, high
heart rates, hypertonic dehydration, and high serum
cortisol concentrations (Owen et al., 1983; Clark et al.,

1993; Friend et al., 1998).

2.4 SUMMARY

Maternal care of the offspring seems to have important
effects on the stress response of adult offspring.
Premature maternal deprivation and transportation are
stressful experiences for many animal species. Stress
reduces the fitness of an animal as measured by parameters
of growth, reproduction, and immune function.

I hypothesize in this thesis that neonatal piglets
will respond to SEW stress as measured by urinary cortisol
as well as behavior indicators such as vocalization,

locomotion, aggression and development of abnormal

26

 

behavior. I also hypothesize that the stressful SEW
experience during neonatal age will be associated with high
levels of aggression, behavioral abnormalities, and
elevated HPA axis responsiveness in pigs when they are

older.

27

Chapter 3 HYPOTHESES AND OBJECTIVES

The overall hypothesis was that neonatal piglets can
respond to segregated early weaning (SEW) stress and that
this stressful experience has long lasting effects on
aggression, behavioral abnormalities, and HPA axis

responsiveness.

The overall objectives were:
1” To investigate the immediate response of neonatal piglets
to SEW stress;

:2.To investigate the long—term effects of SEW stress in

pigs.

EXPERIMENTAL APPROACH
We designed and conducted two experiments. In the

first experiment, we hypothesized that neonatal piglets can
respond to SEW stress as measured by behavioral indicators
and cortisol concentrations. The corresponding objectives
were:
0 To investigate the effects of SEW stress on the behavior

of neonatal piglets shortly after weaning;
0 To determine the effects of SEW stress on cortisol

concentration in neonatal piglets shortly after weaning.

28

The specific aims of this experiment were:

0 To monitor maintenance, locomotion, aggressive, and
abnormal behaviors in neonatal piglets subjected to SEW
stress or left undisturbed with the sow;

0 To monitor urinary cortisol:creatinine ratio in neonatal
piglets subjected to SEW stress or left undisturbed;

0 To monitor growth of pigs as a practical indicator of

production performance under SEW and left undisturbed.

In the second experiment, the SEW piglets were
compared with conventional weaned (CW) piglets that were
removed from their sows at a later age than SEW piglets. We
tested the hypothesis that stressful SEW experience during
neonatal age is associated with high levels of aggression,
behavioral abnormalities, compromised social skills and
elevated HPA axis responsiveness in pigs when they are
older. The objectives of this study were:

0 To investigate the development of behavior abnormalities
in SEW piglets;

0 To determine the HPA axis responsiveness in SEW piglets
at a later age;

0 To investigate the aggression and the capacity to assess

social environment in SEW piglets at a later age.

29

The specific aims of this experiment were:

0 To
or
0 To

monitor the development of abnormal behaviors in SEW

CW piglets until 7 weeks of age;

monitor baseline AM and PM salivary cortisol

concentration in SEW or CW piglets at 5, 6,and 7 weeks of

age;

0 To

to

of

0 T0

in

monitor salivary cortisol concentrations in response
transportation stress in SEW or CW piglets at 9 weeks
age;

monitor the number, duration and outcome of the fights

SEW and CW piglets when they are mixed at 9 weeks of

age;

0 To

monitor piglet growth as a practical indicator of

production performance under SEW and CW system until 8

weeks of age.

30

Chapter 4 IMMEDIATE RESPONSE OF NEONATAL

PIGS TO SEGREGATED EARLY WEANING

4 . 1 INTRODUCTION

Weaning and transportation are stressful to pigs
(Fraser, 1978; Lambooij and van Putten, 1993). The
segregated early weaning (SEW) system, which subjects
piglets to early weaning and transportation has been
rapidly adopted by the North American pig industry.
However, the potential welfare concerns of exposing the
neonatal piglets (as early as 10 days of age) to these two
stressors have not been addressed. Pigs raised in this
system exhibit high levels of behavior abnormalities, such
as belly—nosing, later in their life (Gonyou et al., 1998).

Stress is considered a threat to animal welfare.
Cortisol is the hormone most widely used as an indicator of
stress (Levine, 1985; Shutt et al., 1987; Klemcke and Pond,
1991). During this experiment, I measured cortisol in urine
samples rather than blood samples because urine can be
obtained non-invasively. The validity of using urinary
cortisol:creatinine ratio (UCCR) in a single urine sample
as an indicator of HPA axis activation has been
demonstrated in humans, dogs and sheep (Contreras et al.,

1986; Jones et al., 1990; Miller et al., 1991). In this

31

study, UCCR was used to evaluate the adrenal responses to
SEW stress in piglets.

This study was conducted to address the welfare
concerns by investigating the immediate behavior and
adrenal response of neonatal piglets to SEW stress. I
hypothesized that neonatal piglets can respond to SEW
stress as measured by both behavioral indicators and UCCR.
The expected results were: I) SEW piglets would have a
reduced growth rate; 2) SEW piglets would have increased
activity levels, vocalization rates, aggression and
exploratory behavior; and 3) SEW piglets would have
elevated urinary cortisol levels post weaning compared with

non-weaned animals.

4.2 MATERIALS and METHODS:
4.2.1 Animals and Management

Six Landrace sows were bred using artificial
insemination by the same Yorkshire boar. Sows farrowed
within five days of each other. Within 24 hours after
farrowing, the litter size was standardized to eight
piglets, piglets were tail docked, ear notched, teeth
clipped and the male piglets were also castrated.
Crossfostering was carried out between days 1 and 3

postpartum.

32

The sows and their litters were housed in farrowing
crates. Sows were fed twice a day and water was provided ad
libitum. A nipple drinker was available for the piglets.
Continuous illumination was provided for about 10 hours a
day. The room temperature was set to 18°C. Heating lamps
were used to provide additional heat.

Twenty-three piglets (due to the loss of one piglet)
from three crossfostered litters were weighed, weaned and
transported for 5 minutes to a separate site at 1130 h when
they were 9 to 13 days old (SEW group). Twenty-four piglets
of the other three sows were also weighed, but they were
allowed to stay with the sows (control group). Eight days
later, their weights were measured again.

The weaned piglets were placed in three 1.18 X 1.19 m
pens without regrouping. Artificial lights provided
illumination for 10 hours a day. The room temperature was
set to 27°C. Heating pads were placed in the pen to provide
additional heat. Water was supplied by means of a nipple
drinker ad libitum. SEW phase I diet (Crude Protein 23.4%,
Lysine 1.70%, ME 3429.4 kcal/kg, pellet form) was fed twice
a day ad libitum in a three-hole feeder. In addition, for
the first two days post-weaning, fresh pellets were also
given on a pan in the middle of the floor 5 times during

the day to acquaint the piglets to the new form of diet.

33

 

4.2.2 Behavioral Observations

Piglets were numbered with a livestock marker (LA-CO
Industries, Inc., Elk Grove Village, Illinois) the day
before weaning. Various behavior categories listed in Table
1 were recorded by two trained observers. Direct
observations were carried out on the weaning day (day 0),
and on days 1, 2 and 3 post—weaning. Each animal was
observed continuously for two minutes in each observation
section. The observation was repeated four times between
830 and 1530 h for every animal. Behavior data were logged
onto audio tapes for control animals and directly on a
computer using a behavior software (The Observer® 3.0,
Noldus Information Technology, Wageningen, The Netherlands)
for SEW piglets. The audio tape recording was later
transferred to the computer using the same behavior

software.

4.2.3 urine Sampling

Urine samples were collected one day before weaning
(day —1) and 1, 3 and 5 days post-weaning between 1530 and
1730h. The piglets were placed into individual plastic
boxes (57.15 X 39.37 X 31.75 cm) until they urinated. The
maximum time that a piglet was kept in the box was 10

minutes. In the SEW group, we were able to collect samples

34

Table 1: Description of behavior categories in piglets.

 

 

 

 

 

 

 

 

Behavior . ,
Description
category
Statesz
Lying Body weight is not supported by the limbs.
Standing Supporting its body with four limbs upright
and idle.
Sitting Sitting on the posteriors; fore limbs
stretched; head free from any support.
Moving Four limbs upright and not idle.
In a standing position, the piglet rubs the
Rooting snout on the flooring surface in horizontal
movements.
Two piglets take part in an agonistic
Fighting encounter involving knocking, biting, parallel

and inverse parallel pressing and levering.

A rhythmic up—and—down movement with the snout
Belly-nosing on the belly or soft tissue between the hind
and forelegs of another piglet.

Manipulating Having another piglet’s ear in mouth, may

 

ear suck, chew, or bite.
Manipulating Having another piglet’s tail in mouth, may
tail suck, chew, or bite.

 

Manipulating Sucking, chewing, biting or horizontal
other part movement of snout on another piglet’s body
of the body except ear and tail.
A rhythmic up—and—down movement with the snout

 

 

 

 

 

 

 

Massaging near the sow’s teat, it usually occurs before,
the sow ,
between or after suckling.
Sucking Having sow’s teat in the mouth.
Eating The head is over or in the feed trough.
Drinking Having the nipple drinker in the mouth.
Bar biting Chewing or biting the metal bar.
Event53
Grunting Low tonal vocalization.

 

 

2Behaviors of relatively long duration, such as prolonged
activities, body postures or proximity measures. The
salient feature of states is their duration (mean duration,
or the proportion of time spent performing the activity)
(Martin and Bateson, 1993).

3Behaviors of relatively short duration, such as discrete
body movements or vocalizations. The salient feature of
events is their frequency of occurrence (Martin and
Bateson, 1993).

35

Table 1 (Cont’d)

 

 

Squealing Intense high pitch vocalization.
Fore limbs on top of another piglet’s back,
Climbing hind limbs stretched and in an upright
position.

 

 

from 3, 11, 11, 17 piglets on days —1, 1, 3, 5 post-
weaning, respectively. In the control group, we obtained 6,
10, 15, 10 samples on day —1, 1, 3, 5 post-weaning,
respectively. Urine was collected into 15—ml plastic

conical tube and put on ice immediately. Samples were
stored at -20°C until analysis. Upon defrosting, urine

samples were centrifuged for 15 min (1,300 X g, 4°C) and

the supernatant was used for analysis.

4.2.4 urinary Cortisol and Creatinine Analysis:

Urinary cortisol concentrations were determined using
an 125I radioimmunoassay (Coat-A-Count Cortisol Kit,
Diagnostic Products Corp, Los Angeles, California)
following standard procedures except that samples were not
extracted with CHfiflQ. Urinary creatinine was analyzed using
commercial diagnostic kits (555-A creatinine kit, Sigma
Chemical Company, St. Louis, Missouri).

The inter-assay coefficient of variation for quality

control (std/mean x 100) on two creatinine assays at 113.15

mmol/l and 256.36 mmol/l was 6.91% and 4.72%, respectively.

36

The intra-assay coefficient of variation on duplicate
samples was 8.14% in the cortisol assay and 3.82% in
creatinine assay, and was calculated using the following

formula:

1/2

 

Intra—assay coefficient =

[Md/u x 100?]

2N
where;

d

the difference between duplicate estimates

u the mean of the duplicate estimates

N

the number of duplicate samples

4.2.5 Statistical Analysis:

Behavior data were expressed as either percentage of
observation time (states) or frequency per minute (events
and states). Cortisol in urine was expressed as the ratio
of urinary cortisol concentration to creatinine
concentration (UCCR).

From the viewpoint of data distribution, the data sets
obtained from this research can be classified into three
groups:

1” Some data were normally distributed, including body
weight and % of time spent lying;

1” Some data were not normally distributed. In some of these

cases, distributional assumptions were satisfied by using

37

certain transformations. For the UCCR data, a log—
transformation was applied before data analysis;

2. In the other cases where data were not normally
distributed, transformations did not satisfy the
distributional assumptions. All the rest of the behavior
data fit into this category.

For the first two types of data, a mixed model with
repeated measures from SAS® (SAS institute Inc, Cary, North
Carolina, U.S.A.) was used. Lsmeans 1 standard error of the
means (SEM) were reported or back transformed to the
original scale wherever necessary to facilitate
interpretation and discussion of the results. Differences
between treatment means at various trial days were tested
for significance (when P < 0.05). The experimental unit
used in the analysis was the pen.

For the third type of data, a nonparametric Wilcoxon
rank sum test was chosen. It tests whether the distribution
of a variable has the same location parameter across
different groups based on the simple linear rank
statistics. The assumption is that the samples were
randomly, independently drawn from one population and
received two treatments. Mean rank score i SEM are

reported.

38

Statistical model:

Yijklm = H + (11' + Bj + pkm+ 51 + aBij + eijklm

Where;

Ytfihn = dependent variable (such as body weight, % of time
spend lying, and log-transformed UCCR) observed for
the mth piglet, in the ith treatment, at the jth day,
in the kH‘pen, and from the lu‘litter;

u = overall mean of dependent variable;
a1: the fixed treatment effect (1 = SEW, Control);

Bj= the fixed day effect (j=1,2; or 1,2,3; or 1,2,3,4,
depending on Yukmdi

the random effect of the k“‘(k=1,2,3) pen nested

PM)

within the iU‘treatment, 9M0 ~ NIID(0, 0am“) for all
i and k;
51: the random effect of the lu‘litter (l=l,m,6), 51
~ NIID(0, 0%) for all 1;
aBU = the interaction of treatment and day;

eukn1= random error term, eukn1~ NIID(0,<¥e) for all

i! j, k, l, m.

4.3 RESULTS

The weight of the control and SEW groups were not

39

I SEW
[:1 Control

8-

***

C)
r

   

 

Weight (kg)
A

N
1

 

 

   

 

O
I

 

 

10 18
Age (days)

Figure 1: Mean weight of SEW and control piglets before and
a week after weaning treatment. *** P < 0.001. Comparison
was made between SEW and control piglets within the same

time period.

different (P = 0.7741) before early weaning. The SEW
piglets had significantly lower body weight (P = 0.0002)
than piglets left with sows after one week of weaning (Fig.
1).

Eating and drinking averaged 4% in SEW piglets’ time
budget. Control piglets spent on average, 15% of
observation time sucking and massaging the sow (Table 4 in
Appendix).

On days 0 and 1 post-weaning (PW), SEW piglets

displayed higher levels of vocalizations (P :5 0.0001),

showed higher frequency of climbing (P :s 0.037) and spent

40

Table 2: Mean Wilcoxon rank sum score (iSEM) in SEW piglets
and control piglets (not weaned) for frequency of grunting,
climbing, and duration of moving immediately after and in

the first day post weaning.

 

 

 

 

 

giiiogig Day 0 Post Weaning Day 1 Post Weaning

(;:::?* SEW Control SEW Control
Grunting 30.8i1.6a 17.51215b 57.4i4.0a 38.0:3.9b
Climbing 25.6i0.9a 22.545043b 51.2:3.9a 44.013.23b
Moving 30.01.10a 18.2i1.9b 58.7:5.7a 36.7:5.4b

 

* Comparison was made between SEW and control piglets
within the same time period; different letters indicate a

statistical significant difference (P < 0.05).

more time moving (I>:S 0.002) than undisturbed control

piglets (Table 2).
Time spent rooting and frequency of belly-nosing were
not different in the two treatment groups on day 0 post-

weaning but were consistently higher (P 5; 0.012) throughout

days 1, 2, and 3 post-weaning in SEW piglets than in
control piglets (Table 3). The frequency and time spent
fighting did not differ in the two groups at any
observation days post-weaning (P > 0.15; Table 3).

SEW animals spent less time lying on day 1 (P =

0.0004), but more time on day 3 (P = 0.0057) post-weaning

41

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“UOAHOQ OEHu mEmm man segues muoflmflm Houucoo one 3mm cmm3umn mote mm3 COmHHMQEOU I

 

 

 

o.mIm.ee H.~Im.ee o.mIe.me H.mIm.me m.HIm.me o.mIe.me Asuemzomhnv
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whoom

mcflcmmz umom mcflammz umom mcflcmmz umom Esm xcmm

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paw cofiumuzp out .mcflboou mo coflumHSU .mchOGIxaamQ mo hocmsvmuw HOw Apmcmo3

pony muwamflm Houucoo paw mumamflm 3mm CA Azmmflv ouoom 55m xcmu coxooaflz cmoz "m canoe

42

I SEW

 

 

 

 

 

 

 

I»

-§ 100 - DControl ..
3 30 - I...

c

8 60 -

m

g 40 -

1:7 20 -

“6

°\o O ' l

 

 

1 2 3
Post Weaning (days)

Figure 2: Average percentage of time spent lying from 0830
to 1530 h in SEW piglets over three days post weaning, with
reference to control piglets that stayed with the sows. **

P < 0.01; *** P < 0.001.

 

 

 

 

 

 

 

g c 300 - ISEW
{5. CE) [jControl
g = 200-
h o
9 E
3 E, 100- T
In
-- o
t z:
0 m 0
o *- '
-1 1 3 5

Post Weaning (days)

Figure 3: Mean urinary cortisol:creatinine ratio in SEW and
control piglets during pre— and post-weaning period. * P <

0.05, *** P < 0.001.

43

than control piglets (Figure 2). The morning data
contributed to these differences, while the lying time in
the afternoon was not different in any given day between

the treatments.

The UCCR was elevated in SEW piglets on days 1 (P 5;

0.0001) and 3 (P = 0.0339) post—weaning compared to control

piglets (Fig. 3).

4.4 DISCUSSION

Even when provided with digestible and palatable
diets (Dritz et al., 1996), the SEW piglets had a reduced
growth rate during the first week after weaning compared
with control piglets (Fig. 1). Several elements may
contribute to this phenomenon. First, the adaptation to
solid feed takes time. Gonyou et al. (1998) reported that
the eating behavior in early—weaned piglets (12 days of
age) on SEW diets did not develop adequately in the first
36 hours post—weaning. In contrast, piglets weaned at 21
days of age established eating behavior within 24 hours
post—weaning. Second, the retardation in growth can be a
result of the SEW stress response. The
intracerebroventricular administration of corticotrophin
releasing hormone (CRH), which triggers the activation of

HPA axis during a stressful event, reduces food consumption

44

in rats and mice (Britton et al., 1982; Berridge and Dunn,
1986). The secretion of growth hormone and insulin-like
growth factor-I is also suppressed during stress (Diegez et
al., 1988). Therefore, increased HPS axis activity in the
SEW piglets of this study (Fig. 3) could have contributed
to the reduced body weight gains in the first week post—
weaning.

Vocalization and climbing on other pigs have been
classified as fear-related behavior in response to stress
(Brundige, 1998). The SEW piglets vocalized at a high rate
up to one day post-weaning (Table 2). Weary et al. (1997)
demonstrated that the vocalization of weaned piglets
indicates their needs for resources provided by the sow,
such as heat and milk. Climbing behavior (Table 2), which
was also elevated in SEW piglets on days 0 and 1 post-
weaning, could be an expression of their fear towards an
unpredictable and uncontrollable environment as suggested
by Brundige (1998). I also observed that the newly weaned
animals huddled together, which could be interpreted as a
cold or fear response. Some weaned piglets kept trying to
jump out of the pen immediately post-weaning. Obviously,
the jumping was an escape attempt. My results showed that
SEW piglets spent more time moving than control pigs on

both days 0 and 1 post—weaning (Table 2). Fraser (1978),

45

Metz and Gonyou (1990) concluded that piglets are restless
after weaning when the weaning takes place at two or three
weeks of age. Restlessness in the initial post-weaning
period occurred in the SEW piglets as well, and combined
with the fear and cold responses, may have contributed to
activation of the HPA axis (Fig. 3).

Belly—nosing may be a redirected behavior, which
results from the deprivation of the appropriate
environmental cues for sucking and massaging behavior.
Blackshaw (1981) and Metz and Gonyou (1990) suggested that
a time lag exist before redirected nosing started in weaned
piglets. In contrast, belly—nosing was recorded on day 1
post-weaning in the current group of SEW piglets and the
frequency of belly-nosing was significantly higher from day
1 post-weaning in SEW than in control piglets (Table 3).
The differences across studies might be due to the fact
that piglets were weaned at two to three weeks of age in
the previous studies (Blackshaw, 1981; Metz and Gonyou,
1990) versus day 10 of age in the current study. As
expected, SEW piglets spent more time showing exploratory
behavior (rooting) than undisturbed piglets on days 1, 2
and 3 post—weaning (Table 3). Contrary to my expectation,
however, the time spent and frequency of fighting was not

different between the two treatment groups (Table 3). One

46

explanation could be that all litters were initially
crossfostered. Fighting occurs more often in crossfostered
litters than in natural litters (Robert, 1997). Therefore,
a disturbed basal aggression level in the control piglets
may have made it difficult to detect differences in
aggression between the SEW and control piglets in this
study.

Lying time on the day 1 post—weaning was much shorter
in SEW than in control animals. However, lying time rose
sharply on the following days so that the percentage of
time spend lying was much longer on day 3 post-weaning in
SEW group (Fig. 2). It is unknown what caused this dramatic
increase in lying time, but a depletion of energy and/or
acquaintance to the new environment may be the factor(s).
The piglets spent more time lying in the afternoons than in
the mornings. Surprisingly, the lying time remained
constant in the afternoons, regardless the treatment
groups. It seems that the SEW stress did not affect
activity levels in the afternoon.

As indicated previously, the cortisol concentration as
a parameter of physiological stress in pigs was examined
(Klemcke and Pond, 1991; Becker et al., 1985; Hicks et al.,
1998). The UCCR in SEW animals was 328% and 143% of the

control animals on days 1 and 3 post weaning, respectively

47

(Fig. 3). This demonstrates a sustained activation of the
HPA axis. When male piglets were castrated at two to four
weeks of age, a 291% increase in unbounded plasma cortisol
was observed after an hour of castration. Cortisol
concentration returns to basal levels within 24 hours
(Schénreiter et al., 1999). Therefore, the magnitudes of
cortisol elevation were comparable between castration and
SEW. Since 50% of administrated l4C cortisol excreted
through urine within 6.0 i 0.6 hours in piglets (Kraan et
al., 1986), SEW stress could be more chronic than
castration stress as indicated by the duration of cortisol
elevation. Cortisol could affect carbohydrate metabolism,
providing necessary energy and participating in thermal
regulation, which may be important to the newly weaned
animals. However, high levels of cortisol also have
unwanted effects such as the suppression of immune response
(Owen et al., 1983; Munck et al., 1984; Burton and Kehrli,
1995, 1996). Hence, the sustained high levels of cortisol
in SEW piglets may increase their susceptibility to
infection. If true, it would be very important to reduce
pathogen loads in piglets rearing facilities to a minimum
as well as supplement piglets with appropriate dietary
vitamins and minerals that are known to enhance immuno—

competence. SEW pigs are usually regarded as healthier than

48

conventionally raised animals. However, healthier does not
necessarily mean a better immune function. The improved
health status of SEW animals may partially due to the
cleanness of improved housing. More research is needed to

reach a conclusion on this aspect.

4.5 CONCLUSION

The short—term responses to SEW include reduced growth
rate, altered behavior patterns, and elevated cortisol
levels. These evidences confirmed my hypothesis that the
neonatal piglets could respond to SEW stress. All of the
responses are indicators of an inadequate environment and
difficulties in coping, therefore the welfare of SEW
piglets is probably poor for the first few days post-
weaning. More research is needed to determined whether SEW

risks piglet’s welfare in the long term.

49

Chapter 5 LONG-TERM EFFECTS OF SEGREGATED EARLY

WEANING STRESS ON PIGS

5.1 INTRODUCTION

Premature maternal deprivation imposes profound
changes in behavioral patterns in several animal species.
Examples have been demonstrated in horses, pigs, mink,
mice, monkeys and polar bear (Heleski et al. 1999; Worobec
et al., 1999; Mason, 1994; Wfirbel and Stauffacher, 1997;
Hinde et al., 1966; Poulsen et al., 1996). At six weeks of
age, piglets that were weaned at 6 and 7 days of age
displayed more belly-nosing, and gained less weight than
piglets weaned at 14 or 28 days (Orgeur et al., 1998;
Worobec et al., 1999).

Maternal care during early post—natal age also affects
long term physiological responses to stress in the
offspring. Rat puppies that received higher levels of
maternal care had a reduced corticosterone response to
stress in adulthood (Liu et al., 1997). In contrast, rat
puppies subjected to 3-hour/day maternal separation during
early age (2 to 14 days post-natal) had an increased
corticosterone response to restraint stress compared with

undisturbed offspring (Plotsky and Meaney, 1993).

50

These evidence lead to the hypothesis that premature
maternal deprivation has long-term effects on the welfare
of animals. As a result, the welfare of animals subjected
to early maternal deprivation may at risk in the long-term.

In the previous experiment (chapter 4), I showed that
neonatal piglets can respond behaviorally and
physiologically to SEW stress in the short term. For the
current experiment, conventionally weaned (CW) piglets were
used as a control group to compare welfare indicators of
SEW piglets later in life. I hypothesized that the
stressful SEW experience during neonatal age is associated
with high levels of aggression, behavioral abnormalities,
compromised ability to establish a stable hierarchy, a loss
in circadian pattern of cortisol, and elevated HPA axis

responsiveness in pigs when they are older.

5.2 MATERIAL AND METHODS
5.2.1 Animals and Management

Six Landrace sows were bred using artificial
insemination by the same Yorkshire boar. Sows farrowed
within four days of each other. Each litter consisted of 8
to 12 piglets. The piglets were tail docked, ear notched,
teeth clipped within 24 hours after birth, and the male

piglets were also castrated.

51

Four piglets from each litter were randomly selected,
weighed, weaned and transported 5 min to a separate site
when they were 9 to 12 days old (SEW group). The remaining
piglets were weaned at 20 to 23 days of age. Four piglets
from each litter were randomly selected, weighed and
transported to the same building where the SEW group was
housed (CW group). The animals were then weighed weekly
after weaning until week 8 (except for weeks 4 and 7).

The weaned piglets were placed in twelve 1.17 X 0.98 m
pens without regrouping. The layout of the pen is pictured

in Figure 4. Piglets were exposed to artificial lights from

830 to 1830 h. The room temperature was maintained at 27°C

for newly weaned animals and gradually decreased to 21°C by

9 weeks of age. Heating pads were used to provide
additional heating until the piglets reached the age of 5
weeks. Water was supplied by means of a nipple drinker ad
libitum. During the first week after conventional weaning,
one piglet in the CW group was removed due to severe
diarrhea.

SEW phase I diet (Crude Protein 23.4%, Lysine 1.70%,
MB 3429.4 kcal/kg) in pellet form was provided fresh twice
a day ad libitum in a feeding trough. For the first four
days after SEW and first two days after CW, the pellets

were also given five times a day on a pan placed in

52

" . Nipple drinker

.I
l
I

 

Fig. 4: Size and design of a nursery pen.

the middle of the floor to acquaint the newly weaned
piglets to the new form of diet. SEW piglets were fed phase
I diet for 2 weeks, phase II (Crude Protein 21.48%, Lysine
1.45%, MB 3409.09 kcal/kg) for 2 weeks, and phase III
(Crude Protein 19.64%, Lysine 1.25%, ME 3396.4 kcal/kg)
until weeks 9 of age. The CW piglets were fed phase I diet
for four days, phase II for 2 weeks, and phase III until
weeks 9 of age.

At 9 weeks of age, piglets were mixed, loaded and
transported to a growing unit. Littermates housed in the

same pen after weaning were separated into four groups

53

according to their body weight (i.e. the heaviest to group
1, the lightest to group 4). After the regrouping, each pen
consisted of 12 pigs (one pen had only 11 pigs), six of

them were SEW pigs, the other six were CW pigs.

5.2.2 Behavior Observations

Immediately after weaning, the piglets were numbered
with black hair dye (Clairol Inc., Stamford, Connecticut).
Piglets were video recorded from 830 to 1800 h daily for a
week post—weaning, then weekly up to 7 weeks of age. Nine
o’clock was arbitrarily picked as the start point for the
video decoding. The videotapes were then observed
continuously for 5—min. This 5—min observation was repeated
every 30 min of recording time. Frequency of eight
behaviors, including belly-nosing, bar biting, fighting,
manipulating pen-mates (ears, tails and other body parts),
drinking, lying were collected for all piglets using
behavior software (The Observer® 3.0, Noldus Information
Technology, Wageningen, The Netherlands).

After the pigs were regrouped and transported to the
growing unit, they were video taped for 12 hour/day for
three consecutive days. All agonistic interactions were
monitored. Data were obtained considering the initiator

pig, the opponent pig, the duration of the fight, and

54

whether a clear outcome resulted. An outcome was clear when

a winner could be identified at the end of the fight.

5.2.3 Saliva Sampling

Pig saliva was collected using cotton dental roll
(diameter: 1 cm, length: 4 cm, TIDI Products, Inc., Troy,
Michigan). To retrieve the dental roll from the pig’s mouth
and allow the pig to move freely, approximately 1 m of
dental floss (Johnson & Johnson, Skilman, New Jersey) was
tied at the center of the cotton roll.

During the fifth week of age, piglets were trained to
chew on cotton rolls in three different days. They were
allowed to chew for about a minute. Morning (7:00) and
afternoon (15:00) saliva samples were taken using this
method on Fridays in weeks 6, 7 and 8. At 8 weeks of age,
each pig was placed into a wood box by itself and isolated
for 30 minutes. Saliva samples were obtained before and
after the isolation. Pre- and post-transportation saliva
samples were also collected for the transportation at 9
weeks of age.

Cotton rolls containing saliva were placed into 15 ml
conical tubes, sealed and placed on ice immediately after
collection. These samples were centrifuged within two

hours. A 15-min centrifugation protocol (1,000 X g, 4°C)

55

was used to retrieve the saliva from the cotton rolls into

the conical tubes. Samples were frozen at -20°C, thawed and
centrifuged again (15-min at 1,300 X g, 4°C) to separate
saliva from particulate matter. The supernatant was stored

for subsequent assay.

5.2.4 Salivary Cortisol Analysis

Salivary cortisol concentrations were determined using
an 125I radioimmunoassay (Coat-A-Count Cortisol Kit,
Diagnostic Products Corp, Los Angeles, California, U.S.A.)
following the modified procedure (Determinations in Saliva,
September, 1992) provided by the company.

Gemus (1998) has validated the use of cotton rolls for
the assay of salivary cortisol. Her results showed that the

cotton rolls retained 1.4% of the fluid, and there is no

non-specific binding of cortisol in the cotton rolls.

5.2.5 Statistical Analysis:

Behavior data was expressed in frequency per hour. Two
types of data distribution were identified in this
research:
1.Some data were normally distributed, such as body weight;
2. In the other cases data were not normally distributed,

but distributional assumptions were satisfied by using

56

certain transformations. The data on salivary cortisol
and duration of fights was log—transformed before data
analysis. For the behavior data, a square—root
transformation was applied before data analysis;

A mixed model with repeated measures from SAS®
software (SAS institute Inc, Cary, North Carolina, U.S.A.)
was used (see below). Lsmeans i standard error of the means
(SEM) were reported or back transformed to the original
scale wherever necessary to facilitate interpretation and
discussion of the results. Differences between SEW and CW
treatment means at different time periods were tested for
significance (when P < 0.05). The experiment unit used in
this analysis was the pen.

The data on outcome of agonistic interactions
consisted of frequencies in discrete categories. A chi—
square test was used to examine the significance of
differences among three types of possible encounters (two
CW pigs fight, two SEW pigs fight, and a SEW pig fights

with a CW pig).

Statistical model:
Yijlm = u+0ti+Bj+51 +aBij+eij1m

Where;

57

Y;flm = dependent variable (weight, log-transformed salivary

aBij =

eijlm =

cortisol and duration of fight, and square root

transformed behavior data) observed for the malpig,

in the ifi‘treatment, at the jU‘age, and from the 1th

litter;
overall mean of dependent variable;
the fixed effect of treatment (i=SEW, CW) or

possible encounter (i=SS, SC, CC) depending on Yjflm;

the fixed age or day effect (j=1,2,3; or 1,2
depending on Yiflm);

the random and blocking effect of the lullitter
(l=1,m,6) or the random pen effect (l=1,2,3,4)

depending on Ygflm,ih ~ NIID(0, 0%) for all 1;

the interaction term;

the random error term of the observation, euhn~

NIID(0, 023) for all i, j, 1, m.

5.3 RESULTS

Unfortunately, all the A.M. and P.M. saliva samples

from 47 piglets at 6 and 7 weeks of age, and half of the

samples from the social isolation experiment were

mistakenly discarded by laboratory personnel. Due to the

loss of 236 samples, neither the circadian of cortisol, nor

58

I SEW

 

 

 

 

 

 

 

 

 

25 -
now
A 20 -
3’
V 15 ..
in!
.r:
.9 10 -
a:
3 5 _
T
O " I I _I
5 6 7 8
Age (weeks)

Fig. 5: Average body weight of SEW and CW piglets at weeks

5, 6 and 8 of age. I Weight on weeks 7 was not measured.

the HPA axis response to the social isolation stressor
could be investigated. For this reason, a considerable
amount of physiological data from the SEW and CW animal
models was missing.

There were no differences in the weight gains between
treatment groups across different ages post weaning (P =
0.7085, Fig. 5).

The behavior characteristics of the SEW and CW piglets
are summarized in Figure 6, which consists of four
individual graphs. The frequency of belly—nosing behavior

is presented in Figure 6a. A higher incidence of belly—
nosing (P 5 0.0142) was observed in SEW piglets than in CW

piglets at both weeks 5 and 7 of age. There was no age (P =

59

 

Fig. 6: Average frequency of (a) belly-nosing, (b)
manipulating, (c) fighting, and (d) bar biting from 0900 to
1800 h in SEW and CW piglets at weeks 5, 6 and 7 of age. *

P < 0.05, ** P < 0.01, *** P < 0.001.

60

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A o

 

 

 

I—LI

>>0 D ..
>>wm l

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.:
om
b
em

4 -
e w
INVAI
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a
lmld
>>ODM w.
2mm- em

meennn-nmm in

 

 

 

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(Inoq .Ied) Kouenbaid

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meanemnn in

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(man .Iad) KouenbaIg

 

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CC

 

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(D

mcflmOGIhaamm Am

61

0.1190) or age by treatment interaction (P = 0.1125)
effects on the belly-nosing behavior.

Conventional weaned piglets had a higher frequency of

manipulating other piglets than SEW piglets (P S 0.017) in
weeks 5 and 6 (Fig. 6b). The frequency of this behavior was
affected by age in CW piglets (P = 0.0019), but not in SEW
piglets (P = 0.4219).

Figure 6c shows that CW piglets fought more often (P =
0.0366) than SEW piglets in week 5. There was no age (P =
0.2178) or treatment by age (P = 0.7255) effect on the
fighting behavior.

The result of bar biting behavior is summarized in
Figure 6d. Frequency of this behavior increased as SEW
piglets were getting older (P = 0.0001), while the
performance of this behavior did not change in CW piglets
over the three weeks observation (P = 0.2327). CW piglets
showed more bar biting than SEW piglets in both weeks 5 and
6 (P S 0.0166). In week 7, however, a higher frequency of
bar biting was recorded in the SEW piglets than in the CW
piglets (P = 0.0002).

A twenty—minute transportation experiment at week 9 of
age increased salivary cortisol levels by 834% and 646% in
SEW and CW piglets, respectively. The percentage of

increase was not significantly different (P = 0.45) between

62

ISEW

20- nCW

15-
10-

5‘

ol

Cortisol(nmo|,l)

 

 

 

——|

Pre-transportation Post-transportation

Time

Fig. 7: Mean salivary cortisol concentration before and
after a 20-minute transportation in SEW and CW pigs at 9

weeks of age.

a) Day 1 afier mixing b) Day 2 after mixing c) Day 3 after mixing

   
  

6096 2396

11%

40%

89%

   

77%
I clear El not clear I clear El not clear I clear E] not clear
n=348 n=277 n=189
x2 = 161.849
P = 0.001

Fig. 8: The distribution of clear and unclear outcome of
fights in three days after regrouping when SEW pigs were

mixed with CW pigs at 9 weeks of age.

63

a) SEW pig fight with b) CW pig fight with C) SEW pig fight with
SEW pig CW Pig

   

I clear El not clear 2 clear D not clear Ell clear El not clear

n=186 n=176 n=452
x2 = 10.034
P= 0.007

Fig. 9: The distribution of clear and unclear outcome of
fights in three types of encounters when SEW pigs were

mixed with CW pigs at 9 weeks of age.

[3 SEW fight with SEW

 

 

30 -
E)? El CW fight with CW
3; a El SEW fight with CW
: ab
.2 20 -
H
g b
3
D
d) -
U) 10
(B
h
‘>’
<

0 I 1 l
1 2 3

Post Regrouping (days)
Fig. 10: Average duration of fights when outcomes were not
clear in days 1, 2 and 3 after the mixing of SEW and CW

a

pigs at 9 weeks of age. ' bMeans without a common

superscript differ (P < 0.05).

64

treatments. There were no significant differences (P >
0.18) between treatments in either pre or post-
transportation cortisol concentrations (Fig. 7).

After regrouping at week 9 of age, a total number of
821 fights was observed in the four pens of 12 pigs over a
three—day period. Winners were identified in 40.23% of the
agonistic interactions in the first day. The percentage of
clear outcomes increased (P < 0.0001) in the second and
third day (Fig. 8). Of all the interactions monitored,
fights within SEW pigs (Fig. 9a) were more likely to have
an unclear outcome (P < 0.007) than those within CW pigs
(Fig. 9b), and between CW and SEW pigs (Fig. 9c). The

number of fights was not different between these three

types of encounters (x2 = 0.0007, P > 0.99). When the fight

had a clear outcome, the duration of the fights was not
different (P = 0.58) among the three types of encounters.
However, in a situation where the outcome of a fight was
unclear, SEW pigs fought for a longer period of time (P =
0.0075) than CW pigs in the first day after mixing (Fig.
10). Regardless of the outcomes and types of fights, the
average duration of a fight was significantly longer in the
first day than that of the second and third day after
regrouping (16.61 vs. 7.02 and 7.98 seconds respectively, P

= 0.0001).

65

5.4 DISCUSSION

In Chapter 4 Experiment 1, I demonstrated that SEW
piglets had a reduced growth rate in the first week after
weaning when compared with suckling piglets. However, no
weight differences were observed between SEW and CW piglets
following weaning at weeks 5, 6 and 8 of age (Fig. 5). It
is likely due to that CW piglets also experienced a period
of growth decline immediately after weaning while the SEW
piglets had an opportunity to catch up in weight
development (Table 6 in Appendix). Worobec et al. (1999)
showed that at 6 weeks of age, piglets weaned at 7 days of
age were lighter than those weaned at 14 and 28 days. One
possible explanation for the differences in results could
be that the nutrient requirements of 7-day piglets are
different from those of 10-day old piglets. Alternatively,
the younger piglets (7 days old) may have a stronger bond
with the sow than my 10 days old piglets, making weaning
more difficult to cope with. Piglets under semi-natural
environment start to rest outside their own nest site only
after 8 days of age (Newberry and Wood—Gush, 1988). In

addition, by 8 days of age, piglets play an active role in

66

the reinforcement of sow—piglet relationship (Blackshaw and
Hagelso, 1990).

Previous studies in pigs have shown that the earlier
the weaning took place, the higher the incidence of belly—
nosing (Gonyou et al., 1998; Worobec et al., 1999).
Corroborating these observations, piglets weaned at 10 days
of age in the current study displayed a significantly
higher frequency of belly-nosing in weeks 5 and 7 than CW
piglets (Fig. 6a). Gonyou et al. (1998) also reported that
the highest level of belly-nosing occurred during 2 to 3
weeks post-weaning. My results showed that at 6 weeks of
age when CW piglets have been weaned for 3 weeks (SEW
piglets have been weaned for 4.5 weeks), the frequency of
belly-nosing behavior was not different between SEW and CW
piglets. Early-weaned animals displayed continued elevated
levels of belly—nosing and chewing on other pigs throughout
the growing/finishing phase (Gonyou et al., 1998). It
appears as though a higher baseline of belly—nosing
behavior may be developed as a result of early maternal
deprivation.

Contrary to my initial hypothesis, the frequency of
manipulating pen-mates was higher in CW piglets than SEW
piglets at weeks 5 and 6 of age (Fig. 6b). Previously it

was reported that if weaned piglets were stressed by

67

crowding, they were more likely to engage in the
manipulation of other animals (Dybkjar, 1992). In my study,
behavior was compared at the same age but at different post
weaning times (CW piglets were weaned for 2 or 3 weeks and
SEW piglets were weaned for 3.5 or 4.5 weeks). However, in
the study by Dybkjer (1992), behavior of piglets was
compared at the same ages and post weaning time. This may
be responsible for the discrepancies in the results.

The incidence of fighting remained constant over three
weeks in both groups, indicating that a stable social
relationship was established before week 5. Surprisingly,
the overall agonistic interactions were higher in the CW
piglets. The CW piglets fought more often at 5 weeks of age
than the SEW piglets (Fig. 6c). However, Worobec et al.
(1999) reported no differences in the aggression level in
weeks 4 and 6 between animals weaned at 7, 14 and 28 days.
In their study, instantaneous behavioral samples were
obtained every 5—min from 48 h videotapes. Aggression
included pushing, head thrusting, biting and chasing. I
monitored fighting according to the definition in Table 1
(Chapter 4). Since pushing, head thrusting, and biting do
not necessarily result in fighting, and chasing does not
necessarily result from fighting, Worobec et al.’s (1999)

results and my results are not necessarily conflicting.

68

Besides belly—nosing, another long-term change in the
behavior of SEW was the development of bar biting. While
the occurrence of bar biting in CW piglets remained the
same, it increased in SEW piglets as they grew (Fig. 6d).
Dybkjer (1992) concluded that this type of redirected oral
behavior pattern was one indicator of stress in weaned
piglets. As well as belly-nosing, manipulating penmate and
bar biting are both considered as abnormal behavior.
Animals in both treatment groups displayed these three
types of behavior abnormalities, indicating coping is
difficult in the current intensive housing system
regardless the weaning age. Hence I conclude that the
welfare of pigs in both groups is poorer than free—range
animals. The SEW piglets showed a consistent high frequency
of belly-nosing and an increase in frequency of bar biting,
suggesting that the welfare of SEW animals is poorer than
CW animals. But CW animals directed more oral activities
toward their penmates than SEW animals, suggesting that the
welfare is poorer in CW piglets than in SEW piglets.

As expected, mixing, followed by a 20-minute
transportation, induced a significant increase in salivary
cortisol concentration in 9 week old pigs (Fig. 7). This
observation was consistent with that of Hicks et al.

(1998). However, the pre- and post—transportation salivary

69

cortisol concentrations, and the percentage of increases in
salivary cortisol were not different between SEW and CW
pigs (Fig. 7). Apparently, the SEW stress and the weaning
age of piglets did not alter the response of the HPA axis
to transportation stress later in life. This result
conflicted with the literature on rodents (Plotsky and
Meaney, 1993; Liu et al., 1997). Physiological and
behavioral differences between rodents and pigs may account
for the conflicting results. Firstly, the maternal behavior
of rats or mice is different from sows confined in
farrowing crates. Maternal behavior in rodents includes
licking and grooming of the young (Liu et al., 1997). Sows
display sniffing, grunting, and deliberate nose contact
with the piglets (Blackshaw and Hagelso, 1990). Secondly,
the rats have a stress hyporesponsive period approximately
from day 4 to 14 after birth (Schapiro et al., 1962) not
seen in pigs (Klemcke and Pond, 1991). Thirdly,
transportation can be a very stressful experience for pigs,
which may maximize the response of HPA axis. It may mask
the differences in the HPA axis responsiveness between SEW
and CW pigs.

Every agonistic interaction that resulted in an
unclear outcome denied a piglet from either being

advantaged (if it was declared the winner) or disadvantaged

7O

(if it was declared the loser). According to the results,
pigs from the SEW group were subjected to the most
agonistic interactions that resulted in unclear outcomes
(Fig. 9). This information suggests that SEW pigs were more
unassertive when in combat. When the outcome was not clear,
a longer average duration of fights was also found on the
first day after mixing in SEW pigs than that of CW pigs
(Fig. 10). It requires more energy and may cause more body
damage for an animal to fight longer. This result suggests
that SEW pigs were more aggressive. All the information
imply that the social skills of SEW pigs may be impaired.
It may be due to the fact that they were given a shorter
period of time to interact with dams than CW pigs.
Interestingly, when a SEW pig fought with a CW pig, the
distribution of the outcomes was similar to that of the
fights among CW pigs (Fig. 9). The average duration of
agonistic interactions did not differ from the other two
types of encounters (Fig. 10). Two pieces of information
suggests that a dominant hierarchy became stable over the
three-day period. One was that the percentage of unclear
outcome decreased from 59.77% in the first day to 10.58% in
the third day (Fig. 8). The other was that the average
duration of a fight decreased from 17 seconds in the first

day to 8 seconds in the third day.

71

5 . 5 CONCLUSION

There were no advantages or disadvantages of SEW over
CW in the production performance indicator as measured by
their body weights. The basal cortisol concentration and
the HPA axis response to stress at nine weeks of age was
unchanged comparing SEW practice with CW practice. On the
other hand, the long-term effects of SEW seemed to be
reflected in the differences in behavioral patterns between
SEW and CW piglets. The SEW piglets showed higher frequency
of belly—nosing and lower frequency of manipulating pen-
mates than the CW piglets. The incidence of bar biting
increased with time in the SEW piglets but remained
constant in the CW piglets. In a stable group, the overall
fighting levels were low in the SEW piglets. But when the
environment was disturbed, the SEW pigs were less assertive
and had more difficulties in establishing a stable
hierarchy. These results add to the complexity of comparing
the welfare status of SEW and CW pigs. As my results
indicated, SEW pigs in some aspects may have worse welfare,
while in other cases they may have better welfare than CW
pigs. Overall, it seems that welfare is poorer in SEW pigs
than in CW pigs. I feel the evidence suggest this

conclusion outweigh the rest.

72

Chapter 6 GENERAL DISCUSSION

6 . 1 BEHAVIOR

Maternal behavior is vital to the survival of young
animals, especially in mammals. Domesticated animals are
usually cared for by humans. As a result, it is possible
for the offspring to be nutritionally independent from
their dams earlier than wild animals. Young animals,
however, also learn relevant behaviors from their
parent(s). For example, foals hand—raised by humans do not
know how to interact with other horses (Shelle, personal
communication). Therefore, a sudden deprivation of maternal
care during early development may influence the offspring’s
behavioral pattern and possibly their coping strategies. My
research work found that there were short and long term
changes in the behavioral patterns of SEW animals, most
notably in aggressive behavior and the development of

belly-nosing.

6.1.1 Belly-nosing

Belly-nosing closely resembles a principal component
of normal nursing behavior and its occurrence is clearly
associated with weaning age (Fraser, 1978; Gonyou et al.,

1998, Worobec et al., 1999). Fraser (1980) has described

73

the nursing behavior in detail. Nursing behavior starts
with the piglets vigorously nosing the udder of the sow.
This causes the sow to grunt rhythmically, and the grunts
notify any absent piglets that nursing is in progress. As
the grunting rate increases and oxytocin is released, most
piglets start sucking on the teat. The milk ejection begins
20 s after the release of oxytocin and lasts 15 to 25 8.
After the milk ejection ceases, some piglets continue to
suck, some may change quickly to a different teat, others
resume nosing the udder. It is suggested that vigorous
nosing of the udder before the milk ejection may increase
the flow of oxytocin-rich blood through the mammary gland,
thereby increasing the milk yield (Fraser, 1980). In
addition, the piglets start nosing and pushing any vertical
surface they encounter within the first 20 minutes after
birth (Hartsock and Graves, 1976). These facts lead to the
hypothesis that nosing the udder constitutes a significant
part of suckling behavior and may be critical to piglets’
growth.

The nosing directed to the sow’s udder is probably
some element of appetitive feeding in piglets. A redirected
behavior of nosing the udder, known as belly-nosing, is
developed in some piglets after the appropriate

environmental cue (sow) is removed. Therefore, it may be

74

important for piglets to be able to perform this behavior.
Furthermore, the “nursing posture4” can sometimes be
elicited in very young pigs by rubbing the belly (Fraser,
1976). This behavior response in pigs may facilitate the
development of belly-nosing.

Gardner et al., (1999) showed that the establishment
of feeding and the presence of milk in the diet had no
effect on the performance of belly-nosing in piglets weaned
between 14—18 days of age. It seems that belly—nosing is
connected to the sucking motivation.

Over the first 6 weeks postpartum, piglets increase
their activity and feeding behavior. Piglets’ interest in
solid food becomes apparent at about three weeks of age
(Fraser, 1978). Piglets develop and express more complex
behavioral patterns as they grow (Newberry and Wood-Gush,
1988). This might account for the negative relationship

between the exhibition of belly-nosing and the weaning age.

6.1.2 Aggression
Pigs have a complex social structure. They live in
groups and have hierarchies, which are established through

agonistic interactions. Pigs displayed aggressive behaviour

 

4 Sow lies on one side, exposes both rows of teats and often
thrust its front legs back (Fraser, 1984).

75

throughout their growth period. Aggressive interactions
start with the formation of teat order, which peaks at
about the second hour after birth (Hartsock and Graves,
1976). Once a teat order is established, the piglets settle
into an orderly sequence during suckling with little
fighting at the udder.

Previous studies have showed that, in commercial
settings, aggression level is high at weaning. Producers
commonly sort pigs at several production stages to create
homogenous groups. In general, the first time that pigs are
sorted is at weaning. Fraser (1978) found that artificial
weaning increases the levels of aggression in littermate
piglets. In addition, grouping of strange pigs causes
fierce fighting (Meese and Ewbank, 1973). These studies
strongly suggest that weaning combined with regrouping may
lead to intense aggression. Social stability within a newly
formed group may be observed within 24 h after mixing 3
(McGlone, 1986). Once the hierarchy is formed, fighting is
often replaced by non—contact threats (Ewbank and Meese,
1971).

The behavior sequence of fight is shown in Fig. 11
(Adapted from McGlone, 1985). Normally, fighting begins

with mutual nosing and sniffing, but quickly escalates into

76

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a contest in which two pigs face each other with their
shoulders pressed together. As the fight progresses, more
damaging behaviors occur. Submissive behavior begins with a
180-degree body turning after receiving ear biting. Then
the winner chases and tries to bite the rump and ear of the
loser. The submissive behavior can last several seconds to
a few minutes (McGlone, 1985).

The ability to establish hierarchy is important for
the stability of a group. Maintaining social stability is
important for pig welfare. McGlone (1985) suggested that
the physical damage from aggressive behavior might be
reduced if all fights have a clear outcome and the test of
strength does not have to be repeated. When an unclear
outcome (McGlone, 1985 refers that as “undecided fights”)
occurs, a hierarchy between two animals is undecided. My
result showed that the percentage of unclear outcomes
decreased from 59.77% to 10.58% during the first three days
after mixing, suggesting that a stable dominance hierarchy
was under development. Since longer duration and a higher
percentage of unclear outcome occurred when two SEW

piglets’ fought, mixing of SEW piglets should be avoided.

78

6.2 RESPONSIVENESS OF HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

The HPA axis is activated when homeostasis is
disturbed or threatened. It is hypothesized that the
stress-induced HPA axis activation protects the body by
preventing physiological reactions from overshooting,
thereby helping to restore the homeostasis (Munck et al.,
1984). Thus it is critical for an animal to have a HPA axis
that can respond properly. The responsiveness of HPA axis
can be altered by maternal care (e.g. Liu et al., 1997). An
altered HPA axis may imply a time difference in re-
achieving homeostasis and vulnerability to stress-related
diseases (Seckl and Meaney, 1993).

The development of HPA axis has a distinct pattern in
rats. Corticosterone levels in neonatal rats are high
immediately after birth, then decreases dramatically in two
days and remains low until around 14 days. This period from
4 to 14 days postpartum is known as stress hyporesponsive
period (SHRP)(Levine, 1994). During SHRP, the HPA axis does
not respond to stressors or the administration of ACTH.
However, SHRP is critical for the development of the HPA
axis. Offspring who received more maternal care during this
period demonstrated a reduced response of the HPA axis to
stressors (Liu et al., 1997). A similar period does not

exist in pigs (Klemcke and Pond, 1991). This implies that

79

the development stage of the HPA axis differs during
postnatal age between piglets and rats. Hence, maternal
care or early experience of piglets may not be able to
alter the HPA axis responsiveness in their adult life. My
results support this hypothesis by showing that the
response of HPA axis to transportation is similar between

SEW and CW pigs.

6.3 BEHAVIOR AND HORMONES

A causal relationship between corticotropin releasing
hormone (CRH) and certain behaviors has been suggested. CRH
is a 41 amino acid peptide that is mainly released from the
hypothalamus. CRH triggers the activation of the HPA axis
and also acts as a neurotransmitter within the central
nervous system (CNS) (Owens and Nemeroff, 1991). Previous
studies on CRH strongly suggest that CRH integrates not
only the endocrine but also the autonomic, immunological
and behavioral responses of mammalians to stress (Owens and
Nemeroff, 1991). For example, CRH increases locomotion in
familiar environments and suppresses locomotion and
vocalization in novel, stressful environments (Owens and
Nemeroff, 1991; McInturf and Hennessy, 1996). CRH reduces
food consumption in both familiar and novel environments

(Britton et al., 1982; Berridge and Dunn, 1986). Sexual

80

behavior is also potently inhibited by central
administration of CRH in both males and females (Owens and
Nemeroff, 1991). In addition, research data supported that
CRH may mediate many of the fear—related behaviors of
stress (Britton et al., 1982; Swerdlow et al., 1989).
Results on the relationship between glucocorticoids
and stereotypies are conflicting. Glucocorticoids are the
end products of the HPA axis. Stereotypies are abnormal
behaviors characterized by a repetitive, invariant sequence
of behavior without obvious function. Stereotypies are
often observed in farm and zoo animals housed in a confined
environment. Zanella and Mason (1997) reported a lower
concentration of plasma cortisol in mink with high
stereotypy than mink with low stereotypy. Von Borell and
Hurnik (1991) found that sows showing stereotypies had a
higher cortisol response to ACTH challenge than sows
without stereotypies. However, Terlouw et al. (1991)
reported no correlation between the exhibition of
stereotypies and plasma cortisol level in sows. Due to the
loss of more than 200 saliva samples, I have limited
information on the activity of HPA axis in SEW versus CW
pigs to assess its relationship with abnormal behavior,

including belly-nosing, manipulating, and bar biting.

81

6.4 ANIMAL WELFARE AND SEGREGATED EARLY WEANING
Human—animal relationship changes as society evolves.
Currently, social attitudes have been challenged by the
claims that “the interests of non—human animals are being
neglected, and that we have a moral obligation to consider
them more than we do at present" (Broom and Johnson, 1993).
Since there is a high public demand to address these
concerns, philosophers and biologists promote the study of
animal welfare. Important welfare indicators include growth
rate, behavior patterns, endocrine profile, susceptibility
to disease, body damage, mortality, and reproduction
performance (Blackshaw, 1986; Broom and Johnson, 1993;
Wechsler, 1995). As I mentioned before, welfare is defined
as the state of an individual in regard to its attempts to
cope with its environment (Broom and Johnson, 1993). Since
welfare of an individual depends on the efforts to cope and
the outcome of the coping attempts, this definition implies
that welfare could vary from very poor to very good. For
example, death, failure to grow, or failure to reproduce
are evidences of failure to cope, where welfare is
considered very poor. Sometimes, animal may succeed in
coping but with great difficulty. In this case, the animal

welfare is still poor, but better than if it fails to cope.

82

This research focused on the welfare of SEW piglets.

The results of the welfare study were as follows:

1” SEW caused vocalization, climbing, belly-nosing and an
increase in activity levels within the first three days
post—weaning, and a reduced growth rate in the first week
post—weaning. Taken together, these data suggested that
the welfare of SEW piglets was poor during the initial
post—weaning period.

2.1he urinary cortisol concentration continued to be
elevated for three days post weaning. This result also
indicates that welfare problems may exist for at least
three days post weaning in SEW piglets.

3. SEW animals displayed elevated levels of belly-nosing
throughout the two-month experiment period. Belly-nosing
is an abnormal behavior and can lead to skin lesions in
the piglets who receive it (Worobec, 1999). Thus, welfare
is reduced in animals that are exhibiting or receiving
belly—nosing. As discussed before, nosing warm and soft
surfaces is probably important to piglets. To minimize
the occurrence of belly-nosing, one solution is to wean
the piglets older than 3 weeks of age because of a
negative correlation between weaning age and belly-
nosing. Though no research has been done yet, another

solution may be to provide appropriate environmental

83

enrichment devices, which have warm and soft surfaces, so
that the occurrence of nosing pen-mates’ bellies could be
reduced.

14.The impaired ability to establish a stable hierarchy (as
indicated by the duration of fight and the proportion of
unclear outcome after mixing) also suggests a poor
welfare of SEW pigs, because impaired social skills
implies that coping is more difficult for these pigs in
new social environments. To solve this problem, more
information is needed on the fundamentals of aggressive
behavior, the maternal behavior of sow and its influence
on piglets.

In summary, the results suggest that the welfare of
the SEW piglets may be poor. Therefore the SEW practice
needs to be re—evaluated and modified to accommodate the

nature and biological needs of the animals.

84

APPENDIX

85

 

APPENDIX

Table 4: Summary of basic statistics on behavioral and
physiological data in Experiment 1:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dependent . Geometric Standard . . .
. Unit . . Minimum .Max1mum
variable mean dev1ation
Lying % 66.30 31.00 0 100
Standing % 7.25 9.71 0 45.88
Sitting % 1.58 4.35 0 57.38
Moving % 7.70 13.10 0 80.50
Rooting % 1.36 4.38 0 39.30
Fighting minT 0.08 0.19 0 1.25
Belly-nosing minJ' 0.06 0.27 0 2.75
Manliiiftlng min‘1 0.05 0.13 0 0.75
M°n1521itlng mind' 0.02 0.07 0 0.5
Manipulating
other part min"1 0.13 0.27 0 1.75
of the body
M:::°2::g % 4.00 8.20 0 42.90
Siiijiiwft % 11.28 17.61 0 75.71
Eating % 3.50 7.90 0 48.70
Drinking % 0.6 2.00 0 14.30
Bar biting minq' 0.02 0.11 0 1.25
Grunting min'1 0.17 0.79 0 7.25
Squealing min4 0.03 0.26 0 3.75
Climbing min4' 0.03 0.10 0 0.75
Jumping min‘1 0.03 0.18 0 1.75
Cortisol mol/l 0.25 0.30 0.03 1.69
Creatinine mol/l 2.71 2.54 0.41 12.41
UCCR mmol/mol 94.23 70.37 23.74 405.87

 

 

86

Table 5: Summary of basic statistics on behavioral and
cortisol data in Experiment 2:

 

 

 

 

 

 

 

 

 

 

Dependent . Geometric Standard . . .
. Unit . . Minimum Max1mum
variable mean dev1at1on
Belly-nosing h"1 2.61 3.39 0 19.33
manliiiftlng h“ 1.85 1.96 0 12
Manlg::itlng h‘1 0.95 1.15 0 8
Manipulating
other part rrl 3.90 2.54 0 13.33
of the body
Bar biting h4- 1.51 1.73 0 8
Fighting h*- 1.97 1.57 0 6
Drinking h'1 3.45 2.23 0 12.67
Cortisol mmol/l 7.59 7.78 .38 28.94
Flizidiifer 8 19.63 47.14 1 1047

 

 

87

Table 6: Summary of body weight of SEW and CW piglets in
Experiment 2:

 

 

 

w ' h
?::)t Day 10 Day 18 Day 21 Day 40 Day 46 Day 61
SEW 3.23: 4.18: 9.88: 12.71: 20.83:
0.16 0.21 0.51 0.65 1.07
CW 4.91: 9.12: 11.95: 19.71:
0.25 0.47 0.61 1.01

 

 

88

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89

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