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Use of photoperiod management and microwave toe-treatment to improve carcass quality in broiler chickens

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USE OF PHOTOPERIOD MANAGEMENT AND MICROWAVE TOETREATMENT TO IMPROVE CARCASS QUALITY IN BROILER CHICKENS
by
Baoyan Wang
Submitted in partial fulfillment o f the requirements
for the degree o f Master o f Science
at
Dalhousie University
Halifax, Nova Scotia
and
Nova Scotia Agricultural College
Truro, Nova Scotia
December, 2004
© Copyright by Baoyan Wang, 2004
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TABLE OF CONTENTS
LIST OF TA B LES........................................................................................................................ ix
ABSTRACT................................................................................................................................... xi
LIST OF ABBREVIATIONS AND SY M B O LS................................................................... xii
ACKNOW LEDGEMENTS...................................................................................................... xiv
CHAPTER 1 GENERAL INTRODUCTION...........................................................................1
CHAPTER 2 LITERATURE R EV IE W ................................................................................... 5
2.1
Photoperiod.................................................................................................................... 5
2.1.1
Photoperiodic Response in B irds...........................................................................5
2.1.1.1
The Critical Daylength and Saturation Daylength........................................5
2.1.1.2
Photosensitive Phase......................................................................................... 6
2.1.1.3
Photorefractoriness........................................................................................... 6
2.1.2
Light Reception........................................................................................................ 7
2.1.3
Photoperiod and M elatonin.....................................................................................8
2.1.3.1
Circadian Rhythm o f Melatonin and Its Modulation by Photoperiod
8
2.1.3.2
Photoperiod, Melatonin and Immune Function.......................................... 10
2.1.4
Impact o f Photoperiod on Perform ance.............................................................. 12
2.1.5
Impact o f Photoperiod on Welfare and H ealth .................................................. 14
2.1.6
Light Intensity.........................................................................................................16
2.2
Stress and F ear.............................................................................................................17
2.2.1
Introduction............................................................................................................. 17
2.2.2
Definitions o f Stress and Fear.............................................................................. 18
2.2.3
Biological Response to Stress.............................................................................. 19
iv
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2.2.4 Influence o f Stress and Fear on Perform ance.....................................................21
2.2.5
Assessments o f Stress and F e a r............................................................................ 22
2.2.5.1
The Heterophil/Lymphocyte R a tio .............................................................. 23
2.2.5.1.1
Avian Blood Cell Types and Their Functions....................................23
2.2.5.1.2
Significance o f the Heterophil/Lymphocyte R atio ............................ 24
2.2.5.2
Creatine K inase............................................................................................... 26
2.2.5.3
Tonic Immobility R eaction........................................................................... 27
2.3
The Skin........................................................................................................................ 29
2.3.1
Structure and Function o f the S k in .......................................................................29
2.3.2 C ollagen....................................................................................................................30
2.3.2.1
Structure and Function o f C ollagen............................................................. 30
2.3.2.2
Collagen T ypes............................................................................................... 32
2.3.2.3
The Matrix Metalloproteinase....................................................................... 33
2.3.3
2.4
Skin Strength and C ollagen.................................................................................. 34
Carcass D efects...............................................................................
2.4.1
37
Scabby Hip Syndrom e............................................................................................37
2.4.2 Avian C ellulitis........................................................................................................38
2.5
Toe-treatment............................................................................................................... 39
2.5.1
T oe-treatment T echniques..................................................................................... 39
2.5.2 Effects o f Toe-treatment on Carcass Scratches and Perform ance...................40
2.5.3
2.6
Effects o f Toe-treatment on Stress and Fear R esponse.....................................41
O bjectives.....................................................................................................................42
CHAPTER 3 PERFORMANCE OF MICROW AVE TOE-TREATED BROILER
CHICKENS GROWN WITH TWO PHOTOPERIOD PR O G R A M S
v
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43
3.1
Abstract
.43
3.2
Introduction..................................................................................................................44
3.3
Materials and M ethods................................................................................................ 46
3.3.1
Birds, Diets and H ousing..................................................................................... 46
3.3.2
Microwave Toe-treatment.................................................................................... 47
3.3.3
Experimental Lighting Program s........................................................................ 49
3.3.4
Performance Parameter Determination.............................................................. 49
3.3.5
M ortality............................................................................................................... 50
3.3.6Statistical Analysis...................................................................................................... 51
3.4
Results and D iscussion............................................................................................... 52
3.4.1
Body Weight and Body W eight G ain.................................................................52
3.4.2
Feed Intake and Feed Conversion R atio ............................................................ 62
3.4.3
M ortality............................................................................................................... 68
3.5
Conclusions................................................................................................................... 71
CHAPTER 4 STRESS AND FEAR LEVELS OF MICROWAVE TOE-TREATED
BROILER CHICKENS GROW N WITH TWO PHOTOPERIOD
PROGRAM S.......................................................................................................73
4.1
A bstract..........................................................................................................................73
4.2
Introduction................................................................................................................... 74
4.3
Materials and M ethods................................................................................................ 77
4.3.1
Birds, Diets and H ousing..................................................................................... 77
4.3.2
Microwave Toe-treatment.....................................................................................80
4.3.3
Experimental Lighting Program s........................................................................ 80
4.3.4
Blood Sam pling..................................................................................................... 81
4.3.5
The Tonic Immobility T e st.................................................................................. 82
vi
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4.3.6
Laboratory A nalysis.............................................................................................. 82
4.3.7
Statistical A nalysis.................................................................................................83
4.4
Results and D iscussion............................................................................................... 85
4.4.1
The Activity o f Plasma Creatine Kinase.............................................................85
4.4.2
The Heterophil to Lymphocyte R atio .................................................................87
4.4.3
The Tonic Immobility T e st.................................................................................. 91
4.5
Conclusions................................................................................................................... 96
CHAPTER 5 CARCASS QUALITY AND SKIN INTEGRITY OF MICROWAVE
TOE-TREATED BROILER CHICKENS GROWN WITH TWO
PHOTOPERIOD PROGRAM S.......................................................................97
5.1
A bstract..........................................................................................................................97
5.2
Introduction................................................................................................................... 98
5.3
Materials and M ethods...............................................................................................100
5.3.1
Birds, Diets and H ousing....................................................................................100
5.3.2
Microwave T oe-treatment...................................................................................103
5.3.3
Experimental Lighting Program s.......................................................................103
5.3.4
Blood Sam pling................................................................................................... 104
5.3.5
Skin Sampling....................................................................................................... 104
5.3.6
Carcass Scratches and B ruises........................................................................... 105
5.3.7
Laboratory A nalysis.............................................................................................105
5.3.7.1
Skin Puncture S trength...............................................................................105
5.3.7.2
Zym ography..................................................................................
5.3.7.3
Imm unohistochem istry...............................................................................107
5.3.8
5.4
106
Statistical A nalysis............................................................................................... 108
Results and D iscussion............................................................................................ 110
vii
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5.4.1
Carcass Scratches and B ruises............................................................................110
5.4.2
Skin Puncture S trength........................................................................................113
5.4.3
Zymography and Imm unohistochemistry.........................................................116
5.5
Conclusions................................................................................................................120
CHAPTER 6 OVERALL DISSCUSSION AND CONCLUSION...................................121
REFERENCES .........................................................................................................................125
viii
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LIST OF TABLES
Table 3. L Ingredient composition and calculated analyses o f diets used in the
experim ent................................................................................................................ 48
Table 3. I Two lighting programs used in the experim ent...................................................49
Table 3. 5 Effect o f microwave toe-treatment on body weight o f broiler chickens based
on the traditional weighing method and computerized bird scales at 0, 7, 14,
24, 31, and 38 days o f a g e ...................................................................................... 53
Table 3. 1 Effect o f lighting on body weight o f broiler chickens based on the traditional
weighing method and computerized bird scales at 0, 7, 14, 24, 31, and 38 days
o f a g e ..........................................................................................................................54
Table 3. > Effect o f sex on body weight o f broiler chickens based on the traditional
weighing method and computerized bird scale data at 0, 7, 14, 24, 31, and 38
days o f age.................................................................................................................55
Table 3. i Lighting by sex interaction for body weight o f broilers based on the traditional
weighing method and computerized bird scales..................................................56
Table 3. 7 Effect o f lighting, microwave toe-treatment, and sex on body weight gain o f
broiler chickens at 0-6, 7-13, 14-23, 24-30, and 31-38 day periods based on
the traditional weighing m ethod.......................................................................... 57
Table 3. I Effect o f lighting, microwave toe-treatment, and sex on body weight gain o f
broiler chickens at 0-6, 7-13,14-23, 24-30, and 31-38 day periods based on
computerized bird scale d a ta..................................................................................58
Table 3. ) Lighting by sex interaction for body weight gain o f broilers based on
computerized bird scales........................................................................................ 58
Table 3. 10 Effect o f lighting, microwave toe-treatment, and sex on feed intake o f broiler
chickens at 0-6, 7-13, 14- 23, 31-38, and 0-38 day periods............................. 64
Table 3. II Lighting by sex interaction for feed intake o f b ro ilers....................................65
Table 3. 12 Toe-treatment by sex interaction for feed intake o f b ro ilers..........................65
Table 3. [3 Effect o f lighting, microwave toe-treatment and sex on feed conversion ratio
based on computerized bird scales and the traditional weighing method
during 0-6, 7-13, 14-23, 24-30, 31-38, and 0-38 day periods..........................66
Table 3. 14 Effect o f lighting, microwave toe-treatment and sex on mortality o f broiler
chickens during 0-7 and 0-38 day periods......................................................... 70
ix
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Table 4.
Ingredient composition and calculated analyses o f diets used in the
experim ent.................................................................................................................79
Table 4. 1 Two lighting programs used in the experim ent.................................................. 81
Table 4. i Effect o f lighting, microwave toe-treatment, and sex on the activity o f plasma
creatine kinase o f broiler chickens at 21 and 35 days o f a g e ............................ 86
Table 4. 1 Effect o f lighting, microwave toe-treatment, and sex on the
heterophil/lymphocyte ratio o f broiler chickens at 21 and 35 days o f age
88
Table 4. i Effect o f lighting, microwave toe-treatment, and sex on the duration o f tonic
immobility o f broiler chickens at 10, 22, and 36 days o f a g e ........................... 92
Table 5.
Ingredient composition and calculated analyses o f diets used in the
experim ent...............................................................................................................102
Table 5. ’ Two lighting programs used in the experim ent................................................ 104
Table 5. > Effect o f lighting, microwave toe-treatment and sex on carcass scratches and
bruises at 38 days o f age........................................................................................111
Table 5. 1 Effect o f lighting, toe-treatment and sex on skin puncture strength in broiler
chickens at 38 days o f age.....................................................................................114
Table 5. > Effect o f lighting, microwave toe-treatment, and sex on the activity o f plasma
pro-MMP-2 and active MMP-2 in broiler chickens at 21 and 35 days o f age
...................................................................................................................................117
Table 5. > Means o f the scores o f skin type I and III collagen staining intensity...........118
x
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ABSTRACT
This study was conducted to evaluate microwave toe-treatment in combination with
increasing photoperiod as a means to improve carcass quality in broiler chickens. The toe
tips o f day-old broilers were exposed to microwave energy to restrict claw growth. Birds
were grown under two photoperiod programs known to influence bird activity and growth
rates and suspected o f altering collagen metabolism. Bird performance and the level o f
stress and fear were measured. Zymography and immunohistochemistry were employed
to determine the activity o f plasma collagenase and skin type I and type III collagen
content. The results indicate that the microwave toe-treatment improved carcass quality
by reducing carcass scratches and increasing skin strength without detrimental effects on
growth performance and wellbeing o f the birds. The increasing photoperiod program
decreased mortality and reduced feeding costs, as well as tended to increase type I
collagen content in the skin without reducing overall performance o f the birds.
XI
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LIST OF ABBREVIATIONS AND SYMBOLS
ACTH
Adrenocorticotropic hormone
AA-NAT
Arylalkylamine N-acetyltransferase
ABC
Avidin and Biotinylated horseradish peroxidase macromolecular Complex
BW
Body weight
BWG
Body weight gain
BSA
Bovine serum albumin
CaCl2
Calcium chloride
C a 2+
Calcium ions
cm
Centimeter
CL
Continuous/Constant lighting
CS
Corticosterone
CK
Creatine kinase
d
Day
°C
Degrees Celsius
df
Degrees o f freedom
DAB
3, 3’-diaminobenzidine
EDS
Ehlers-Danlos syndrome
FCR
Feed conversion ratio
FI
Feed intake
g
Gram
FI/L
Heterophil/lymphocyte
h
Hour
HC1
Hydrochloric acid
h 2o 2
Hydrogen peroxide
fflOMT
Hydroxyindole- O-methyltransferase
HPA
Hypothalamic-pituitary- adrenocortical
IL
Increasing lighting
IT
Intact-toe
kcal
Kilocalorie
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kg
Kilogram
LSMeans
Least squares means
L/D
Light/Darkness
Log
Logarithm
LH
Luteinizing hormone
MMP
Matrix metalloproteinase
ME
Metabolizable energy
MCP
Microwave claw processor
pg/pm
Microgram/micrometer
mg/ml/mm
Milligram, milliliter, millimeter
mM
M illim ole
min
Minute
N
Newton
PSE
Pale, soft, and exudative
%
Percent
PBS
Phosphate buifer saline
PAGE
Polyacrylamide gel electrophoresis
KC1
Potassium chloride
KH2PO4
Potassium phosphate monobasic
p
Probability
s
Second
N aN 3
Sodium azide
NaCl
Sodium chloride
SDS
Sodium dodecyl sulfate
N a 2HP 0 4
Sodium phosphate
SEM
Standard error o f the mean
SAM
Sympathetic-adrenal-medullary
TT
Toe-treated
TI
Tonic immobility
USDA
The United States Department o f Agriculture
wk
Week
xiii
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ACKNOWLEDGEMENTS
I would like to express my sincere thanks to everyone who put time and effort
toward helping me complete this project and thesis. First o f all, I would like to thank my
supervisor, Dr. Bruce Rathgeber, for his constant guidance and never ending support as
well as his kindness. I would also like to thank Dr. Kirsti Rouvinen-Watt and Dr. Taijei
Tennessen for serving on my supervisory committee and bringing their extensive
knowledge and expertise o f animal research into this project. Special thanks to Dr. Tess
Astatkie for his assistance with statistical methodologies and interpretation. Thanks to Dr.
Leslie MacLaren for her advice on immuohistochemistry and generously loaning the use
o f equipment required for this analysis. Thanks to Dr. Jim Duston and Dr. Henry Classen
for acting as external examiner o f m y Admission to Candidate (ATC) examination and
thesis defense, respectively.
Big thanks go to Janice Maclsaac for her technical support and management for
this project. I am forever grateful to Hai Choo Smith and Judy Grant for lending their
technical e xpertise o n i mmunohistochemistry and b lood c ell c ounting. T hanks t o A lex
Oderkirk for helping with blood sampling. I would like to extend m y sincere gratitude to
the staff o f Nova Scotia Agricultural College (NSAC) poultry unit for their technical
help. I also would like to thank faculty, staff and graduate students at the Department o f
Plant and Animal Sciences o f NSAC for their assistance and friendship. I very much
appreciate the help and encouragement from Ms. Jill Rogers throughout m y M.Sc.
program. Thanks to Nova Tech Engineering Inc. for loaning the use o f the microwave
toe-treatment unit.
Funding support provided by ACA-Cooperative Ltd., Nova Scotia Department o f
Agriculture & Fisheries, and Poultry Industry Council was greatly appreciated.
I would like to thank m y family and friends for their encouragement and support
through everything I do. Finally, to my husband, Hongyong, thank you for always
encouraging, understanding and helping me throughout m y M.Sc. Program.
xiv
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1
CHAPTER 1
GENERAL INTRODUCTION
M odem commercial broiler chickens have been genetically selected for rapid
growth and muscle mass. Selection for rapid growth and efficient feed conversion has
resulted in a broiler with such a high rate o f metabolism that its heart and lungs can be
inadequate to sustain the body (Julian, 1990). These birds often develop problems with
their cardiovascular system, as they are susceptible to pulmonary hypertension and heart
failure, which have become some o f the leading causes o f mortality and whole carcass
condemnations in modem broiler flocks throughout the world (Olkowski et al., 1996;
Jakob et al., 1998; Maxwell and Robertson, 1998). The most consistent method for
reducing the incidence o f these diseases in broiler flocks is to reduce the rate o f early
growth in order to reduce the demand for oxygen.
This m ay be accomplished by
restricting the feed available to the birds or giving low density feed. However, a more
practical method is to manipulate the photoperiod length the birds are exposed to.
Photoperiod management in broiler production has evolved over years, and a wide
range o f lighting programs have been used in the broiler industry. Broiler chickens have
traditionally been kept on a continuous or nearly continuous lighting schedule (24 or 23
h light per day) so as to maximize feed intake and growth rate (Morris, 1967; Beane et
al., 1979). However, some research suggests that continuous lighting disrupts the
diurnal rhythm and has serious welfare consequences for growing broiler chickens, such
as decreased sleep, higher physiological stress and decreased immune response
(Gordon, 1994; Rozenboim et al., 1999; Sanotra et al., 2002). Additionally, it has been
shown t hat r earing b roilers u nder 1ighting p rograms w ith 1ong d arkness r educed s kin
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2
tearing at the processing plant compared to continuous lighting programs (Hammershoj,
1997). Downgrading problems associated with skin integrity such as cuts and tears
cause substantial economic losses to the broiler industry (Bilgili et al., 1993).
Christensen et al. (1994) found that birds with lower skin strength exhibited an
increased incidence o f skin tears during slaughter in the commercial processing plant.
Lighting programs that provide short photoperiod early in life and increase
daylengths as a broiler ages have been described as increasing lighting programs
(Gordon, 1994). Rearing broiler chickens with increasing daylengths has been shown to
reduce the incidence o f heart disease and leg problems compared to continuous lighting
programs (Classen et al., 1991; Rozenboim et al., 1999). Olkowski et al. (2001)
demonstrated that broilers raised at cool temperature increased the incidence o f heart
disease
and
those
birds
with
heart
disease
had
elevated
levels
o f matrix
metalloproteinase-2 (MMP-2) in the cystolic fraction o f cardiac tissue. Matrix
metalloproteinases (MMPs) are responsible for extracellular collagen degradation and
remodeling (Spinale et al., 1999). Collagen is the principal protein in the skin and is a
major determinant o f skin strength (Granot et al., 1991a). Collagen has also been
proposed to be a major determinant o f skin tears (Granot et al., 1991b). So if continuous
lighting also increases the activity o f plasma MMPs or influences the synthesis o f
collagen, this may reduce the structural integrity o f collagen and increase susceptibility
o f the broiler's skin to bruises and tears.
In spite o f the fact that continuous photoperiod programs increase heart disease
and cause welfare consequences in broilers, producers continue to use this lighting
program. One reason is to reduce the incidence o f carcass scratches. Birds on increasing
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3
lighting or light-dark programs have been reported to have an increased incidence o f
carcass scratches due to increased bird activity (Blair et al., 1993; Sanotra et al., 2002).
During the broiler’s short life, scratches may develop into an inflammation o f the skin
or dermatitis known as scabby hip syndrome (Proudfoot and Hulan, 1985), a severely
afflicted flock o f which may have the number o f Grade A carcasses reduced by as much
as 5 0% ( Proudfoot and Hulan, 1 985). S cratches a Iso c an 1ead t o t he d evelopment o f
cellulitis, which is the most common cause for carcass condemnation in Canada (Kumor
et al., 1998). The primary cause for carcass scratches has been shown to be likely the
injury inflicted by toenails o f other broilers (Hargis et al., 1989).
A potential solution for reducing skin scratching damage is to treat toes o f day-old
broiler chickens with microwave energy. Toe-treatment is a common practice in the
turkey industry to reduce downgrading due to scratching (Moran, 1985; M cEwen and
Barbut, 1992). Traditionally, a hot blade or surgical scissors was used to trim the
toenails o f turkeys (Owings et al., 1972; Greene and Eldridge, 1975). Currently, the
turkey industry uses microwave technology to restrict claw growth and reduce carcass
scratches. The cost o f this new technology has prohibited the use o f it with short lived
broiler chickens until now. Documentation o f the effects o f toe-treatment on carcass
scratches and growth o f birds is limited and inconsistent (Proudfoot et al., 1979; Moran,
1985; Hargis et al., 1989; Newberry, 1992). With animal welfare issues at the forefront
o f animal production it has become important to evaluate the effect o f toe-treatment on
bird wellbeing as well as growth performance. Toe-treatment m ay pose an initial stress
to birds (Compton et al., 1981); however, the physical insult caused by toe-treatment
early in life may easily outweigh the chronic pain o f lacerations to the skin o f birds later
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4
in the rearing period. Honaker and Ruszler (2004) found that microwave toe-treated
layers displayed a lower level o f fearfulness to humans as measured by the tendency o f
birds to not panic in the presence o f a human. Compton et al. (1981) and Satterlee et al.
(1985) also reported toe-clipped birds to be less active and fearful. If microwave toetreatment is beneficial in reducing the level o f fearfulness in broiler chickens, this
aspect could have positive impact on welfare as well as growth performance o f broilers.
This study was conducted to evaluate microwave toe-treatment in combination
with increasing photoperiod as a means to improve carcass quality in broiler chickens.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
5
CHAPTER 2
LITERATURE REVIEW
2.1 Photoperiod
2.1.1 Photoperiodic Response in Birds
2.1.1.1
The Critical Daylength and Saturation Daylength
The vast majority o f avian species including chickens are diurnal (daylight
dwelling). The cycle o f light to dark is known as the circadian cycle and the sunrise to
sunset segment is a photoperiod or daylength. The minimum daylength (photoperiod)
that will induce reproductive development is the critical daylength (Lofts, 1970).
Plasma luteinizing hormone (LH) concentration is low when daylength is below the
critical daylength for the chicken (Leeson and Summers, 2000). Increases in daylength
above the critical daylength stimulate increased LH release; however, a point is reached
when any further increase in daylength does not give further increase in plasma LH, this
is called the saturation daylength (Lofts, 1970). Reproduction is best stimulated when
the daylength is greater than the critical daylength and less than the saturation
daylength. Critical and saturation daylengths are about 10 h and 14 h respectively for
chickens (North and Bell, 1990). A turkey hen will begin and continue to lay when
provided with daylength that exceeds the critical daylength (Proudman and Siopes,
2004). This varies by season and for optimum egg production is approximately 11-11.5
h in winter and at least 14 h in summer (Siopes, 1994).
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6
2.1.1.2 Photosensitive Phase
Birds differentiate night from day because o f the effect o f light stimulating the
hypothalamus in the brain. Light energy is converted into neural transmissions that
ultimately guide the pituitary in releasing all important gonadotrophin releasing
hormones (Leeson and Summers, 2000). However, birds are really not "stimulated" by
the e ntire p eriod o f 1ight, b ut r ather b y t wo i mportant p arts o f t his p eriod. B irds a re
sensitive to the time o f initial “light-on" and subsequently during a period 11-13 hours
later (Leeson and Summers, 2000). This latter period is called the photosensitive phase,
and essentially indicates whether or not the bird perceives the day as being “long” or
“short” (Beck, 1963). A short day is not stimulatory, w hereas a long day initiates or
maintains the cascade o f hormonal release that controls ovulation or spermatogenesis
(Robinson and Renema, 1999). Therefore, if lights are on during the photosensitive
period, then the light is stimulatory for reproduction, in detail, if birds perceive light
during the photosensitive phase which occurs 11-13 hours after initiation o f natural
dawn or “light on”, then the ovary or testes can be functional (Shanawany, 1993). This
pattern o f dawn/dusk or light on/light off sets the circadian rhythm o f the bird, which is
essentially an inherent biological clock (Bunning, 1967).
2.1.1.3 Photorefractoriness
Switching “on”and “o f f ’the seasonal responses in many species is regulated by
the development o f photorefractoriness - a phenomenon when the organism no longer
remains capable o f a stimulatory response to a photoperiod (Rose, 1997). Photosensitive
avian species undergo neuroendocrine changes during a reproductive season that cause
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7
them to gradually become unresponsive to a photoperiod that initially stimulated
reproduction
(Siopes
and
Proudman,
2003).
In
photoperiodic
species,
photorefractoriness is dissipated by providing sufficient weeks o f a photoperiod that is
below the critical daylength (Proudman and Siopes, 2004). The development o f
photorefractoriness is a natural process that assures that reproductive activity will occur
at times o f the year that maximizes chances for survival o f the young in nature (Nicholls
et al., 1988). Photorefractoriness may be qualitatively different between species.
Broadly,
two
categories
of
photorefractoriness
are
described:
absolute
photorefractoriness and relative photorefractoriness. Prolonged exposure to long days
will shift the critical and saturation daylengths upwards, this is called relative
photorefractoriness (Siopes and Proudman, 2003). Absolute photorefractoriness means
that the critical and saturation daylengths are fixed (Siopes and Proudman, 2003).
Chickens are a species with relative photorefractoriness; juvenile birds will eventually
reach sexual maturity even if they are kept on short days (Lofts, 1970). Turkeys exhibit
both relative and absolute photorefractoriness (Proudman and Siopes, 2002).
2.1.2 Light Reception
In birds, photic information can be received by photoreceptors in the retina and
the pineal gland, as well as in the brain (Brandstatter, 2003). The avian eye is also a
functional photoreceptor; it contains a circadian clock and produces a circadian rhythm
of melatonin release (Binkley et al., 1980). The avian circadian system has at least three
elements - pineal, eyes, and hypothalamic pacemaker (Dawson et al., 2001). The pineal
o f birds not only produces circadian rhythms but also combines stored information o f
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8
previously experienced light-dark cycles to influence its pacemaker output (Dawson and
King, 1994; Brandstatter et al., 2000). A memory o f the previous photoperiod allows
the animal to compare it with the present photoperiod to determine whether photoperiod
is decreasing or increasing, and could smooth out photoperiodic noise caused by
weather conditions affecting dawn or dusk light intensity (Dawson et al., 2001).
The key role o f light intensity relates to the bird's perception o f night and day.
Chickens appear to need a minimum o f a 10-fold difference in light intensity between
night and day to properly distinguish them (North and Bell, 1990). The quantity o f light
necessary for chickens to see and eat is unusually low, and after some training, the birds
will find their way to feeders and eat when the light intensity is less than 2.5 lux (North
and Bell, 1990). However, it requires from two to four times this amount o f light to
stimulate the pituitary (North and Bell, 1990). It seems reasonable to assume that a
minimum o f 5-6 lux is needed to distinguish day from night, as well as to generate the
maximum photoperiodic response (North and Bell, 1990).
2.1.3 Photoperiod and Melatonin
2.1.3.1
Circadian Rhythm of Melatonin and Its Modulation by Photoperiod
Melatonin (N-acetyl-5-methoxytryptamine) is a hormone that is synthesized and
secreted during the dark phase o f the light-dark cycle in virtually all species, regardless
o f whether they are day active or night active (Pevet, 1998). This makes melatonin
rhythm an endocrine marker o f night. Duration o f the melatonin increase is controlled
by photoperiod, the longer the night, the longer the duration o f secretion in most species
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9
(Arendt, 1995). Stated differently, long-duration melatonin is equivalent to short days
and short-duration melatonin is equivalent to long days, so melatonin is called the
darkness hormone. The fact that blinding did not obviously affect pineal melatonin level
nor did pinealectomy affect ocular melatonin levels indicates that melatonin is
synthesized in both eyes and pineal glands in birds (Underwood et al., 1984). Cockrem
(1991) proposed that circadian rhythms o f melatonin secretion in birds are influenced
not o nly b y d aylength b ut a Iso b y 1ight i ntensity. C ockrem ( 1991) i nvestigated d aily
patterns o f melatonin secretion in adelie penguins under natural continuous daylight at
Cape Bird, Antarctica, during the summer season and noted that some birds had periods
o f increased melatonin levels that tended to occur during the time o f day w hen light
intensity was least. A single daily light pulse o f suitable intensity and duration in
otherwise constant darkness is sufficient to phase shift and to synchronize the melatonin
rhythm to 24 h in animals (Illnerova, 1988). The nocturnal melatonin increase is,
however, abolished or substantially reduced in animals maintained in constant light. In
animals, exposed to light during night, even a short light pulse, melatonin synthesis is
inhibited, and melatonin concentration declines rapidly (Vanecek and Illnerova, 1982).
Melatonin synthesis from serotonin is controlled by two enzymes: arylalkylamine
N-acetyltransferase
(AA-NAT)
and hydroxyindole-O-methyltransferase
(HIOMT)
(Greve et al., 1993), where the key regulatory enzyme is AA-NAT. M elatonin and AANAT exhibit dark-time peak, while HIOMT has but small daily changes (Greve et al.,
1993). In vivo, AA-NAT activity in chicken retina photoreceptor cells exhibits a
circadian rhythm that peaks at night in darkness, AA-NAT activity is low during the
daytime, and suppressed by light exposure at night (Ivanova and Iuvone, 2003a;
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10
Ivanova and Iuvone, 2003b). The above results suggest that melatonin’s daily rhythm is
due to AA-NAT. Melatonin is metabolized primarily in the liver by hydroxylation to 6hyroxymelatonin, which is then converted to a sulfate or a glucuronide (Kopin et al.,
1961). In summary, light controls melatonin: light sets the phase o f the melatonin
rhythms and determines the duration o f melatonin synthesis. By this means, circadian
rhythms (e.g. locomotor activity and body temperature) and seasonal rhythms (e.g.
reproduction) are controlled.
2.1.3.2 Photoperiod, Melatonin and Immune Function
A relationship between melatonin and the immune system was discovered 30
years ago
and in virtually
all cases, melatonin has been proven to have
immunoenhancing effects (Reiter, 2003). The fact that melatonin secretion mediated by
photoperiod, d irectly i nfluences i mmune f unction i s w ell-documented i n h umans a nd
animals (Hotchkiss and Nelson, 2002). Typically, this immunoenhancement has been
examined in immuno-suppressed animals. Moore and Siopes (2002) conducted an
experiment to determine if transient and/or continuous melatonin treatments could
enhance immune functions in Japanese quail without prior immunosuppression. All
quail were kept on an 8:16 LD photoperiod through the entire study; 50.0 pg/ml
melatonin was provided ad libitum in the drinking water either continuously or for 3 h
per day. They found that the cellular and humoral immune responses were significantly
elevated in transient (3 h) and continuous (24 h) melatonin treatment groups as
compared to the control group (0 h). Nelson and Drazen (2000) reviewed a number o f
laboratory studies that consistently reported enhanced immune function on short
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11
daylengths. Enhanced immunity function in short days is correlated with increased
duration o f nightly melatonin secretion. Studies indicate that melatonin can act directly
on immune cells to enhance immune function (Drazen and Nelson, 2001). Moore and
Siopes (2000) placed adult Japanese quail and juveniles under three light conditions:
short day (8:16 LD), long day (16:8 LD), and constant light (CL). They demonstrated
that for adult birds, both short day and long day treatments produced similar cellular
and humoral immune responses. But these responses were significantly greater than
those observed in CL. The juvenile birds held under short daylength also had
significantly greater cellular and humoral immune responses a s compared to juvenile
birds held in CL. Effect o f different photoperiod regimens on T- and B-lymphocyte
activities in broiler chickens was investigated by Kliger et al. (2000). The photoperiod
regimens used in their study were: constant lighting (23:1 LD ); intermediate lighting
(12:12 LD) and intermittent lighting (1:3 LD). They found that splenic T- and Blymphocytes from 6-wk-old chickens grown in intermittent lighting had higher
activities than those from chicken grown in constant lighting.
Based on the above discussion, melatonin seems to be an integral part o f the
immune system, by exerting direct or indirect stimulatory effects on both cellular and
humoral immunity. In addition, melatonin has been functionally linked in various
species to regulation o f tumor inhibition and most recently, melatonin has also been
found to act as a free radical scavenger and antioxidant (Reiter, 2003).
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12
2.1.4 Impact of Photoperiod on Performance
The important goal o f photoperiod management in the poultry industry is to
improve performance o f birds. Morris (1967) found growth to be maximized with a near
continuous daylength; body weights o f the broilers were 5-10% heavier with 23-h light
than grown under an 8 or 12-h photoperiod. Beane et al. (1979) also suggested that
optimum growth and feed conversion were attained with chickens kept on continuous
lighting as compared to another three intermittent light treatments. However, a number
o f researchers have proposed that short daylengths have positive or neutral effects on
the performance o f broilers. Blair et al. (1993) reported that birds with an increasing
daylength were lighter in weight at 3 wk than those on continuous light; but by market
age (6 wk) they had similar body weights. Shah and Petersen (2001) reared broilers
under three different lighting programs: traditional program (23:1 LD), increasing
photoperiod (from 14 to 23 L) and decreasing photoperiod (from 23 to 14 L) in a 42-day
period. They found only an initial reduction in body weight and body weight gain in
birds reared under the increasing photoperiod compared to the nearly continuous
daylight. Furthermore, they noted that the difference in body weight between male and
female broilers were highest under increasing photoperiod. The female broilers did not
respond with a compensatory growth during the last two weeks o f growth to the extent
that the males did. This study revealed significantly lower mortality in birds reared
under increasing daylength (2.8%) followed by the birds reared under decreasing
daylength (5.0%) compared to birds reared under 23:1 LD (7.8%) throughout the
experimental period. Altan et al. (1998) placed broilers with three lighting treatments in
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13
a 40-day growing period and they did not find lighting effects for body weight, feed
conversion ratio, mortality, or carcass weights and proposed that it is possible to use a
restricted lighting schedule after three days o f age in broilers that are raised in curtain­
sided and conventional poultry houses without any detrimental effect on broiler
performance. Intermittent lighting for growing broilers has been shown to result in
equal or improved feed efficiency compared with continuous lighting (Buyse et al.,
1996a). The improvement in feed conversion with an intermittent lighting schedule was
related to higher metabolizability and lower energy expenditure on physical activity,
compared to continuous lighting (Apeldoom et al., 1999).
The application o f supplementary lighting was studied by Stanley et al. (1997).
Five treatments o f broilers in a 7-wk rearing period received 6 wk o f continuous
lighting (CL), 5 wk o f CL, 4 wk o f CL, 3 wk o f CL, 2 wk o f CL in the later life
respectively, the former weeks were on daylight and it was found that the birds which
received 2 wk o f CL had the highest mean body weight compared to the birds with 6 wk
o f CL, an improvement o f approximately 7.2%. It was also found that the absence o f
supplementary light up to 4 wk o f age did not suppress body weight, suggesting there is
an age-related response to photostimulation. Body weights o f chickens exposed to
continuous light after 4 wk o f age were larger than those that received 7 wk (control) o f
continuous light. The influence o f light on body weight may be due to its effect on
feeding activity (Weaver and Siegel, 1968). It is also possible that endogenous (such as
genotype and sex) and exogenous (such as dietary composition, feeder space) factors
interact with lighting schedule (Buyse et al., 1996b).
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14
2.1.5 Impact of Photoperiod on Welfare and Health
There appears to be many potential welfare benefits for growing broiler chickens
on short or moderate daylengths, including increased sleep, lower physiological stress,
improved immune response and the establishment o f activity rhythms (Gordon, 1994).
A few studies have examined the effect o f photoperiod on physiological stress response
in broilers, as indicated by elevated plasm a corticosterone or increased heterophil to
lymphocyte (H/L) ratios, which are the m ost accepted indicators o f stress in birds
(Gross and Siegel, 1983; Jones et al., 1988; El-Lethey et al., 2003). Information
regarding photoperiod on fear response in birds is often indicated by tonic immobility
(TI) reaction.
Zulkifli et al. (1998) found that broiler chickens illuminated 24 h had greater H/L
ratios and TI duration, indicating continuous light was stressful and caused higher level
o f fearfulness for growing broilers. Vo et al. (1998) also demonstrated an increase in
H/L ratio in broilers housed under continuous photoperiod compared to the birds with
12 or 16 h o f light. Buckland et al. (1976) reported that higher concentrations o f plasma
corticoids in broilers housed under continuous daylengths than in birds exposed to
another two intermittent light patterns. Continuous lighting reduces the opportunity for
rest and sleep, lack o f sleep with continuous daylengths may increase physiological
stress (Gordon, 1994). In addition, stress also increases as the birds' age because
disturbance under continuous light is likely to be greater as stocking density increases
(Lewis and Humik, 1990). Some studies, however, do not support the concept that the
birds under continuous lighting are more stressed. Campo and Davila (2002) examined
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15
the H/L ratio in 36-wk-old hens o f three Spanish breeds o f chickens exposed to lighting
programs o f 23:1 LD, 14:10 LD or 18.5:5.5 LD. They proposed that there was no
evidence o f an increased stress response in broilers with continuous lighting. Blair et al.
(1993) presented similar results; they reported that the H/L ratio in broilers was not
affected by the lighting pattern used. Likewise, Renden et al. (1994) reported that
plasma corticosterone concentration was not associated with photoperiod treatments,
indicating continuous lighting did not cause higher stress in broilers (four light
treatments in this experiments were: 23:1 LD; 1:3 LD from start to 56 day o f ages; 6:18
LD from start to 14 day o f ages, 1:3 LD from 15 to 56 day o f age; 6:18 LD from start to
14 day o f age, 23:1 LD from 15 to 56 day o f age).
Sanotra et al. (2002) raised broilers under two light-dark programs (program 1: 24
h light from day 0 to day 3, from day 4 to day 7, light was reduced by 2 h daily, down to
16 h light until 25 d o f age, light was increased by 2 h daily from days 26 to 29, 24 h
light was restored on d 30; program 2: 24 h light from day 0 to day 3, 16 h light from
day 4 to day 30, light was 24 h from day 31 until the end o f the trial) and one
continuous lighting program, and the TI test was conducted on days 21 and 35. They
found that both light-dark programs reduced the duration o f TI compared with
continuous lighting. The duration o f TI with continuous light was 426 s, and for lightdark programs the durations were 309 and 269 s, respectively. Campo and Davila
(2002) measured duration o f TI in 36-wk-old hens o f a synthetic breed exposed to
lighting programs o f 23:1 LD or 14:10 LD. They observed that the duration o f TI o f
hens housed under 23:1 LD was longer (236±32 s) than that o f hens housed under 14:10
LD (137±32 s).
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16
Rearing broiler chickens with the lights on continuously or near continuously has
been shown to increase the incidence o f heart disease and sudden death syndrome
compared to increasing lighting programs (Classen et al., 1991; Riddell and Classen,
1992; Blair et al., 1993). Birds on increasing lighting programs were also found to have
fewer leg problems and lower mortality than birds on continuous lighting (Riddell and
Classen, 1992; Renden et al., 1993; Rozenboim et al., 1999).
2.1.6 Light Intensity
The roles that light intensity plays in poultry production have been evaluated in
several ways. The possibility o f a relationship between intensity o f light and the
problem o f feather pecking and cannibalism has been studied by Kjaer and Vestergaard
(1999). Leghorn chickens were reared under 3 or 30 lux on litter floor, it was observed
that gentle pecks were approximately 20 times more frequent in 3 than 30 lux, whereas
severe pecks were 2-3 times more frequent in 30 lux. The preferences o f broiler and
layer strains for different light intensities were investigated by Davis et al. (1999). It
was found that for both strains, most o f the time was spent in the bright environment
(200 lux) at 2 wk o f age, but in the dim environment (6 lux) at 6 wks o f age.
Prayitno et al. (1997) suggested that it is preferable to provide bright light early in
the rearing period for the bird’s behavior needs. Bright light can assist young birds in
finding feed and water (Deaton et al., 1981). Deaton et al. (1981) attempted to
determine the amount o f light needed during the brooding period (0 to 3 wks) o f
broilers. The results o f their study showed that broilers, which received continuous
lighting a t an intensity o f 75 lux, had significantly less mortality than those brooded
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17
under a continuous lighting o f 5 lux. Newberry et al. (1986) found that light intensity
(0.5, 10, 20 and 30 lux in experiment 1 and 0.1, 1, 10 and 100 lux in experiment 2) had
no effect on the 3, 6, or 9-wk body weight in a 9-wk growing period o f roaster chickens.
A few research teams have reported that reducing light intensity increased broiler body
weights (Hooppaw and Goodman, 1972; Quarles and Kling, 1974; Cherry et al., 1980).
It has been suggested that performance is better under low light intensity because the
chickens are less active and therefore, use less energy exercising (Proudfoot and Sefton,
1978). C herry and B arwick (1962) proposed th a t light intensities above 10.8 lu x are
unnecessary and probably depress growth. Light intensities below 1.08 lux make
routine operations difficult and may lead to a decline in bird performance under
commercial conditions (Cherry and Barwick, 1962). It is generally recommended that
an intensity o f 5 lux be provided for broiler chickens after the first days o f life (Buyse et
al. 1996a).
2.2
Stress and Fear
2.2.1 Introduction
W ith growing concern regarding the welfare o f birds, stress and fear have
received much attention in recent years because both o f them represent major welfare
issues facing the poultry industry (Siegel, 1995; Jones, 1996). The rearing environment
and husbandry have changed frequently and dramatically with the introduction o f
intensive farming. These changes may make it difficult for the birds to cope with their
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18
environment and management, resulting in stress or fear. Considerable evidence shows
that factors such as group size, stocking density, social stability, genetic background,
housing systems, human handling, transportation and lighting regimes influence the fear
and stress response in birds (Siegel, 1995; Jones, 1996). These factors not only have
consequences on the welfare o f birds, but also on bird performance and the profitability
o f the poultry industry (Siegel, 1995; Jones, 1996). Some researchers have advocated
that regular gentle handling is a potent and reliable method o f reducing chickens’ fear o f
humans (Jones and Waddington, 1992; Jones, 1993).
2.2.2 Definitions of Stress and Fear
Stress has proven to be a complex phenomenon in animals. In trying to provide a
definition o f stress, some researchers look at the stimulus/input side, some at the
behavioral output and others at intermediate physiology (Toates, 1995). Moberg (2000)
defined stress as the biological response elicited when an individual perceives a threat
to its homeostasis, with the threat being the “stressor”. Stress was defined by Allen et al.
(1973) as: “ ... a collection o f diverse stimuli which damage or potentially damage the
organism and have in common an ability to stimulate adrenocorticotropic hormone
(ACTH) secretion. This results in increased glucocorticoid secretion that enables the
organism to better adapt to potential or actual life-threatening challenges”. Fraser et al.
(1975) proposed the definition o f stress as “an animal is said to be in a state o f stress if
it is required to make abnormal or extreme adjustments in its physiology or behavior in
order to cope w ith adverse aspects o f its environment and management”. Stress has
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also been defined as a condition in the animal that results from the action o f one or
more stressors that m ay be o f either external or internal origin (Ewing et al., 1999).
A widely accepted definition o f fear given by Jones (1996) is “the adaptive
psychophysiological r esponse t o p erceived d anger”. Fear i s a v ital component o f t he
stress response (Beuving et al., 1989). However, it is different from stress in some
ways. Fear is considered one o f the primary emotions, which governs the way in which
human and other animals respond to their social and physical environment (Jones,
1996). Campo and Redondo (1996) found that there was no consistent correlation
between fear and stress as indicated by the tonic immobility duration and the heterophil
and lymphocyte ratio. Similar results were reported by Campo et al. (2001), when they
examined association between plumage condition and fear and stress levels in five
breeds o f chickens. They found that hens with very poor plumage were less fearful and
more stressed than hens with perfect plumage.
2.2.3 Biological Response to Stress
Considerable am ount o f studies have been conducted to exam ine the effects o f
stress. Despite the large variety o f stressors used, overall effects are often similar. This
phenomenon is explained by the fact that different forms o f stress are directly or
indirectly translated to the body by the same pathways. A stress response begins with
the central nervous system perceiving a potential threat to homeostasis (Moberg, 2000).
W hether or not the stimulus is actually a threat is not important, it is only the perception
o f a threat that is critical; that is why psychological stressors can be so devastating
(McEwen and Stellar, 1993). Once the central nervous system perceives a threat, it
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20
develops a biological response that consists o f some combination o f the four general
biological defense responses: th e behavioral response, th e autonomic nervous system
response, the neuroendocrine response and the immune response (Moberg, 2000).
The behavioral response is the first and most biologically economical response.
The animal may be successful in avoiding the stressor by simply removing itself from
the threat (Moberg, 2000). The animal’s second line o f defense during stress is the
autonomic nervous system. During stress, the autonomic nervous system affects several
biological systems, such as the cardiovascular system, the gastrointestinal system, the
exocrine glands and the adrenal medulla (Moberg, 2000). There are two important
neurological and endocrinological pathways associated with stress response. They are
the
sympathetic-adrenal-medullary (SAM)
axis
and the hypothalamic-pituitary-
adrenocortical (HPA) axis (Post et al., 2003).
The adrenal glands play an important role in the response to aversive stimulation,
which form part o f both o f these axes. The adrenal glands are made up o f two distinct
components: the adrenal medulla (forming part o f the SAM axis) and the adrenal cortex
(forming part o f the HPA axis) (Toates, 1995). The primary stress response is a rapid
release o f catecholamines (adrenaline and/or noradrenaline) from the adrenal medulla
followed by activation o f the SAM axis (Post et al., 2003). These hormones have the
effect o f preparing the animal for “flight or fight” (Freeman, 1985). The second
pathway HPA is responsible for the release o f glucocotricoids in the form o f
corticosterone during the adaptation stage (Maxwell, 1993). This hormone is presumed
to help, in the adaptation o f the animal to its new set o f environmental conditions
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(Freeman, 1985). If the animal fails to respond because o f adrenal insufficiency, it
enters a stage o f exhaustion and dies (Freeman, 1985).
The assumption that stress influences host immunity arises from observations o f
increased disease occurrence in animals exposed to extreme, stressful environment
(Blecha, 2000). Exactly how the immune system responds to stress, however, is not
very c lear yet. A cute t hermal s tressors h ave b een s hown t o r educe concentrations o f
circulating antibodies and suppress cell-mediated immunity (Thaxton and Siegel, 1972;
Regnier and Kelley, 1981). Social or behavioral environm ents also activate stress
responses in bird, and like physical stressors, they are capable o f depressing immune
response (Siegel, 1995).
2.2.4 Influence of Stress and Fear on Performance
Both stress and fear may have deleterious effects on bird wellbeing and
performance, especially those that affect energy and mineral metabolism and
interactions with the immune system (Siegel, 1995). There is mounting evidence o f a
negative relationship between stress and fear and several indices o f reproductive
performance, growth, and meat quality. Mills and Faure (1990) reported that the
imposition o f frightening and stressful procedures, such as translocation from one
environment to another, handling, or capture and crating, often disrupts egg laying.
Fearful layers showed poorer egg production as well as lower body w eight than less
fearful birds (Jones, 1996). Egg mass production was lower for birds that showed
increased fear o f humans (Bredbacka, 1988). Eggshell quality and hatchability are also
reduced in fearful birds (Shabalina, 1984; Mills et al., 1991). M edium hybrids with
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22
history o f laying eggs with abnormal shells were more fearful in a range o f tests than
normal layers (Mills et al., 1991). The hatchability o f eggs from broiler breeders was
reduced in the breeding groups containing cockerels characterized as fearful rather than
calm (Shabalina, 1984).
Seasonal heat exposure has been reported to cause undesirable changes in turkey
breast meat quality (McKee and Sams, 1997). This meat is described as pale, soft and
exudative (PSE) (McKee and Sams, 1997). Tankson et al. (2001) examined the
relationship between stress and nutritional quality o f broilers and found that both ACTH
injection and heat treatment caused reductions in body weight, carcass weight, carcass
protein, and muscle calorie content. They concluded that the decreased meat yield and
detrimental changes in meat quality suggest that stress, whether induced hormonally or
by exposure to over-heating, caused losses that were as severe as those associated with
PSE under field conditions. Kannan et al. (1997) proposed that higher preslaughter
stress levels in broilers could influence the color o f thigh meat, although overall meat
quality was not affected under the conditions o f their study. M ashaly et al. (2004) found
that not only body weight and feed consumption were significantly reduced in hens in
the h eat s tress group, b ut a Iso t otal w hite b lood c ells c ount a nd antibody p roduction
were significantly inhibited in hens in the heat stress group.
2.2.5 Assessments of Stress and Fear
Puvadolpirod and Thaxton (2000a) developed a model to study stress response in
chickens. A variety o f parameters were proposed including plasma corticosterone,
glucose, cholesterol, triglycerides, high-density lipoprotein, total protein, and heterophil
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23
to lymphocyte (H/L) ratios as well as body weight, liver weight, and relative weights o f
the major immunobiological organs (i.e., spleen, thymus, and bursa o f Fabricius). As for
the assessment o f fear in birds, there are many ways. These include the open field, novel
environment, emergence or hole-in-the wall tests, home cage avoidance tests, box plus
experimenter, approaching human test and the tonic immobility test (Jones, 1996). The
literature on the methods used to measure the level o f stress and fear in this study were
reviewed as follows.
2.2.5.1
The Heterophil/Lymphocyte Ratio
2.2.5.1.1 Avian Blood Cell Types and Their Functions
It is appropriate to define the functions o f the different blood cells before
describing the importance o f the heterophil/lymphocyte (H/L) ratio, in particular
leucocytes involved in the stress response. Unlike mammals, avian red blood cells
(erythrocytes) are nucleated. The role o f the erythrocyte is to transport oxygen and
carbon dioxide (Maxwell, 1993). The thrombocyte, unlike the non-nucleated platelet, is
very active and plays a role in phagocytosis in the bird (Awadhiya et al., 1980), but it
has no role to play in the clotting mechanism (Gross, 1989). The leucocytes that
constitute the immune system can be classified into three groups: granulocytes,
lymphocytes and monocytes (Dieterlen-Lievre, 1988). Granulocytes are considered to
be the first line o f defense as they respond m ost rapidly to an invading organism
(Dieterlen-Lievre, 1988). The granulocytes are subdivided into three cell lines:
heterophils, eosinophils and basophils. These names are based on their staining affinity
for Romanowsky dyes. The heterophils are responsible for the defense against bacteria,
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24
eosinophils against parasites, while basophils are less understood (Maxwell, 1993). One
o f the functions o f basophils appears to be a mediator in the early inflammatory
response ( Maxwell, 1 993). T he 1ymphocyte i s t he m ost n umerous t ype o f 1eucocytes
and is the main cell type o f the immune system. Lymphocytes assist in the recognition
and destruction o f many types o f pathogens while monocytes are crucial in the defense
against intracellular parasites such as viruses and certain bacteria (Maxwell, 1993).
2.2.5.1.2 Significance of the Heterophil/Lymphocyte Ratio
The heterophil to lymphocyte (H/L) ratio has been established and widely
accepted a s an i ndicator f or d etermining c hronic s tress i n p oultry ( Gross an d S iegel,
1983). In response to environmental stressors, in chicken blood samples, the number o f
lymphocytes decreases and the number o f heterophils increases, thus increasing the H/L
ratio (Jones et al., 1988; El-Lethey et al., 2003). These blood samples also had an
increased level o f corticosterone (CS), a hormone known to be immunosuppressive and
to be the major stress hormone o f chickens (Jones et al., 1988; El-Lethey et al., 2003).
Plasma CS concentrations have been used as an indicator o f early stress more often than
the H/L ratio, but this response cannot be maintained when the stressor is present for a
long period (Jones et al., 1988). When the bird is subjected to environmental stressors,
hypothalamic-pituitary-adrenocortical (HPA) axis is activated and increases the plasma
CS concentrations. It has been found that long-term elevation o f plasma CS
concentrations may impair leucocytic responsiveness (Jones et al., 1988). The activation
o f the HPA inhibits humoral and cell mediated immunity (Davison et al., 1983; Siegel
et al., 1983). McFarlane and Curtis (1989) subjected chicks to multiple concurrent
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25
stressors, such as aerial ammonia, beak trimming, coccidial infection, heat stress,
intermittent electric shock and continuous noise. The plasma CS concentrations did not
change as a result o f any stressor or stressor combination; but aerial ammonia, heat
stress, and intermittent electric shock all increased the H/L ratio. The H/L ratio
increased linearly from 0.53 to 0.86 as the number o f concurrent stressors increased.
The authors concluded that in the chick, leucocyte changes in response to stress are less
variable and more enduring than those resulting from the corticosterone response. They
also indicated that the H/L ratio appears to be appropriate for evaluating stressful
animal environments typical o f agricultural production settings.
Alodan and Mashaly (1999) found an increase o f the H/L ratio in laying hens with
induced molting. Maxwell et al. (1992) reported that restricted-fed broilers showed an
increase in heterophil and basophil numbers, together with a corresponding decrease in
lymphocytes. Road transportation o f broiler chickens resulted in raised H/L ratios and
plasma creatine kinase values suggesting the presence o f physiological stress in these
birds (Mitchell et al., 1992). Campo et al. (2000) found that female chickens had H/L
ratios ranging from 0.28±0.02 to 0.58±0.04 while males had higher H/L ratios ranging
from 0.43±0.04 to 0.66±0.04.
Gross and Siegel (1986) demonstrated that H/L ratios were reduced after repeated
fasts. This indicates that chickens become habituated, that is, the repeat fast is less
stressful than the initial one. K atanbaf et al. (1988) reported a similar response in early
and late feathering broiler breeder hens reared under different feeding regimes. This
adaptation response for H/L ratios has also been recognized by Maxwell et al. (1990) as
well. They identified significantly higher H/L ratios in broilers compared with layers, o f
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26
both sexes, but failed to show significantly higher H/L ratios in broilers maintained on a
prolonged program o f food restriction. It was suggested that, with time, birds adjust to
new experiences.
Gross (1990) was interested in establishing how quickly the H/L ratio responded
and fell after stimulation from a noise stressor lasting 30 s. Chickens exposed to a single
incident o f 104 decibels showed a rise in H/L ratios 18 h later and the ratios reached
their maximum value in 20 h before returning to pre-stress values after 30 h.
temporal
pattern
of
stress
responses
following
continuous
infusion
The
of
adrenocorticotropic hormone (ACTH) for 7 day in broilers were studied by
Puvadolpirod and Thaxton (2000b). They found that the first response was elevated
plasma corticosterone (2 h, lasting 6 days); the elevated H/L ratio was found by day 2,
lasting 10 days. Another study conducted to investigate the H/L ratio and tonic
immobility reaction to preslaughter management in broiler chickens treated with
ascorbic acid indicated that subjecting chickens to a brief handling procedure resulted in
an increase in the H/L ratio within 20 h (Zulkifli et al., 2000).
2.2.5.2 Creatine Kinase
An increased activity o f plasma creatine kinase (CK) is an indicator o f stress
(Dzaja et al., 1996). CK is an important component o f voluntary muscle tissue and
several conditions such as bruising, exercise, and frequent penicillin injection can
elevate blood CK activities (Hollands et al., 1980). In both mammals and birds, there
are four isoenzymes: cytosolic brain type (B-CK); cytosolic muscle type (M-CK);
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27
mitochondrial ubiquitous, acidic type (M ia-CK); and mitochondrial s arcomeric, b asic
type (Mib-CK) (Muhlebach et al., 1996).
In broiler chickens, CK is released into the circulation following changes in the
permeability o f the sarcolemma in response to various pathologies and exposure to
environmental stressors (Mitchell and Sandercock, 1995). In studies on broilers, Dzaja
et al. (1996) examined the effects o f histamine application and water-immersion stress
on gizzard erosion and fattening o f broiler chicks and they concluded that CK activities
were increased in the stressed chicks. Increases in plasm a CK occurred in heat-stressed
broilers (Mitchell and Sandercock, 1995). Plasma CK activity also increases with age in
broilers and it has been proposed that genetic selection for rapid growth rate induces
alterations in membrane integrity and elevates the efflux o f intracellular enzymes
(Mitchell and Sandercock, 1994; Hocking et al., 1998).
2.2.5.3 Tonic Immobility Reaction
Tonic immobility (TI) reaction is an unlearned state o f reduced responsiveness to
external stimulation and is induced by physical restraint (Beuving et al., 1989). As a
logical extension o f the fear hypothesis, it was suggested that TI might be a terminal
reaction o f a n anti-predator response w hich w as supposed to include freezing, flight,
fight, and immobility (see review Jones, 1986). Some characteristics o f TI include
temporary suppression o f the righting response, reduced vocalization, intermittent eye
closure, rigidity, Parkinsonian-like muscle tremors in the
extremities,
altered
electroencephalographic patterns and changes in heart rate, respiration and core
temperature (Wallnau, 1981). Gallup (1979) examined a variety o f methods for
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28
measuring fearful behavior in the chicken and proposed that TI induced by physical
restraint was the best. Jones (1986, 1987) has examined various aspects o f this
phenomenon and reported that TI is a particularly useful and reliable index o f general,
underlying fearfulness in domestic fowls and the variation has been said to reflect
different levels o f fear. High susceptibility to TI and a long duration o f immobility are
signs o f a high level o f fear (Jones, 1986; 1987).
Ferrante et al. (2001) studied the fear reaction o f two strains o f chickens (an eggtype strain and a meat-type strain) and found that the meat-type birds showed a
significantly lower duration o f TI, indicating a lower level o f fear specifically towards
humans. Individuals vary in their susceptibility to as well as in the duration o f TI
(Erhard et al., 1999). Chicks showed longer TI when they could see the experimenter,
and response duration was prolonged if the latter maintained direct eye contact with the
bird rather than averting its gaze (Gallup et al., 1972). The TI response was strongly
affected by age. A study aimed to investigate the ontogeny o f TI using White Leghorn
male chicks showed that TI was poorly developed during the first 3 days o f life, when
the median TI duration in chicks was 10 s and the mean number o f inductions was
2.3±0.3. After the third day o f life, TI duration increased by up to 15 times and
susceptibility by about two (Heiblum et al., 1998). El-Lethey et al. (2003) found that
chickens housed on slats showed prolonged TI duration suggesting that depriving
chickens o f foraging material was shown to induce fearfulness. Jones (1986), in a
review, summarized some general factors affecting TI including an age effect, and the
influence o f the experimenter, regular handling, genetic factors, social factors and
housing conditions.
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29
2.3 The Skin
2.3.1 Structure and Function of the Skin
The skin is a complex, m ultilayered organ, which produces several specialized
derivative structures called appendages and consists o f heterogeneous cell types and
extracellular components (Haake et al., 2001). There is regional variation in the skin.
This variation is manifested primarily in terms o f skin thickness, composition and
density o f appendages, and, in som e c ases, b iochemical differentiation (Haake e t al.,
2001 ).
The skin consists o f three layers: epidermis, dermis and hypodermis (Haake et al.,
2001). The outer surface o f the skin is the epidermis, which itself contains several layers
- the basal cell layer, the spinous layer, the granular cell layer, and the stratum comeum
(Bouwstra and Honeywell-Nguyen, 2002). Approximately 90-95% o f epidermis cells
are keratinocytes, which are stacked on top o f each other, forming different sub-layers
(Bouwstra and Honeywell-Nguyen, 2002). Melanocytes and Langerhans cells are other
important cells found in the epidermis which have special functions (Haake et al.,
2001). The dermis consists mostly o f connective tissue and is much thicker than
epidermis. The dermis is less cellular than the epidermis and primarily composed o f a
fibrous and amorphous extracellular matrix surrounding the epidermally derived
appendages, neurovascular networks, sensory receptors and dermal cells (Haake et al.,
2001). The dermis is the connective tissue component o f the skin and provides its
pliability, elasticity and tensile strength (Haake et al., 2001). Collagen and elastic
connective tissue (elastin) are the main types o f fibrous connective tissue o f the dermis
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30
(Haake et al., 2001). Collagen is the basic polypeptide in connective tissue responsible
for the structural integrity o f all muscle and skin tissues (Smith et al., 1977). Elastic
fibers return the skin to its normal configuration after being stretched or deformed
(Haake et al., 2001).
The natural function o f skin is to protect the body from unwanted influences o f
the environment. The general functions o f skin are to provide an external covering o f
the body, prevent tissue fluid loss from the body, help to regulate body temperature, and
help to protect the body from dirt and infection (Bouwstra and Honeywell-Nguyen,
2002). The main barrier o f the skin is located in the outermost layer o f the skin, the
stratum comeum (Bouwstra and Honeywell-Nguyen, 2002). Stratum comeum is
considered as a composite material and a biopolymer with properties so unique as to
consider it a “smart material”. Stratum comeum, together with stratum granulosum
responds to environmental signals with appropriate modulations in its barrier properties
(Menon, 2002).
2.3.2 Collagen
2.3.2.1
Structure and Function of Collagen
Collagen, the most abundant mammalian and avian protein, has been found in
many different tissues and organs such as muscles, bones, tendons, placenta, cartilage,
blood vessels, teeth, cornea, intervertebral disks, vitreous bodies and skin (Nimni,
1988). It is a broad family o f structural proteins which function as an extracellular
framework in tissues such as blood vessels and most organs (McCormick, 1999).
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31
Collagen gives many different tissues and organs substantial, stout and elastic properties
(M erlinoet al., 1983). Besides th e structural ro le in m ature tissues, collagen plays a
regulating role in developing tissues as well (Nimni, 1988). It is the major dermal
constituent, accounting for approximately 75% o f the skin’s dry weight (Haake et al.,
2001 ).
The collagen molecule consists o f three helical polypeptide chains (a chains)
containing the repeating amino acid sequence o f Gly-X-Y bond into a stable triple helix
(Nimni, 1988). It contains a relatively high proportion o f hydroxyproline and
hydroxylysine, which are almost unique to collagen (Nimni, 1988). Collagen contains
35% glycine, 12% L-proline, and 10% hydroxyproline (Christensen et al., 1995).
Several models o f the substructure o f collagen fibrils based on theoretical and
experimental consideration have been proposed. The microfibril, the smallest structural
unit, contains from 4 to 8 collagen molecules in cross-section (Silver and Trelstad,
1980). Several ultrastructural techniques are able to resolve collagen fibrils as bundles
o f tightly packed microfibrils, which quite often follow a right-hand helical course
(Ottani et al., 2001). The molecular packing arrangement within collagen fibrils has a
significant effect on the tensile properties o f tissues (Cameron et al., 2002). Collagen
fibrils were proposed to belong to two different forms indicated as “T-type” and “Ctype” (Ottani et al., 2001). The first class, consisting o f large, heterogeneous fibrils,
parallely tightly packed, subjected to tensile stress along their axis, is found in highly
tensile structures such as tendons, ligaments and bone. The other class, consisting o f
small, homogeneous fibrils, helically arranged, resisting multidirectional stresses, is
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32
mostly present within highly compliant tissues such as blood vessel walls, skin and
nerve sheaths (Ottani et al., 2001).
Collagen is synthesized in fibroblasts (McCormick, 1999). Biosynthesis follows
the same basic principle as synthesis o f any other protein, but the modifications o f the
molecule posttranslationally are complex and unique to the collagen molecule
(McCormick, 1999). Procollagen is known as a precursor to synthesize collagen
(Burgeson and Nimni, 1992). Tensile strength and functionality o f collagen fibrils are
due mainly to the formation o f intermolecular crosslinks (McCormick, 1999).
2.3.2.2 Collagen Types
To date, 19 distinct collagen forms have been described and characterized
constituting a family o f proteins that have a wide variety o f roles in biological systems;
these forms are more commonly referred to as collagen types (McCormick, 1999). They
are distinguished on the basis o f their chemical differences. These 19 types differ in the
way they are associated with one another and the way they interact with other
molecules. The most important types are collagen types I, II, III, IV and V. Type I
collagen is the most prevalent, found in bone, tendon, and skin, accounting for about
80-90% o f total collagens (Cameron et al., 2002). Type II collagen is the major collagen
type present in cartilage and it is also present in significant amounts in other connective
tissues (Burgeson and Nimni, 1992). Type III collagen is similar in structure to type I
but less abundant and is often encountered in areas o f rapid new collagen synthesis
(Cameron et al., 2002). Type III collagen is located in blood vessels, uterus, and skin
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33
(Cameron et al., 2002). Type IV and V are for the most part found in various basement
membranes (Nimni, 1988).
The skin o f th e m odem broiler chickens contains close to 5 0% fat and around
3.5% collagen (Cliche et al., 2003); approximately 75% o f the collagen is type I and
15% type III (Abedin and Riemschneider, 1984). Cliche et al. (2003) used pepsin and
ethylene diamine processes and fractionated by precipitation with NaCl to determine the
relative proportion o f the different types o f collagen solubilized in the skin o f broilers
and found that in both cases, the isolated extract contained mainly type I collagen
(74.4% for pepsin and 62.4% for ethylene diamine) and a smaller, variable proportion
o f type III collagen (19.8% for pepsin and 31.7% for ethylene diamine). In contrast, the
proportion o f type IV and V collagen was much lower and amounted to a little less than
6% o f the collagen extracted in both cases.
2.3.2.3 The Matrix Metalloproteinase
Matrix
metalloproteinases
(MMPs),
an
endogenous
enzyme
system
are
responsible for extracellular collagen degradation and remodeling (Spinale et al., 1999).
MMPs belong to a family o f zinc-dependent peptidases (Aimes and Quigley, 1995).
Collagen degradation in connective tissue is mediated mainly by MMPs, with the 72kDa gelatinase (MMP-2) playing a key role in this process (Creemers et al., 1998). The
gelatinase belonging to a MMP subfamily specifically cleaves native triple helical
collagens, yielding collagen fragments as a result o f the hydrolysis o f a single Gly-X-Y
bond in each a chain o f the collagen molecule (Aimes and Quigley, 1995). MMP-2 has
been identified in a wide range o f normal and malignant cells in several species (Aimes
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34
and Quigley, 1995) and both human and chicken MMP-2 are capable o f cleaving type
I, IV and V collagen (Aimes and Quigley, 1995). Olkowski et al. (2001) demonstrated
that the activity o f this enzyme appears to be considerably higher in preparations from
broilers, particularly from the left ventriculum o f fast growing broilers, in comparison to
Leghorn hens or slow growing broilers. They proposed that the pathogenesis o f heart
failure and ascites in fast growing broilers may be associated w ith the increased activity
o f MMPs.
2.3.3 Skin Strength and Collagen
Downgrading problems associated with skin integrity such as cuts and tears cause
substantial economic losses to the broiler industry (Bilgili et al., 1993). Skin integrity in
broilers is generally evaluated by determining skin strength (Angel et al., 1985). Skin
strength o f broilers is important to growers and processors. During rearing, weakened
skin may result in an increase in mechanical skin tears, which could lead to an increase
in mortality due to infection and a general decrease in overall performance (Christensen
et al., 1994). During processing, because fragile skin increases the incidence o f
downgrades, it also causes economic losses (Kafri et al., 1985; Christensen et al., 1994).
In humans, genetic and acquired disorders o f collagen metabolism are the cause
o f the Ehlers-Danlos syndrome (EDS) manifested by skin hyper extensibility and a
tendency to split during minor trauma (Krane, 1984). In broilers, changes in the
physical and chemical properties o f the skin collagen result in skin weakening and
tearing (Ramshaw et al., 1986). Collagen is the principal protein in the skin and is a
major determinant o f skin strength (Granot et al., 1991a). Kafri et al. (1985), however,
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35
found that the differences in breaking strength were not consistently associated with
collagen content o f the skin. Other factors such as the rate o f cross-linking (Granot et
al., 1991b) and the state o f maturation o f the collagen (Crosley et al., 1992) and the ratio
o f t ype I an d t ype III m ay b e a Iso p lay a r ole (Burgeson and N imni, 1 992). C ertain
minerals, vitamins, and amino acid are directly or indirectly involved in collagen
synthesis (Pinion et al., 1995).
Skin collagen is significantly influenced by sex in broilers (Granot et al., 1991b).
A number o f publications have reported that the skin o f males had a higher resistance to
tearing (Smith et al., 1977; W einberg et al., 1986; Pinion et al., 1995). Smith et al.
(1977) postulated that high levels o f fat, accompanied by a reduction in total collagen
concentration, made the skin o f females more susceptible to tearing. Yal<?in et al. (1998)
found that sex effect was significant for skin dry matter and fat content, being higher in
females. C hristensen e t al. (1994) f ound t hat m ales h ave a t hicker d ermal 1ayer than
females even though total skin thickness is less in males. This supports the conclusion
o f Ramshaw et al. (1986), who proposed that skin tensile strength is a direct function o f
the collagenous dermal layer o f the skin. Pines et al. (1996) suggested the higher tensile
strength o f male skin than female skin m ay be due to the elevated skin collagen content
that resulted from increased expression in collagen type I genes and from higher
amounts o f various collagen cross-links.
Skin strength o f broilers increases with age (Bilgili et al., 1993). One possible
reason is that skin collagen content increases with age (Pines et al., 1996). The breast
skin is stronger than the skin from the thigh and the skin, collagen content as a fraction
o f weight or total protein is considered higher in the breast than in the back in broilers
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36
(Kafri et al., 1984; Granot et al., 1991b). There is genetic effect on skin strength, for
example, low-weight female chicken strains had weaker skins when compared to highweight strains (Kafri et al., 1984).
Dietary energy, protein, and fat have been shown to be contributors to skin
strength. Granot et al. (1991b) showed that high dietary protein increased skin collagen
but the feeding o f a low-energy diet led to reduced growth rate, and did not affect skin
tearing or skin collagen. Kafri et al. (1985) reported that the skin strength and collagen
content decreased as the dietary energy concentration increased. Christensen et al.
(1994) performed two experiments to evaluate whether dietary and environmental
temperature affected skin tensile strength o f commercial broilers. They found that the
anticoccidial halofuginone interferes with collagen synthesis, causing decreased
collagen formation and reduced skin strength; neither added dietary fat nor ambient
temperature were involved. They also noted that birds with weakened skin exhibited an
increased incidence o f skin tears during slaughter in a commercial processing plant. The
increase o f skin tearing during processing, induced by halofuginone, is caused by direct
suppression o f skin collagen synthesis (Granot et al., 1991a). A negative correlation
between skin collagen and fat content was reported by Kafri et al. (1985). However,
when comparing ages within the females, skin strength increased with age despite the
fact that fat increased with age as well (Weinberg et al., 1986).
It has been reported that raising broilers on lighting programs with long dark
periods reduces skin tearing at the processing plant (Hammershoj, 1997). Collagen was
proposed to be a major determinant o f skin tears (Granot et al., 1991b). To the author’s
knowledge, the association between photoperiod and skin collagen content has not been
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37
studied. Further research is needed to investigate the effect o f photoperiod on skin
integrity in birds.
2.4 Carcass Defects
2.4.1 Scabby Hip Syndrome
Scabby hip syndrome is a skin condition characterized by inflammation o f the
skin of the hips and caudal parts o f the back o f broilers (Frankenhuis et al., 1988).
Scabby hip syndrome can result in substantial downgrading and trimming at the
processing plant (Page, 1974). Factors related to be associated with the incidence and
severity o f the skin lesions included stocking density, warm humid environments, high
ammonia levels, and old litter (Peterson, 1974; Harris et al., 1978; Proudfoot and Hulan,
1985).
Proudfoot and Hulan (1985) investigated the role o f stocking density on the
incidence o f scabby hip syndrome and observed an increase o f 7 to 40% in males and
12 to 35% in females when floor space was reduced from 840 to 454 cm2 bird’1.
Frankenhuis et al. (1988) found that gentle scratching with chicken claws twice a week
resulted in skin lesions that could not be distinguished from clinical scabby hip
syndrome at slaughter. They proposed that interaction between birds is a prerequisite
for the skin condition to develop. They also reported that clipping o f the claws at day 25
could almost prevent scabby hips when the birds were slaughtered at day 45
(Frankenhuis et al., 1988). Clinical observations have indicated that over 70% o f scabby
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38
hip lesions may be associated with scratches and that the majority o f these scratches are
located on the caudal dorsal convexity o f the carcasses (Hargis et al., 1988). Scanlan
and Hargis (1989) failed to identify any common bacteriologic etiology associated with
such lesions. Scabby hip syndrome reportedly results from toe scratches inflicted as
broilers climb on one another (Hargis et al., 1989).
2.4.2 Avian Cellulitis
Cellulitis can be broadly thought o f as a bacterial infection under the skin
(Norton, 1997). Escherichia coli are the primary causative agent o f this condition
(Singer et al., 1999). Avian cellulitis, sometimes referred to as inflammatory process,
was first systematically described in Great Britain (Randall et al., 1984). Since that
time, cellulitis has been reported and established as a condem nation category in North
America. Cellulitis in broiler chickens is characterized by subcutaneous lesions that
result in economic losses because o f the partial or complete condemnation o f carcass at
processing. Kumor et al. (1998) studied trends in the incidence o f cellulitis using
Canadian National Condemnation Records. They reported that steady increments in
cellulitis condemnations were observed. Between 1986 and 1996, the percentage o f
cellulitis condemnations increased 11.8-fold. In 1996, more than 2.6 m illion broilers
(0.6% o f total slaughter) were condemned due to cellulitis; this constituted 30.1% o f
total condemnations, making it the number one condemnation category in 1996.
Elfadil et al. (1996) examined the problem o f cellulitis in Southern Ontario using
a mailed survey to gather information about the association between cellulitis and
management risk factors. They reported that cellulitis was positively associated with
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39
male and mixed flocks, use o f straw as litter, certain feed companies, use o f zinc
bacitracin as a growth promoter, and other diseases diagnosed at the processing plant.
Cellulitis has also increased along with genetic advances in growth rate (Julian, 1997).
High yielding, slow feathering birds are more susceptible to cellulitis than are slower
growing heavier feathered breeds (Norton et al., 1999).
Avian cellulitis is frequently described as consisting of two types. Type I cellulitis
is described as occurring in the navel region o f the bird and is reported to be hatchery
related (Hill, 1995). Type II cellulitis is described as occurring in other locations on the
body and most frequently associated with scratches occurring during growth (Hill,
1993). Norton et al. (1999) proposed that “Type I” cellulitis lesions or those previously
thought to be due to hatchery-borne infection could be induced with scratches.
2.5 Toe-treatment
2.5.1 Toe-treatment Techniques
Toe-treatment is a practice designed to decrease the occurrence o f carcass
scratches in turkeys (Moran, 1985; McEwen and Barbut, 1992). In 1949, turkey toetreatment with an electric beak trimmer was first recommended by Marsden and Martin
(Owings et al., 1972). Traditionally, a hot blade or surgical scissors was used to trim the
toes o f turkeys (Owings et al., 1972; Greene and Eldridge, 1975). Currently, the turkey
industry uses microwave energy to restrict the claw growth and reduce carcass
scratches. A microwave claw processor (MCP) has been generally used to conduct this
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40
procedure. Toe-treatment levels are determined by both microwave time and percent
trim set in MCP. Microwave time controls the length o f time that the claws are being
treated. A higher setting indicates a longer duration, and a lower setting indicates a
shorter duration. The percent trim controls the amount o f toe that is inserted into the
waveguide for processing. A higher setting indicates a smaller amount o f toe insertion,
and a lower setting indicates a larger amount o f insertion (Nova-Tech engineering, Inc.,
2003).
2.5.2 Effects of Toe-treatment on Carcass Scratches and Performance
Documentation o f the effects o f toe-treatment has not been consistent. Hargis et
al. (1989) found that toe-trimming broilers resulted in a 3.7 to 4.8-fold reduction in
subjective lesion scores and a 7 to 10-fold increase in percentage o f USDA Grade A
carcasses at a commercial processing plant. Proudfoot et al. (1979), using large
experimental pens, however, did not observe any benefit effect o f toenail removal on
carcass quality in broiler turkeys. Owings et al. (1972) and M oran (1985) reported that
toe-treatment did not reduce the final body weight o f the turkeys; while Newberry
(1992) found a significant reduction in body w eight o f turkeys that w ere toe-treated.
This observed reduction in body weight is believed to be the result o f a reduction in
feed consumption early in the bird's life. Some researchers have reported an increase in
the incidence o f mortality among toe-treated turkeys (Owings et al., 1972; Newberry,
1992). However, Ruszler and Kiker (1975), using a hot blade beak trimmer to remove
the toenails o f chicks, found that toe-treated hens had about 2.5% lower mortality and
10 more eggs per hen during the lay cycle as compared to the birds with intact toes.
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41
Martin et al. (1976) reported that toe-treatment significantly increased rate o f egg
production and improved feed conversion.
2.5.3 Effects of Toe-treatment on Stress and Fear Response
Compton et al. (1981) used a hot blade to treat the toes o f cage reared W hite
Leghorn chicks and as a result o f examining blood corticosterone concentrations,
proposed that toe-treatment posed an initial stress to the birds. They also observed that
toe-treated birds were less active and fearful. It has been found that toe-treated turkeys
were quieter, fought less and had fewer skin tears and bruises (Owings et al., 1972).
Honaker and Ruszler (2004) reported that toe-treated layers displayed a lower level o f
fearfulness to humans as measured by the tendency o f birds not to panic in the presence
o f a human. Compton et al. (1981) postulated that the rationale for toe-treatment is to
reduce the level o f stress among birds by effectively reducing the “personal space” (the
physical and social space occupied by an individual) required by each bird.
W ith the development o f microwave toe-treatment technique and decreased cost
o f this new technology, it is possible for microwave toe-treatment to become an
optional management practice in the broiler industry. It is therefore important to
evaluate the effect o f this procedure on carcass quality, growth performance as well as
wellbeing in broiler chickens.
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42
2.6 Objectives
This experiment was conducted to improve carcass quality o f broiler chickens by
the use o f microwave toe-treatment and an increasing photoperiod program. The
detailed objectives o f this study were:
1)
to examine the influence o f photoperiod in combination with microwave toetreatment on body weights, weight gains, feed consumption, feed conversion ratio
and mortality in broiler chickens,
2)
to evaluate the effect o f photoperiod in combination with microwave toetreatment on stress and fear response in broiler chickens indicated by heterophil to
lymphocyte ratios, the activity o f plasma creatine kinase and tonic immobility
reaction,
3)
to determine the effect o f microwave toe-treatment in combination with
photoperiod on carcass scratches and bruises in broiler chickens, and
4)
to investigate the impact o f photoperiod in combination with microwave toetreatment on the integrity o f skin in broiler chickens by examining skin puncture
strength, the activity o f plasma matrix metalloproteinases, and type I and III
collagen content in the skin.
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43
CHAPTER 3
PERFORMANCE OF MICROWAVE TOETREATED BROILER CHICKENS GROWN WITH TWO
PHOTOPERIOD PROGRAMS
3.1 Abstract
Increasing photoperiod programs reduce the incidence o f heart disease and leg
problems. H owever, b irds g iven th is t ype o f p rogram h ave b een r eported t o h ave a n
increased incidence o f carcass scratches due to increased bird activity. The primary
cause for carcass scratches is injury inflicted by toenails o f other broilers. One potential
solution for reducing skin scratching damage is to treat the toes o f day-old broiler
chickens w ith m icrowave e nergy. T he p resent s tudy was conducted t o d etermine t he
impact o f microwave toe-treatment in combination with photoperiod on performance o f
broilers. Two replicate trials were conducted with 728 female and 728 male broilers in
each trial. H alf o f the birds from each sex were toe-treated with microwave energy upon
delivery from the hatchery. The birds in each trial were randomly assigned to four
sections o f two rooms with a total o f 32 floor pens. Two sections were given the
continuous lighting program from start to finish and the other two were on an increasing
lighting program with short photoperiod after the first three days, which increased to 23
h by day 30. The traditional weighing method and computerized bird scales were used
to determine body weight (BW). For the traditional weighing method, the birds were
removed from the pen and weighed as a group. For h alf the pens, the BW was measured
this way on days 7, 14, 24 and 31. On day 38, the birds from all the pens were removed
and weighed. For six pens per section, computerized bird scales were installed to
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44
continually monitor bird weight. Body weight gain (BWG), feed intake (FI) and feed
conversion ratio (FCR) were calculated for each 0-6, 7-13, 14-23, 24-30, 31-38, and 038 day periods. BW was reduced on day 7 based on the traditional weighing method
and days 24 and 31 based on computerized bird scales for the toe-treated birds (p<0.05).
For both sources o f data, the birds given the increasing lighting (IL) were lighter than
those on the continuous light (CL) on days 7, 14, 24 and 31 (p<0.05). By day 38, there
were no lighting and toe-treatment effects. For both sources o f BW data, the males were
heavier than the females on days 24, 31 and 38 (p<0.05). Overall, the males consumed
more feed than the females; the birds with CL had higher FI than birds with IL and toetreated birds had less FI than the birds with intact-toes (p<0.05). The microwave toetreatment, lighting, and sex did not affect overall FCR based on two sources o f data. In
the 38-day growing period, the toe-treated birds, the birds with CL and the females had
greater mortalities (p<0.05). The results suggest that the microwave toe-treatment or the
increasing photoperiod program did not reduce growth performance o f broilers at the
market age.
3.2 Introduction
Light manipulation including source, wavelength, intensity and photoperiod plays
an important role in the management practices for poultry (Manser, 1996). Photoperiod
management i s 1ikely t he m ost i mportant a spect o f 1ight i n b roiler p roduction. S ome
researchers have advocated that the optimum growth rate and feed conversion were
obtained with chickens k ept on continuous or nearly continuous lighting (23 or 24 h
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45
light/d) (Morris, 1967; Beane et al., 1979). Others, however, indicated that a short
photoperiod enhanced growth and feed efficiency (Deaton et al., 1970; Buyse et al.,
1996a). Lighting programs which provide a short photoperiod early in life, increasing
daylengths as a broiler ages have been described as increasing lighting programs
(Gordon, 1994). The major reasons for adopting increasing lighting programs in broiler
production are to decrease the incidence o f heart disease and leg problems (Classen and
Riddell, 1989; Classen et al., 1991; Rozenboim et al., 1999). There are also lower
electrical costs for the producer and improved feed efficiency (Gordon, 1994).
However, birds given this type o f program may have an increased incidence o f carcass
scratches due to increased bird activity (Blair et al., 1993). The prim ary cause for
carcass scratches is injury inflicted by toenails o f other broilers (Hargis et al., 1989).
Scratches m ay develop into an inflammation o f the skin known as scabby hip syndrome,
a severely afflicted flock o f which may have the number o f Grade A carcasses reduced
by as much as 50% (Proudfoot and Hulan, 1985). Scratches also can lead to the
development o f cellulitis, the most common cause for carcass condemnation in Canada
(Kumor et al., 1998).
One potential solution for reducing skin scratching damage is to treat the toes o f
day-old broiler chickens with microwave energy. Toe-treatment is a practice commonly
used in the turkey industry to reduce carcass downgrading due to scratching (Moran,
1985). Traditionally, this procedure was conducted in turkeys by trimming the toes with
a hot blade or surgical scissors (Owings et al., 1972; Greene and Eldridge, 1975).
Currently, the turkey industry uses microwave energy to restrict claw growth and
reduce carcass scratches. The cost o f this new technology has prohibited the use o f it
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46
with short lived broiler chickens until now. Documentation o f the effect o f toetreatment on growth o f turkeys has not been consistent (Owings et al., 1972; Moran,
1985; Newberry, 1992). Information on the effect o f microwave toe-treatment on
growing broiler chickens is lacking. The present study was conducted to determine the
impact o f microwave toe-treatment in combination with photoperiod on performance o f
broilers.
3.3 Materials and Methods
3.3.1 Birds, Diets and Housing
Two replicate trials were conducted with 728 female and 728 male broilers in
each trial. The first trial was performed in the fall and the other in the winter. Sexed,
day-old broilers (Ross 308) were obtained from a commercial hatchery and were placed
in sex-separated pens in a confinement house with untreated wood shavings as litter.
Animal care was provided according to the standards o f the Canadian Council on
Animal Care (1993) and the protocol was approved by Nova Scotia Agricultural
College Animal Care and Use Committee. Two room s were used in each trial; each
room was separated by a black plastic divider into two sections for two photoperiods. In
each trial, the birds were randomly assigned to four sections o f the two rooms with 8
floor pens per section, giving 4 replications o f each photoperiod, toe-treatment and sex
combination treatment. The stocking density was 0.07 m 2 bird' 1 . Feed and water were
available ad libitum with one suspended tube feeder per pen and a nipple drinker system
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47
in each room. There were ten nipples per pen. Box feeders were also used to provide
feed in the first week. All the birds were given the same starter, grower, and finisher
rations. The starter diets contained 23% crude protein and 3050 kcal ME kg'1 and were
fed as mash from day 0 to 13 days o f age. The grower diets contained 20% crude
protein and 3150 kcal ME kg'1 and were fed as mash from day 14 to 23 days o f age. The
finisher diets contained 18% crude protein and 3200 kcal ME k g '1 and were fed as
pellets from day 24 to 38 days o f age. The ingredients used in the diets and the
calculated analyses are shown in Table 3.1. The brooding temperature was set at 3032°C from day 0 to day 7 after which it was reduced 3°C per week until it reached 21°C
where it remained for the rest o f the trial.
3.3.2 Microwave Toe-treatment
H alf o f the birds from each sex were toe-treated using a Microwave Claw
Processor (MCP, Nova-Tech Engineering Inc., Willmar, MN, USA) upon delivery from
the hatchery. To conduct the microwave toe-treatment, the chicks were turned upside
down and their legs were inserted into shackles o f the MCP. The ventral side o f the bird
was toward the operator. Each o f the three front toes were pulled into place by a
vacuum and treated with microwave energy for 0.8 s.
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48
Table 3.1 Ingredient composition and calculated analyses of diets used in the
experiment
Ingredients (%)
Com
Wheat
Soybean meal
Poultry grease
Limestone
Dicalcium phosphate
Iodized salt
DL-Methionine
Amprolium11
BMDV
Pel-Stikw
Vit/Min Premixx,y
Calculated content
Crude protein, %
Metabolizable energy, kcal kg"1
Calcium, %
Available phosphoms, %
Lysine, %
Methionine + Cystine, %
Starter
(0-13 d)
Grower
(14-23 d)
Finisher
(24-38 d)
42.90
10.00
38.69
3.86
1.97
1.10
0.32
0.103
0.05
0.0044
0.50
0.50
50.08
10.00
31.29
4.47
1.85
0.95
0.30
0.002
0.05
0.0044
0.50
0.50
55.74
10.00
26.23
3.98
1.84
0.91
0.30
23.00
3050
1.00
0.45
1.37
0.95
20.00
3150
0.92
0.40
1.15
0.76
18.00
3200
0.90
0.38
1.00
0.70
0.50
0.50
u Amprolium: Merck & Co., Inc., Whitehouse station, NJ, USA.
VBMD: a dried precipitated germentation product obtained by culturing Bacillus subtilis
tracy on media adapted for microbiological production o f bacitracin;calcuim carbonate.
Alpharma, Fort Lee, NJ. USA.
w Pel-Stik: Uniscope, Inc., Johnstown, CO, USA.
x Supplied per kg starter diet: vitamin A, 19,500 IU; vitamin D3, 4 0 0 0 IU; vitamin E, 2.97
mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.046; niacin, 29.7; folic
acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg; thiamine, 5.8 mg;
manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50 mg; wheat
middlings, 1432 mg; ground limestone, 500 mg.
y Supplied per kg grower & finisher diet: vitamin A, 19,500 IU; vitamin D3, 4000 IU;
vitamin E, 2.97 mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.024;
niacin, 29.7; folic acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg;
thiamine, 5.8 mg; manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50
mg; wheat middlings, 1543 mg; ground limestone, 500 mg.
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49
3.3.3 Experimental Lighting Programs
Light was provided by incandescent bulbs mounted at approximately 2.0 m above
the floor level. All birds were subjected to the same light intensity. Light intensity was
measured at the bird head level with a light meter (Cal-Light 400, The Cooke
Corporation) and adjusted by rheostat. All broilers were given 24 h light for the first
three days. Then in two sections o f each trial, the lighting schedule was 23 h per day
until the end o f the trial. The other two sections were on an increasing photoperiod
program. The two lighting programs used in the experiment are in Table 3.2.
Table 3. 2 Two lighting programs used in the experiment
Age (d)
Lighting
Increasing
Continuous
Light intensity
(lux)
0-3
24L!:0D2
24L:0D
20
4-6
23L:1D
10L:14D
20
7-9
23L:1D
10L:14D
5
10-16
23L:1D
12L:12D
5
17-22
23L:1D
14L:10D
5
23-29
23L:1D
18L:6D
5
30-38
23L:1D
23L:1D
5
!L: light
2D: darkness
3.3.4 Performance Parameter Determination
Feed was weighed in as required and was weighed back on days the birds were
manually weighed and as mortality occurred. Feed consumption was measured for each
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50
pen unit at the following intervals: 0-6, 7-13, 14-23, 24-30, 31-38 and 0-38 days o f age.
Analysis o f feed intake was performed on the average grams o f feed consumed per bird
in each pen for the above intervals. To determine bird weight, the traditional weighing
method and computerized bird scales were used. For the traditional weighing method,
the birds were removed from the pen and weighed as a group. For h alf the pens, the
body weight was measured this way on days 7, 14, 24 and 31. All the pens were
weighed this way on days 0 and 38. To decrease human contact, for 6 pens per section,
the body weight o f the birds was monitored by computerized bird scales (Optilink
6.0.14, Tartan opticon, Lockport, MB) left in the pens with the birds. We assumed that
this would benefit welfare o f broilers by decreasing the level o f stress and the incidence
o f scratches and bruises due to catching and weighing birds by hand. These platform
scales were interfaced with a data collection unit to facilitate continuous monitoring o f
the birds that step on the scale. Body weight gain was calculated at the intervals o f 0-6,
7-13, 14-23, 24-30, 31-38, and 0-38 days o f age based on both methods for weighing
birds. Feed conversion ratios, expressed as the average feed consumption divided by the
average body weight gain, were calculated for each pen unit at the same intervals. Both
methods o f weighing were used for feed conversion ratios.
3.3.5 Mortality
The birds’ health was checked twice daily around 8:00 AM and 4:00 PM. Dead
birds were removed from the pen upon discovery and the body weight was measured.
Feed for the pen was weighed back as mortality occurred. All mortalities were sent to a
veterinary pathologist for postmortem examination in order to determine cause o f death.
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51
3.3.6 Statistical Analysis
The experiment was a split-plot factorial design. Four room by trial combinations
(2 rooms in each trial and 2 trials) were the blocks, two sections o f a room were the
whole plots and individual pens within each section were subplots. Two photoperiods
(continuous and increasing) were whole plot treatments and a factorial o f toe-treatment
(treated-toe and intact-toe) and sex (male and female) were subplot treatments. The
response variables including body weight, body weight gain, feed intake and feed
conversion ratio were analyzed with Repeated Measures Analysis using Proc Mixed
procedure in SAS® software (SAS Institute, 1999). The statistical significance was
determined using a (level o f significance) o f 5%. The following model was employed
for statistical analysis o f data for a given time point:
y mm = a + « , + P} + (aP)9 + yk + (a y )lk + ( Py) jk + (aP y)ijk + <r, + M s + ( p s ) fl
+ (a p a )y, + (yS )kI + (ccyS)ikl + (P yS)ilk + {a p 8 y )ijkl + s ijklm
Where y ijklm is the response o f interest; ju is the overall mean; a t is the main
effect o f blocking factor; p . is the effect o f t h e / h photoperiod factor (/= 1,2); y k is the
effect o f the £th toe-treatment (£=1,2); 8l is the effect o f /th sex factor (/=1,2); (P y )jk is
the effect o f the interaction between photoperiod and toe-treatment; (/?8 ) 7 is the effect
o f the interaction between photoperiod and sex; (yS )kl is the effect o f the interaction
between toe-treatment and sex; (PyS) jkl is the effect o f the interaction between
photoperiod, toe-treatment and sex; s ijklm is the random error component assumed to be
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52
normal, independent and have constant variance. Note that the interactions with the
blocking factor are used as errors for testing the effects o f photoperiod, toe-treatment
and sex. For Repeated Measures Analysis, the repeated statement in Proc M ixed was
used, and time was added as another factor.
The assumptions o f above model were tested in SAS®. Transformation was
conducted when the distribution o f the error terms was not normal. W hen a significant
difference was found among main effects or their interactions, Least Squares Means
(LSmeans) comparison tests in SAS® were used to compare these means.
Mortality data were analyzed by Chi-square test in Minitab®. The statistical
significance was determined using a (level o f significance) o f 5%.
3.4 Results and Discussion
3.4.1 Body Weight and Body Weight Gain
Based on the traditional weighing method, the toe-treated (TT) birds had lower
body weight (BW) on day 7 (Table 3.3). BW was reduced on days 24 and 31 for TT
birds based on computer bird scales (Table 3.3). There was no microwave toe-treatment
effect on body weight at other ages measured based on either method o f weighing birds
(Table 3.3). For both sources o f data, the birds given the increasing lighting (IL) were
lighter than those with the continuous light (CL) on days 7, 14, 24 and 31 (Table 3.4).
By day 38, the birds with IL and those with CL had similar body weight (Table 3.4).
For both sources o f data, the males were heavier than the females on days 24, 31, and
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53
38 (Table 3.5). Lighting by sex interaction was found to be significant based on two
sources o f data (Table 3.6). The difference in BW under two lighting treatments was
larger for the males than for the females (76g vs 45g for the males and females under
the continuous and increasing lighting based on computerized bird scales; 69g vs 44g
for the males and females the continuous and increasing lighting based on the
traditional weighing method).
Table 3. 3 Effect of microwave toe-treatment on body weight of broiler chickens
based on the traditional weighing method and computerized bird scales
at 0, 7,14, 24, 31, and 38 days of age
Body weight (g)
Age
Traditional weighing method
Computerized bird scales
Intact-toe
Treated-toe
Intact-toe
Treated-toe
0
38.6
38.3
38.6
38.3
7
138.3a
127.4b
129.4
126.0
14
367.9
353.3
358.2
330.2
24
1002.2
967.8
971.5C
920.5d
31
1566.8
1518.7
1504.2°
1468.4d
38
2150.1
2143.1
2119.4
2090.0
(d)
a’b Means in rows based on traditional weighing method, c"d means in rows based on
computerized bird scales are significantly different in Repeated Measures Analysis o f
Proc M ixed (p^0.05).
Body weight data based on both methods for weighing birds were transformed by cube
root, and means were transformed back and reported.
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54
Table 3. 4 Effect of lighting on body weight of broiler chickens based on the
traditional weighing method and computerized bird scales at 0, 7,14,24,
31, and 38 days of age
Body weight (g)
Age
(d)
Traditional weighing method
Continuous-light
Increasing-light
Computerized bird scales
Continuous-light
Increasing-light
0
38.5
38.4
38.5
38.4
7
147.1a
124.4b
137.4C
118.7d
14
402.9a
322.2b
379.3C
311.7d
24
1041.0a
926.l b
982.8C
909.9d
31
1607.8a
1479.7b
1518.0°
1454.3d
38
2144.1
2149.1
2136.2
2073.5
a~b Means in rows based on the traditional weighing method, c’d means in rows based on
computerized bird scales are significantly different in Repeated Measures Analysis o f Proc
Mixed (p<0.05).
Body weight data based on both methods for weighing birds were transformed by cube
root, and means were transformed back and reported.
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55
Table 3. 5 Effect of sex on body weight of broiler chickens based on the traditional
weighing method and computerized bird scale data at 0, 7,14, 24, 31, and
38 days of age
Body weight (g)
Age
Traditional weighing method
Computerized bird scales
(d)
Male
Female
Male
Female
0
38.5
38.4
38.5
38.4
7
137.3
133.4
126.6
128.9
14
363.7
357.3
348.2
339.8
24
1010.8a
954.l b
982.8°
909.9d
31
1620.9a
1467.7b
1569.8°
1405.9d
38
2272.9a
2025.7b
2230.3°
1985.4d
a"b Means in rows based on the traditional weighing method, c"d means in rows based on
computerized bird scales are significantly different in Repeated Measures Analysis o f
Proc Mixed (p^0.05).
Body weight data based on both methods for weighing birds were transformed by cube
root, and means were transformed back and reported.
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56
Table 3. 6 Lighting by sex interaction for body weight of broilers based on the
traditional weighing method and computerized bird scales
Traditional weighing method
Computerized bird scales
Lighting
Sex
BW (g)
Lighting
Sex
BW (g)
Continuous
Female
654b
Continuous
Female
720b
Increasing
Female
610°
Increasing
Female
675c
Continuous
Male
717a
Continuous
Male
788a
Increasing
Male
648b
Increasing
Male
712b
a"c Means in columns with no common superscript are significantly different in Repeated
Measures Analysis o f Proc M ixed (p^0.05).
Body weight data based on both methods for weighing birds were transformed by cube
root, and means were transformed back and reported.
Body weight gain (BWG) results are presented in Table 3.7 and Table 3.8 for the
traditional weighing method and computerized bird scales, respectively. There were
lighting, sex, lighting by time period and sex by time period effects on BW G based on
the traditional weighing method (Table 3.7). The toe-treatment did not significantly
affect BWG based on the traditional weighing method (Table 3.7). Based on
computerized bird scale data, there were lighting, toe-treatment,
and sex effects on
BWG; lighting by time period, toe-treatment by time period, and sex by tim e period
interactions were found to be significant for BWG (Table 3.8). An interaction between
lighting and toe-treatment was found to be significant for BWG based on computerized
bird scales (Table 3.9). The difference in BWG was 16.9 g for IT birds and was 59.5 g
for TT birds under continuous and increasing lighting programs.
The increasing lighting decreased BWG significantly in the first two weeks based
on both sources o f data (Table 3.7 and Table 3.8). The birds with IL had larger BWG
during 31-38 day period based on the traditional weighing method (Table 3.7). Based
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57
on computerized bird scales, during the second week, the IT broilers gained
significantly more weight than the TT birds (Table 3.8). The males had significantly
greater weight gain during 14-23, 24-30 and 31-38 day periods than the females based
on two weighing methods for (Table 3.7 and Table 3.8).
Table 3. 7 Effect of lighting, microwave toe-treatment, and sex on body weight gain
of broiler chickens at 0-6, 7-13,14-23,24-30, and 31-38 day periods based
on the traditional weighing method
Body weight gain based on traditional weighing method
Treatment
0-6 d
7-13 d
14-23 d
24-30 d
31-38 d
Continuous-light
104.9a
259.4a
675.0
567.4
553.7b
Increasing-light
82.2b
199.9b
644.8
538.4
636.5a
Intact-toe
92.4
236.3
682.9
577.5
594.3
Treated-toe
91.0
221.3
637.0
558.2
594.4
Female
95.4
225.5
619.2b
533.7b
516.2b
Male
91.1
231.8
701.7a
572.3a
678.l a
a‘b Means in columns within a treatment at the same time period are significantly
different in Repeated Measures Analysis o f Proc M ixed (p<0.05).
Body weight data were transformed by square root, and means were transformed back
and reported.
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58
Table 3. 8 Effect of lighting, microwave toe-treatment, and sex on body weight
gain of broiler chickens at 0-6, 7-13,14-23, 24-30, and 31-38 day
periods based on computerized bird scale data
Body weight gain based on computerized bird scales
Treatment
0-6 d
7-13 d
14-23 d
24-30 d
31-38 d
Continuous-light
96.9a
261.5a
620.4
540.1
644.1
Increasing-light
78.8b
204.3b
606.3
541.3
619.9
Intact-toe
88.5
248.4a
630.5
527.3
650.2
Treated-toe
86.8
216.2b
596.5
554.2
630.8
Female
90.5
223.4
577.2b
494.9b
603.9b
Male
84.7
240.8
650.7a
588.5a
660.7a
a~b Means in columns within a treatment at the same time period are significantly
different in Repeated Measures Analysis o f Proc Mixed (p<0.05).
Body weight data were transformed by square root, and means were transformed
back and reported.
Table 3. 9 Lighting by sex interaction for body weight gain of
broilers based on computerized bird scales
Lighting
Toe
BWG*(g)
Continuous
Intact
588.9ab
Increasing
Intact
572.0b
Continuous
Treated
596.6a
Increasing
537.1°
Treated
* BWG: body weight gain.
a’° Means in the column with no common superscript are significantly
different in Repeated Measures Analysis o f Proc M ixed (p<0.05).
Data were transformed by square root, and means were transformed
back and reported.
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59
The TT birds were lighter than the IT birds on day 7 based on the traditional
weighing method. However, they had similar BW on day 7 based on computerized bird
scales. This difference might have been due to lighter TT birds with more discomfort at
this age which m ay affect their ability to step on the platform. The difference in toetreatment effects on BW on days 24 and 31 between two methods for weighing birds
may be due to inactivity o f heavier toe-treated birds at those ages which may be less
likely to step on the platform. The lower feed intake o f toe-treated birds early in life
could have been contributed to the lighter BW o f the TT birds early in life. Reduced
early BW o f the TT also could be the result o f the initial physical insult and pain o f the
toe-treatment. Toe-treatment may cause initial stress in birds as suggested by Compton
et al. (1981). The physiological change in the stress response could be associated with a
reduction in body weight. The reduction in early growth caused by the toe-treatment is
in agreement with the findings o f Compton et al. (1981) who used a hot blade to cut the
toe tips o f White Leghorn chicks at 1 day o f age and found that birds with intact toes
were generally heavier than those that were toe-treated until 14 wk o f age. At 20 wk o f
age, there was no significant difference in BW between the two treatment groups.
Honaker and Ruszler (2004) employed microwave energy to restrict claw growth o f two
strains o f Leghorn chicks at hatch. They reported that TT birds had lower BW than the
IT group for the most o f the growout period; however, this difference was never more
than 30 g throughout the growing period. Moran (1985) treated toes o f large type
turkeys with a hot-blade debeaker at 1 day o f age or 12 days o f age and reported that
toe-treatment had no effect on body weight at any stage o f production during 119 or
131-day growing period for hens or toms, but feed conversion was improved for a short
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60
time immediately following the operation at each treatment age. In our study, for two
sources o f data, by market age, toe-treated broilers were the same weight as the
controls. This result agrees with Owings et al. (1972) who clipped three toes on each
foot with sharp scissors in turkeys at 1 day o f age and observed that toe-treatment did
not reduce the final body weight o f the turkeys raised to 24 wk o f age (10.91 kg and
11.08 kg for toe-treated and untreated turkeys). The similar BW at market age between
the TT birds and IT birds is not in agreement with Newberry (1992). She clipped the
inner two toes o f each foot in turkeys at the hatchery with a hot blade and found a
significant reduction in final body weight o f toe-treated turkeys in a 17-wk growing
period (turkeys with intact toes were 0.44 kg heavier than turkeys with clipped toes at
17 wk).
Our findings that broilers with the increasing lighting program exhibited reduced
early grow th rate and did not reduce final body w eights are consistent with previous
studies shown as follows. Riddell and Classen (1992) reported that broilers under
increasing lighting treatments showed significantly lower weight gains from 0 to 21
days o f age than birds under continuous lighting but significantly higher weight gains
from 42 to 63 days o f age. Overall, the increasing lighting programs increased gain
from 0 to 63 days o f age compared with chickens reared under constant lighting. Blair
et al. (1993) found that broiler chickens raised on an increasing light pattern were
lighter in weight at 3 wks than those on continuous light; but by market age (6 wks)
they had the same body weight. Renden et al. (1993) reported that broilers with 23 h
light had greater B W than other light treatments including 16 h light, 14 h light and
increasing lighting from 7 to 35 days. The BW for birds on each light treatment was not
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61
different by 48 days in their study. Rozenboim et al. (1999) found that at 32 days o f age
birds in group 23:1 LD were significantly heavier than the increasing light group;
however, at 49 days broilers reared under increasing daylength were heavier than those
with 23:1 LD. Shah and Petersen (2001) also reported that there was only an initial
reduction in body weight and body weight gain in birds reared under the increasing
photoperiod compared to the nearly continuous daylight. The reduction in early growth
rate is presumably caused by the extended scotophase during the early stage o f a
broiler’s life (Classen et al., 1991). The influence o f light on body weight m ay be due to
its effect on feeding activity (Weaver and Siegel, 1968). Short daylength given early in
life may restrict time o f access to feed and water. These birds, however, would have the
establishment o f activity rhythms, increased sleep, lower physiological stress and
improved immunoresponsiveness (Gordon, 1994). W ith increasing daylength during
broiler life cycle, the birds have more time to eat and drink and grow faster in later life.
Classen and Riddell (1989) speculated that the role o f androgenic hormone production
is important to increase growth rate in increasing lighting programs. It is also possible
there is difference in metabolism in the light and darkness. However, Morris (1967)
found growth to be maximized with a near continuous daylength; body weights o f the
broilers were 5-10% heavier with 23 h light than grown under an 8 or 12 h photoperiod.
Beane et al. (1979) also suggested that optimum growth and feed conversion were
attained with chickens kept on continuous lighting as compared to light regimens o f less
than 24 h o f light per day.
The difference in BW between sexes could be related to physical differences o f
both sexes and the release o f sex hormones. The growth curve o f both sexes in our
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62
study is consistent with a standard growth curve. We found an interaction with sex and
lighting. Buyse et al. (1996b) also presented that sex may interact with lighting
schedule. Shah and Petersen (2001) reared broilers under traditional program,
increasing photoperiod and decreasing photoperiod in a 42-day period. They found that
difference in body weight between male and female broilers were the highest under
increasing photoperiod. The female broilers did not show compensatory growth in body
weight during the last two weeks o f growth to the extent seen in the male broilers.
In our study, generally the BW based on the traditional weighing method were
heavier than those obtained from the computerized bird scale, this could be that the
heavy birds for all treatments were less active and stepped less on the scale than the
light birds. The difference in BW between the traditional weighting method and
computerized bird scales may also due to different sample sizes o f two methods.
3.4.2 Feed Intake and Feed Conversion Ratio
The microwave toe-treatment reduced feed intake (FI) during 7-13 and 0-38 day
periods (Table 3.10). The toe-treated (TT) birds and the birds with intact toes had
similar FI in other time periods (Table 3.10). The intact-toe birds consumed 83.7 g more
feed than the toe-treated birds during the 38-day growing period. The birds on the
continuous lighting (CL) consumed more feed during the 0-6, 7-13 and 14-23 day
periods (Table 3.10). The birds with the increasing lighting (IL) had significantly higher
FI during the 31-38 d period (Table 3.10). The CL decreased overall FI significantly
(Table 3.10). The birds with CL consumed 131.4 g more feed than the birds with IL in
the 38-day growing period. The males had significantly higher FI during 14-23, 24-30,
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63
31-38, and 0-38 day periods (Table 3.10). The males consumed 434.4 g more feed than
the females in the 38-day growing period. It was found that lighting interacted with sex
and toe-treatment interacted with sex on FI (Table 11 and Table 12). The difference in
FI o f the birds between CL and IL was 20.8 g for the females, and 86.2 g for the males.
The difference in FI o f the birds between IT birds and TT birds was 1.1 g for the
females, and 52.9 g for the males.
Neither the microwave toe-treatment nor the lighting programs had a significant
effect on the feed conversion ratio (FCR) at any time period based on the traditional
weighing m ethod ( Table 3.13). Based o n t he t raditional w eighing m ethod, th e m ales
had better FCR during 31-38 day period, but both sexes had a similar overall FCR
(Table 3.13). Based on computerized bird scales, FCR o f the birds with IL was
significantly lower during 14-23 day period and higher during 31-38 day period (Table
3.13); lighting did not influence this parameter in other time periods (Table 3.13); the
females had better FCR during 31-38 day period, but both sexes had a similar overall
FCR (Table 3.13).
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Table 3.10 Effect of lighting, microwave toe-treatment, and sex on feed intake of broiler chickens at 0-6,7-13,14- 23,31-38,
and 0-38 day periods
__________________________________________ Feed intake (g)__________________________________________
Treatment
0-6 d
7-13 d
14-23 d
24-30 d
31-38 d
0-38 d
Continuous-light
125.53
3 5 0.4a
1037.63
1069.8
1170.2b
3758.2a
Increasing-light
94.0b
282.8b
919.3b
1043.2
1293.0a
3626.8b
Intact-toe
113.1
327.0a
990.7
1070.7
1226.1
3734.2a
Treated-toe
105.4
304.5b
964.5
1042.2
1235.7
3650.5b
Female
111.5
310.4
939.7b
987.6b
1128.9b
3478.2b
Male________________10&9___________ 321.0____________ 1016.1a___________ 1127.63__________ 1337.2a__________ 3912.6a
a"b Means in columns within a treatment at the same time period are significantly different in Repeated Measures Analysis o f Proc
M ixed (p<0.05).
Data were transformed by square root, and means were transformed back and reported.
O-v
4^
Table 3.11 Lighting by sex interaction for feed intake of
broilers
Lighting
Sex
Feed intake (g)
Continuous
Female
939.0°
Increasing
Female
918.2°
Continuous
Male
1076.4a
990.2b
Increasing
Male
a’° Means in the column with no common superscript are
signifcantly different in Repeated M easures Analysis o f Proc
Mixed (p<0.05).
Data were transformed by square root, and means were
transformed back and reported.
Table 3.12 Toe-treatment by sex interaction for feed intake
of broilers
Toe
Intact
Sex
Feed intake (g)
Female
928.0°
Treated
Female
929.1°
Intact
Male
1039.5a
Treated
Male
1006.6b
a’° Means in the column with no common superscript are
signifcantly different in Repeated Measures Analysis o f Proc
Mixed (p<0.05).
Data were transformed by square root, and means were
transformed back and reported.
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Table 3.13 Effect of lighting, microwave toe-treatment and sex on feed conversion ratio based on
computerized bird scales and the traditional weighing method during 0-6, 7-13,14-23,24-30, 3138, and 0-38 day periods
Feed conversion ratio based on computerized bird scales (g:g)
Treatment
0-6 d
7-13 d
14-23 d
24-30 d[
31-38 d
0-38 d
Continuous-light
1.30
1.34
1.68a
2.01
1.82b
1.74
Increasing-light
1.19
1.38
1.51b
1.92
2.09a
1.76
Intact-toe
1.26
1.31
1.57
2.04a
1.94
1.75
Treated-toe
1.24
1.41
1.61
1.89b
1.97
1.75
Female
1.23
1.37
1.61
2.00a
1.87b
1.73
Male
1.26
1.35
1.57
1.93b
2.04a
1.76
Feed conversion ratio based on traditional weighing data (g:g)
Continuous-light
1.24
1.38
1.54
1.89b
2.19
1.74
Increasing-light
1.13
1.46
1.44
2.00a
2.06
1.72
Intact-toe
1.22
1.42
1.47
1.98
2.10
1.72
Treated-toe
1.14
1.41
1.51
1.90
2.15
1.73
Female
1.19
1.44
1.54
1.89
2.27a
1.75
Male
1.17
1.40
1.44
1.99
1.99b
1.71
a'b Means in columns within a treatment in the same time period are significantly different in Repeated Measures
Analysis o f Proc Mixed (p<0.05).
Feed conversion ratio data were transformed by square root, and means were transformed back and reported.
Os
Os
67
It is apparent that the lower feed consumption in the first 3 weeks had contributed
to the lower overall feed intake for the toe-treated birds in this experiment. This is
consistent with the behavior observation that the toe-treated birds were less active to
access feed and water early in life (Peralta, personal communication, 2004). The
reduction in FI early in life for the toe-treated birds agrees with Compton (1980), who
found t hat W hite Leghorn c hickens w ith i ntact t oes c onsumed s ignificantly m ore f eed
during the first 3 weeks o f growout phase, while there were no significant differences in
FI between the TT and IT groups during the latter stages o f the growout period. On the
other hand, the FCR data o f their study indicated that there was no difference between the
two groups during the initial growout period (1 to 8 wks); however, during the latter
portion o f this growout phase (8 to 20 wks), the TT birds demonstrated a significant
improvement in FCR over the intact-toe birds. I n our study, the TT birds had a lower
overall FI, but the TT birds and IT birds had the same overall FCR in the 38-day growing
period. This finding is consistent with the report o f Honaker and Ruszler (2004), they
found that IT Leghorn hens consumed significantly more feed than the TT birds from 8
wk until the end o f the growout period (18 wks), but they had similar FCR from 6 to 18
wks. Goodling et al. (1984) postulated that the greater feed usage by the IT birds was
probably not being deposited as fat but they were using that higher feed consumption to
support their higher level o f activity. M artin et al. (1976), on the other hand, reported that
toe-treatment improved feed conversion in layers.
The birds with CL consumed more feed than the birds on IL, however, the birds
w ith CL and those with IL had similar BW by 38 days o f age. This indicates that more
feed consumption under CL did not convert into BWG; but this extra energy m ay have
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68
been used to maintain the bird’s body temperature and their higher level o f activity in the
light. Riddell and Classen (1992) reported that the increasing lighting treatment produced
improved feed efficiency from 0 to 21 days o f age but did not affect this parameter for
other time periods. Intermittent lighting for growing broilers has been shown to result in
equal or improved feed efficiency compared with continuous lighting (Classen et al.,
1991; Buyse et al., 1996a). Improved feed conversion with an intermittent lighting was
related to higher metabolizability and lower energy expenditure on physical activity,
compared to continuous lighting (Apeldoom et al., 1999).
The conflicting results o f the effect o f sex on FCR during 31-38 day period based
on both methods for weighing birds is hard to explain. Overall, the males consumed more
feed and gained more weight, so both sexes had similar overall FCR.
The findings that toe-treated birds or the birds on IL had a lower FI, but similar
overall FCR to the birds with intact toes or on CL in this study indicates that the
microwave toe-treatment or the increasing lighting would have economiccal benefits for
broiler production. It was especially noted that the birds with IL consumed less feed until
day 24 and more feed in the last week. This may decrease the cost o f feeding more
because early feeding is more expensive due to higher protein content.
3.4.3 Mortality
The mortality in the first week was 3.33%, accounting for nearly 70% o f total
mortality (4.95%) in the 38-day growing period. The TT birds had significantly higher
mortality t han IT groups i n t he first w eek, w hich w ere 4.74% a nd 1.92% r espectively
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69
(Table 3.14). The females had higher mortality than the males in the first week (Table
3.14). There was no lighting effect on mortality in the first week (Table 3.14). In the 38day growing period, the TT birds had higher mortality than IT birds, the birds with IL had
lower mortality than those with CL, and the females had significantly greater mortality
than the males (Table 3.14).
The major cause for mortality o f the toe-treated birds in the first week was found to
be dehydration, accounting for about 87% o f mortality in the first week. This confirms
that microwave toe-treatment could influence the bird’s ability to get access to water and
feed. The microwave toe-treatment resulted in higher overall mortality due to higher
mortality in the first week. For the rest o f growing period, the IT and TT birds had similar
mortality. This suggests that the birds could overcome the initial physical insult after first
week. Owings et al. (1972) reported that toe-treated turkeys averaged 9.9% mortality
versus 1.9% for the birds with intact toes during the first week, and there was no
difference in mortality after the first week between two groups, being 8.1% and 8.7% for
toe-treated birds and intact-toe birds. Newberry (1992) found that mortality to 4 wk was
lower for turkeys with intact toes than treated-toes. However, Ruszler and Kiker (1975),
using a hot blade debeaker to treat the toes o f chicks, reported that toe-treated hens had
about 2.5% lower mortality as compared to the birds with intact toes. Compton (1980)
found that toe-treatment had no significant effect on mortality o f caged Leghorn
chickens.
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70
Table 3.14 Effect of lighting, microwave toe-treatment and sex on mortality of
broiler chickens during 0-7 and 0-38 day periods
Treatment
Age (d)
0-7
0-38
Lighting
Toe
Sex
Continuous Increasing
Intact Treated
Female Male
Live birds
1405
1410
1428
1387
Dead birds
51
46
Total birds
1456
Mortality (%)
3.50
1397
1418
28
69
59
38
1456
1456
1456
1456
1456
3.16
1.92
4.74
4.05
2.68
X2
0.267
17.927
4.673
Probability
0.606
<0.001
0.031
Live birds
1368
1400
1405
1363
1369
1399
Dead birds
88
56
51
93
87
57
Total birds
1456
1456
1456
1456
1456
1456
Mortality (%)
6.04
3.85
3.50
6.39
5.93
3.91
X2
7.481
12.887
6.575
Probability
0.006
<0.001
0.010
Lower m ortality o f t he birds o n t he i ncreasing 1ighting p rogram i s i n a greement
w ith the findings o f Rozenboim et al. (1999), who reported that the mortality o f broilers
was lower in the increasing lighting group than the 23:1 LD group. In our study, around
8% mortality o f the birds on CL was due to sudden death syndrome, but no sudden death
syndrome was found in the IL group. This confirms the findings o f Classen et al. (1991),
who demonstrated that increasing lighting decreases the incidence o f sudden death
syndrome. Other causes o f mortality in the flock were omphalitis, conjunctivitis, trauma,
autolysis, and leg deviation. The evidence supports the concepts that darkness, and more
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71
specifically the melatonin hormone, are associated with increased immune function and
reduced mortality (Hotchkiss and Nelson, 2002). Melatonin is synthesized and secreted
during the dark phase o f the light-dark cycle in virtually all species (Pevet, 1998).
Duration o f the melatonin increase is controlled by photoperiod, the longer the night, the
longer the duration o f secretion (Arendt, 1995). Kliger et al. (2000) reported that splenic
T and B lymphocytes from 6-wk-old chickens grown in short daylength had higher
activities than those from chicken grown in constant lighting.
The female broilers had higher mortality than the male birds in the 3 8-day growing
period, which was a result from higher mortality o f the females in the first week. This
may suggest that the male chicks could tolerate the negative effect caused by toetreatment better than the females. This could be related to physical difference in toes
between males and females, being thicker for the males. The sex difference in mortality
rates o f two Leghorn strains were investigated and it was found that rate o f loss
significantly lower in males than females but did not differ between crosses (Lowe and
Garwood, 1976).
3.5 Conclusions
The microwave toe-treatment reduced early body weight (BW) o f the broilers, but
did not reduce the BW o f the birds at market age (day 38) proves that the birds can
overcome this negative effect with age. The toe-treatment reduced feed intake (FI) and
had no effects on final body weight o f the birds and feed conversion ratios (FCR). The
toe-treated (TT) group had higher overall mortality than the intact-toe (IT) birds due to
the higher mortality o f the TT birds in the first week. The birds on the increasing lighting
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72
program (IL) were significantly lighter than the birds on the continuous lighting program
(CL) during most o f the growout phase. The reduction in early growth rate w ith IL was
expected. This benefited the bird’s health and resulted in decreased mortality in later life.
The birds with IL consumed less feed, especially starter and grower diets which are more
expensive due to higher protein percentage, but had the same feed conversion ratio as the
CL birds. This suggests that the increasing lighting would be more econom ic than the
continuous photoperiod program for growing broilers. The males had a higher FI, greater
BW and weight gains, but had lower mortality than the females in this study. It was found
that the male broilers had a larger BW difference with continuous and increasing light
than the females. The same trend was found between sexes for the IT and TT birds. There
was a higher FI difference for the males than females under CL and IL. The IT and TT
females consumed similar amount o f feed, but the IT males consumed more feed than the
TT males. Collectively, the present results demonstrate that the microwave toe-treatment
or the increasing photoperiod program did not have a detrimental effect on growth
performance o f broilers by market age. In order for microwave toe-treatment to become a
viable commercial procedure, the level o f exposure to microwave energy may need to be
reduced in effort to reduce related mortality. Additional projects to determine the
optimum level for broiler chickens is recommended. The results also prove that the
increasing lighting program has benefits to decrease mortality.
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73
CHAPTER 4
STRESS AND FEAR LEVELS OF MICROWAVE
TOE-TREATED BROILER CHICKENS GROWN W ITH TWO
PHOTOPERIOD PROGRAMS
4.1 Abstract
Stress and fear have received much attention in the poultry industry in recent
years because there is a growing concern regarding the welfare o f birds. Stress and fear
also have consequences on performance o f birds and the profitability o f poultry
production. Increasing lighting programs are currently o f interest for growing broilers
because this type o f photoperiod adjustment has been shown to be a ssociated with a
reduction in heart disease and leg problems. However, birds raised under this type o f
program may have an increased incidence o f carcass scratches inflicted by toenails o f
other broilers. One potential solution for reducing scratch damage is to treat the toes o f
day-old broiler chickens with microwave energy. The present study was conducted to
investigate the influence o f microwave toe-treatment and photoperiod on the activity o f
plasma creatine kinase (CK), the heterophil to lymphocyte (H/L) ratio and tonic
immobility (TI) reaction in order to evaluate the level o f stress and fear in broilers. Two
replicate trials were conducted with 728 female and 728 male broilers in each trial. H alf
o f the birds from each sex were toe-treated with microwave energy upon delivery from
the hatchery. The birds i n each trial were r andomly assigned to four sections o f two
rooms with a total o f 32 floor pens. Two sections were given the continuous lighting
from start to finish and the other two were on an increasing lighting program with short
photoperiod after the first three days, which increased to 23 h by day 30. Blood samples
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74
were collected at 21 and 35 days o f age to measure the activity o f plasma CK and H/L
ratios. The tonic immobility (TI) test was conducted on days 10, 22 and 36. The
microwave toe-treatment did not affect the activity o f plasma CK, the H/L ratios and TI
response o f the birds. The birds on the continuous lighting program had a higher
activity o f plasma CK (p<0.05), but similar H/L ratios compared to birds with the
increasing light at both 21 and 35 days o f age. The increasing lighting program
decreased the duration o f TI on day 10 (p<0.05); did not influence this parameter on day
22; whereas increased duration o f TI at 36 days o f age (p<0.05). The males had a lower
activity o f plasma CK (p<0.05) and H/L ratios than the females (p<0.05). There was no
difference in TI response between the males and the females. These results suggest that
the microwave toe-treatment did not influence the level o f stress and fear o f the birds.
The increasing lighting program could reduce the level o f stress, and decrease the level
o f fear early in life, but may increase the level o f fear in later life. The males showed a
lower level o f stress and similar level o f fear compared to the females.
4.2 Introduction
Stress and fear have received much attention in the poultry industry in recent
years because there is a growing concern regarding welfare o f birds. Both stress and
fear m ay also have deleterious effects on growth performance, reproductive
performance and meat quality o f birds (McKee and Sams, 1997; Thankson et al., 2001;
Mashaly et al., 2004). Fear is a vital component o f the stress response (Beuving et al.,
1989), which begins with the central nervous system perceiving a potential threat to
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75
homeostasis (Moberg, 2000). Once this occurs, the organism develops a biological
response that consists o f a combination o f the four general biological defense responses:
the b ehavioral r esponse, t he a utonomic n ervous system r esponse, t he n euroendocrine
response and the immune response (Moberg, 2000).
In the broiler industry, birds have traditionally been kept on a continuous or
nearly continuous photoperiod schedule (24 or 23 h light/day) throughout the growing
period so as to maximize feed intake and growth rate (Morris, 1967; Beane et al., 1979).
However, considerable evidence suggests that there are benefits to bird welfare when
broilers are on short or moderate daylengths; these include lower physiological stress
and a lower level o f fearfulness (Gordon, 1994; Davis et al., 1999; Rozenboim et al.,
1999). Increasing photoperiod programs, which provide short photoperiod early in life
and increase daylengths as a broiler ages, are currently o f interest for growing broilers
because t hey h ave b een s hown t o b e associated w ith a r eduction i n t he i ncidence o f
heart disease and leg problems (Classen and Riddell, 1989; Classen et al., 1991; Renden
et al. 1991; Rozenboim et al., 1999). However, broilers given this type o f program may
have an increased incidence o f carcass scratches In flicted by toenails o f other broilers
(Hargis et al., 1989). The increased incidence o f carcass scratches is due to increased
bird activity under increasing photoperiod programs (Blair et al., 1993). A potential
solution for reducing scratch damage is to treat the toes o f day-old broiler chickens with
microwave energy. Currently, the turkey industry uses this technology to restrict claw
growth and reduce carcass scratches. Previous to this procedure, researchers
experimented with hot blade beak trimming equipment to remove the tip o f the toe.
Compton et al. (1981) used a hot blade to trim the toes o f W hite Leghorn chicks and
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76
proposed that toe-trimming posed an initial stress indicated by increased plasm a
corticosterone. However, the initial pain caused by toe-treatment early in life may easily
outweigh the chronic pain o f lacerations to the skin o f birds later in the rearing period.
Honaker and Ruszler (2004) reported that microwave toe-treated Leghorn layers
displayed a lower level o f fearfulness to humans as measured by the tendency o f birds
to not panic in the presence o f a human. Satterlee et al. (1985) also found toe-treated
hens to be less active and fearful.
There are a number o f measurements that have been used to examine the level o f
stress and fear. The heterophil to lymphocyte (H/L) ratio has been established as a
widely accepted indicator for determining chronic stress in poultry (Gross and Siegel,
1983). In response to environm ental stressors, in chicken blood samples, the number o f
lymphocytes decreases and the number o f heterophils increases, thus increasing H/L
ratios (Jones et al., 1988; El-Lethey et al., 2003). W hen the bird is subjected to
environmental stressors, the hypothalamic-pituitary-adrenocortical (HPA) axis is
activated and increases plasma corticosterone concentrations. It has been found that
long-term elevation o f plasma corticosterone concentrations may impair leucocytic
responsiveness (Jones e t al., 1 988). T he activation o f H P A inhibits hum oral an d c ell
mediated immunity (Davison et al., 1983; Siegel et al., 1983). In recent years, the
elevated activity o f plasma creatine kinase (CK) has been found in the stressed birds
(Mitchell and Sandercock, 1995). In broiler chickens, CK is released into the circulation
following changes in the permeability o f the sarcolemma in response to various
pathologies and exposure to environmental stressors (Mitchell and Sandercock, 1995).
Tonic immobility (TI) reaction has been considered as a reliable index o f fearfulness in
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77
birds (Gallup, 1979). High susceptibility to TI and a long duration o f TI are signs o f a
high level o f fear (Jones, 1986; 1987). The objective o f this study was to investigate the
influence o f microwave toe-treatment and photoperiod on stress and fear levels in
broiler chickens. Activities o f plasma CK, the H/L ratio and TI tests were employed in
the experiment.
4.3 Materials and Methods
4.3.1 Birds, Diets and Housing
Two replicate trials were conducted with 728 female and 728 male broilers in
each trial. The first trial was performed in the fall and the other in the winter. Sexed,
day-old broilers (Ross 308) were obtained from a com m ercial hatchery and were placed
in sex-separated pens in a confinement house with untreated wood shavings as litter.
Animal care was provided according to the standards o f the Canadian Council on
Animal Care (1993) and the protocol was approved by Nova Scotia Agricultural
College Animal Care and Use Committee. Two room s were used in each trial; each
room was separated by a black plastic divider into two sections for two photoperiods. In
each trial, the birds were randomly assigned to four sections o f the two rooms with 8
floor pens per section, giving 4 replications o f each lighting, toe-treatment and sex
combination treatment. The stocking density was 0.07 m2 bird'1. Feed and water were
available ad libitum with one suspended tube feeder per pen and a nipple drinker system
in each room. There were ten nipples per pen. Box feeders were also used to provide
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78
feed in the first week. All the birds were given the same starter, grower, and finisher
rations. The starter diets contained 23% crude protein and 3050 kcal ME k g '1 and were
fed as mash from day 0 to 13 days o f age. The grower diets contained 20% crude
protein and 3150 kcal ME kg'1 and were fed as mash from day 14 to 23 days o f age. The
finisher diets contained 18% crude protein and 3200 kcal ME k g '1 and were fed as
pellets from days 24 to 38 days o f age. The ingredients used in the diets and the
calculated analyses are shown in Table 4.1. The brooding temperature was set at 3032°C from day 0 to day 7 after which it was reduced 3°C per week until it reached 21°C
where it remained for the rest o f the trial.
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79
Table 4 .1 Ingredient composition and calculated analyses of diets used in the
experiment
Ingredients (%)
Com
Wheat
Soybean meal
Poultry grease
Limestone
Dicalcium phosphate
Iodized salt
DL-Methionine
Amprolium11
BMDV
Pel-Stikw
Vit/M in Premixxy
Calculated content
Crude protein, %
Metabolizable energy, kcal kg'1
Calcium, %
Available phosphorus, %
Lysine, %
M ethionine + Cystine, %
Starter
(0-13 d)
Grower
(14-23 d)
Finisher
(24-38 d)
42.90
10.00
38.69
3.86
1.97
1.10
0.32
0.103
0.05
0.0044
0.50
0.50
50.08
10.00
31.29
4.47
1.85
0.95
0.30
0.002
0.05
0.0044
0.50
0.50
55.74
10.00
26.23
3.98
1.84
0.91
0.30
23.00
3050
1.00
0.45
1.37
0.95
20.00
3150
0.92
0.40
1.15
0.76
18.00
3200
0.90
0.38
1.00
0.70
0.50
0.50
“ Amprolium: Merck & Co., Inc., Whitehouse station, NJ, USA.
VBMD: a dried precipitated germentation product obtained by culturing Bacillus subtilis
tracy on media adapted for microbiological production o f bacitracin;calcuim carbonate.
Alpharma, Fort Lee, NJ. USA.
w Pel-Stik: Uniscope, Inc., Johnstown, CO, USA.
x Supplied per kg starter diet: vitamin A, 19,500 IU; vitamin D3, 4000 IU; vitamin E, 2.97
mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.046; niacin, 29.7; folic
acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg; thiamine, 5.8 mg;
manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50 mg; wheat
middlings, 1432 mg; ground limestone, 500 mg.
y Supplied per kg grower & finisher diet: vitamin A, 19,500 IU; vitamin D 3, 4000 IU;
vitamin E, 2.97 mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.024;
niacin, 29.7; folic acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg;
thiamine, 5.8 mg; manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50
mg; wheat middlings, 1543 mg; ground limestone, 500 mg.
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80
4.3.2 Microwave Toe-treatment
H alf o f the birds from each sex were toe-treated using a Microwave Claw
Processor (MCP, Nova-Tech Engineering Inc., Willmar, MN) upon delivery from the
hatchery. To conduct the microwave toe-treatment, the chicks were turned upside down
and their legs were inserted into shackles o f the MCP between the knee and the ankle.
The ventral side o f the bird was toward the operator. Each o f the three front toes were
pulled into place by a vacuum and treated with microwave energy for 0.8 s.
4.3.3 Experimental Lighting Programs
Light was provided by incandescent bulbs mounted at approximately 2.0 m above
the floor level. All birds were subjected to the same light intensity. Light intensity was
measured at the bird head level with a light meter (Cal-Light 400, The Cooke
Corporation) and adjusted by rheostat. All broilers were given 24 h light for the first
three days. Then in two sections the lighting schedule was 23 h per day until the end o f
the trial. The other two sections were on an increasing photoperiod program. The two
lighting programs used in the experiment are in Table 4.2.
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81
Table 4. 2 Two lighting programs used in the experiment
Age (d)
Lighting
Continuous
Increasing
Light intensity
(lux)
0-3
24L1:0D2
24L:0D
20
4-6
23L:1D
10L:14D
20
7-9
23L:1D
10L:14D
5
10-16
23L:1D
12L:12D
5
17-22
23L:1D
14L:10D
5
23-29
23L:1D
18L:6D
5
30-38
23L:1D
23L:1D
5
*L: light
2D: darkness
4.3.4 Blood Sampling
At 21 and 35 days o f age, blood samples were collected from 3 randomly selected
birds from each o f the 32 pens per trial. The blood sampled birds were wing-banded on
day 21 and the same birds were used for blood sampling on day 35. Approximately 3.5
ml blood was collected via venipuncture from the brachial vein into a 7.0 ml
Vacutainer® tube containing sodium heparin as an anticoagulant. The blood samples
were immediately placed on ice until they were ready for centrifugation. At the same
time two drops o f blood from each bird was collected using heparinized micro-capillary
tubes from two o f the three birds and was smeared onto each o f two glass slides for
determining the H/L ratio. The blood samples were centrifuged at 1600 x g at 4°C for
15 min and plasma samples were transferred into microcentrifuge tubes and frozen at 80°C until analyzed.
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82
4.3.5 The Tonic Immobility Test
On days 10, 22 and 36, two birds from each pen were randomly chosen to
perform tonic immobility (TI) tests between 9:00 AM and 12:00 noon. The birds used
for blood sampling the day before were not used for TI test. TI was induced by
inverting t he b ird o n i ts b ack i n a p lastic b ox c ontaining a 10 c m 1ayer o f u ntreated
wood shavings. The same handler conducted this test throughout the trial. To induce
immobility one hand o f the handler rested lightly on the bird's breast and the other
covered its head. The bird was restrained for 15 s. The handler stood back in full view
o f the bird, but avoided eye contact with the bird. If the bird remained immobile for 10
s, TI was considered to be induced. If the bird righted itself before this minimum time
expired, the procedure was repeated until the immobility response was induced. A
maximum o f 10 inductions was performed. If the bird failed to be induced, another bird
was used. If the bird did not show a righting response over the 10-min period, the bird
was returned to the pen and a maximum duration o f 600 s was given for righting time.
The number o f times needed to induce immobility and the duration o f immobility were
recorded.
4.3.6 Laboratory Analysis
The activity o f plasma creatine kinase was measured using a commercial assay kit
produced by Bayers Diagnostic (Bayers Inc., Toronto, Ontario, Canada). This test was
conducted by the Veterinary Diagnostic Laboratory o f the Nova Scotia Department o f
Agriculture and Fisheries.
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83
dt)
•
•
•
•
Blood smears were fixed and stained using Hema 3 stam set (Fisher Diagnostics,
Fisher Scientific Company L.L.C. Middletown, VA) and observed under a light
microscope. One hundred leukocytes, including granular (heterophils, eosinophils,
basophils) and nongranular (lymphocytes, monocytes) ones, were randomly selected
and counted on one slide. The H/L ratio was calculated by dividing the number o f
heterophils by lymphocytes. Two slides were counted per bird and the mean H/L ratio
was calculated.
4.3.7 Statistical Analysis
The experiment was a split-plot factorial design. Four room by trial combinations
(2 rooms in each trial and 2 trials) were the blocks, two sections o f a room were the
whole plots and individual pens within each section were subplots. Two photoperiods
(continuous and increasing) were whole plot treatments and a factorial o f toe-treatment
(treated-toe and intact-toe) and sex (male and female) were subplot treatments. The
response variables including the activity o f plasma CK, the H/L ratio, the duration and
induction o f TI were analyzed with Repeated Measures Analysis using Proc M ixed
procedure in SAS® software (SAS Institute, 1999). The statistical significance was
determined using a (level o f significance) o f 5%. The following model was employed
for statistical analysis o f data for a given tim e point:
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84
yijum = M + a t + Pj + ( a p ) y + r k + 0ocy)ik + (P y ) Jk + ( aP r )m + ^ + (« cr)« + ( ^ ) /7
+ ipcPar)ffl + { y S ) kl + ( a y S ) ikl + (P yS )iJk + ( a p S y ) ijkl + s ijklm
Where y ijklm is the response o f interest; pi is the overall mean; a t is the main
effect o f blocking factor; /?;. is the effect o f t h e / h photoperiod factor (/'= 1,2); y k is the
effect o f the kth toe-treatment (£=1,2); S, is the effect o f /th sex factor (/=1,2); (P y )jk is
the effect o f the interaction between photoperiod and toe-treatment; {P8) jt is the effect
o f the interaction between photoperiod and sex; (yS)kl is the effect o f the interaction
between toe-treatment and sex; {J3yS) jkl is the effect o f the interaction between
photoperiod, toe-treatment and sex; e Jklm is the random error component assumed to be
normal, independent and have constant variance. Note that the interactions with the
blocking factor are used as errors for testing the effects o f photoperiod, toe-treatment
and sex. For Repeated Measures Analysis, the repeated statement in Proc Mixed was
used, and time was added as another factor.
The assumptions o f above model were tested in SAS®. Transformation was
conducted when the distribution o f the error terms was not normal. When significant
difference was found among main effects or their interactions, Least Squares Means
(LSmeans) comparison tests in SAS® were used to compare the means.
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85
4.4 Results and Discussion
4.4.1 The Activity of Plasma Creatine Kinase
The microwave toe-treatment did not influence the activity o f plasma creatine
kinase (CK) (Table 4.3). The birds on the continuous lighting program (CL) had a
higher activity o f plasma CK than birds on the increasing light program (IL) at both 21
and 35 days o f age (Table 4.3). There was a significant difference in plasma CK
activities between sexes; the males showed a lower plasma CK activity at both 21 and
35 days o f age (Table 4.3). All birds had a significantly higher activity o f plasma CK at
35 days o f age than at 21 days o f age (Table 4.3).
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86
Table 4. 3 Effect of lighting, microwave toe-treatment, and sex on
the activity of plasma creatine kinase of broiler
chickens at 21 and 35 days of age
Treatment
Activity o f plasma creatine kinase (U/L)1
Day 21
Day 35
Continuous-light
2195a
7533c
Increasing-light
1706b
6394d
Intact-toe
2036
6928d
Treated-toe
1866
6998c
Female
2135a
7630C
Male
1867b
6297d
1U/L: Unit per liter.
a'b’ c'd Means in columns within a treatment are significantly different
in Repeated Measures Analysis o f Proc M ixed (p<0.05).
There was a significant difference in the activity o f plasma creatine
kinase between 21 and 35 days o f age (p^0.05).
Data were transformed by Log, and means were transformed back
and reported.
Marked elevations in plasma CK activity have been reported in several
pathological muscle conditions in poultry including growth-associated and stressinduced myopathies (Mitchell et al., 1992; Mitchell and Sandercock, 1994; 1995). In
studies on broilers, Dzaja et al. (1996) examined the effects o f histamine application
and water-immersion stress on gizzard erosion and fattening o f broiler chicks and
concluded that CK activities were increased in the stressed chicks. Increases in plasma
CK also occurred in heat-stressed broilers (Mitchell and Sandercock, 1995). The
increased activity o f plasma CK on the continuous lighting program at both 21 and 35
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87
days o f age in our study may suggest that the continuous lighting program could have
detrimental effects on the meat quality o f broilers because elevated activity o f CK in
plasma or serum is well established as a reliable indicator o f skeletal muscle damage in
birds (Hamburg et al., 1992). The increased CK activity on CL may also be related to
increased stress level, fast growth rate or heart muscle damage o f the birds on CL.
Fast growing birds have elevated plasma CK activities compared to other strains
(Mitchell and Sandercock, 1995; Mitchell and Sandercock 1996). Genetic selection for
rapid growth rate induces alterations in membrane integrity and elevates the efflux o f
intracellular enzymes (Mitchell and Sandercock, 1995). Our findings that broilers at 35
days o f age had a significantly higher activity o f plasma CK than at 21 days o f age are
consistent with Hocking et al. (1998) who found that plasma CK activities were
significantly greater in the older, larger broilers and proposed that increase in the
plasma CK activity in the older birds may reflect age- or size-dependent increases in
muscle cell metabolism and turnover (Hocking et al., 1998).
4.4.2 The Heterophil to Lymphocyte Ratio
Neither the microwave toe-treatment nor the lighting program had any significant
effect on the heterophil to lymphocyte (H/L) ratio at either day 21 or day 35 (Table 4.4).
There was a significant difference in the H/L ratio between sexes; the females had a
greater H/L ratio than the males (Table 4.4).
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88
Table 4. 4 Effect of lighting, microwave toe-treatment, and sex on
the heterophil/lymphocyte ratio of broiler chickens at
21 and 35 days of age
Treatment
Heterophil/Lymphocyte ratio
Day 21
Day 35
Continuous-light
0.43
0.46
Increasing-light
0.41
0.43
Intact-toe
0.44
0.45
Treated-toe
0.46
0.49
Female
0.45a
0.46a
Male
0.39b
0.43b
a"b Means in columns within a treatment are significantly different in
Repeated Measures Analysis o f Proc M ixed (p<0.05).
Data were transformed by Log, and means were transformed back
and reported.
To the author’s knowledge, there have been no reports on the effect o f toetreatment on H/L ratios. The microwave toe-treatment did not influence the H/L ratio at
either 21 or 35 day o f age suggesting that the microwave toe-treatment did not influence
the stress level o f birds at those ages. However, this does not exclude the possibility that
stress occurs at an early age due to the microwave toe-treatment. Compton et al. (1981)
found that declawing posed an initial stress to the cage reared Leghorn chickens by
examining the blood corticosterone concentrations at 7, 13, and 19 wks o f age. In our
study, an attempt was made to collect blood samples at 7 days o f age for the analysis o f
the H/L ratio to evaluate if the toe-treatment procedure influenced the stress level early
in life. It was very hard to collect adequate volumes o f blood without sacrificing the
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89
birds. Therefore this was omitted. Further research is recommended to examine the
influence o f microwave toe-treatment on stress early in life. If the H/L ratios were
increased e arly in 1ife due t o a n i nitial s tress resulting from th e t oe-treatment i n this
experiment, the increased H/L ratios were not maintained by 21 days o f age when the
first blood samples were taken. In our study, the toes o f microwave-treated broilers
were healed well before 21 days o f age. Gross (1990) was interested in establishing how
quickly the H/L ratio responded and fell after stimulation from a noise stressor lasting
30 s. Chickens exposed to a single incident o f 104 decibels showed a rise in H/L ratios
18 h later and the ratios reached their maximum value in 20 h before returning to pre­
stress values after 30 h. The temporal pattern o f stress responses following continuous
infusion o f adrenocorticotropic hormone (ACTH) for 7 days in broilers was studied by
Puvadolpirod and Thaxton (2000b). They found that the first response was an elevated
plasma corticosterone level (2 h, lasting for 6 days); the elevated H/L ratio was found
by day 2, lasting for 10 days.
Compton
(1980)
demonstrated
that
declawed
hens
had
lower
plasma
corticosterone concentrations during the latter phases o f the laying period indicating a
lower stress level at this time period. Gildersleeve et al. (1981) reported that plasma
glucocorticoid levels were significantly higher in control hens than in toe-treated hens
indicating that the toe-treatment ameliorated stress. The rationale for the reduced stress
level o f caged layers with treated-toes is that the toe-treatment may effectively reduce
the “personal space” (the physical and social space occupied by an individual) required
by each bird. This would reduce the number and intensity o f physical interactions
among cage mates, thereby abating the stress associated with high density caging
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90
(Compton, 1980). The difference in the effect o f toe-treatment on the stress response
between our findings and previous studies could be associated with the type o f birds
used in the experiment and housing conditions for the birds. In our study, the broilers
were reared in floor pens, and in studies cited, caged hens were employed. Different
indicators for measuring stress levels used in the experiments and different ages for
sampling might also play roles.
The finding that the continuous lighting program did not result in increased H/L
ratios in the current study supports the results o f Campo and Davila (2002). They
measured H/L ratios in 36-wk hens exposed to lighting programs o f 23:1 LD, 14:10 LD
or 18.5:5.5 LD and proposed that there was no evidence o f an increased stress response
in broilers with continuous lighting. Blair et al. (1993) also reported that the H/L ratio in
broilers was not affected by photoperiod. However, Zulkifli et al. (1998) found that
broiler chickens illuminated 24 h per day had greater H/L ratios. Vo et al. (1998) also
presented an increase in H/L ratio fo r broilers housed under continuous photoperiod.
Buckland et al. (1976) found higher concentrations o f plasma corticoids in broilers
housed under continuous daylengths than in birds exposed to short daylengths
suggesting a higher level o f stress with CL. Renden et al. (1994), however, showed that
plasma corticosterone concentration was not associated with the photoperiod treatments,
indicating continuous lighting did not cause a higher level o f stress in broilers.
Continuous lighting has been postulated to reduce the opportunity for rest and sleep,
while the lack of sleep with continuous daylengths is assumed to increase physiological
stress (Gordon, 1994). Stress might also increase as the bird ages because disturbance
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91
under continuous light is likely to be greater as stocking density increases (Lewis and
Humik, 1990).
The higher H/L ratio in the females in our study disagrees with Campo et al.
(2000), who reported that female chickens had H/L ratios ranging from 0.28±0.02 to
0.58±0.04 and males had higher H/L ratios ranging from 0.43±0.04 to 0.66±0.04. The
discrepancy in these results might be due to different treatments and different genetic
background o f the birds used in the experiments.
4.4.3 The Tonic Immobility Test
Age had a significant effect on the number o f attempts required to induce
immobility, with day 10 values longer than days 22 and 36 (p<0.05). The number o f
attempts required to induce immobility were 2.6, 2.1 and 2.2 on days 10, 22, and 36.
Photoperiod, microwave toe-treatment, or sex did not affect this parameter. The
induction o f TI on day 10 was significantly larger than on days 22 and 36 (p<0.05), and
there was no difference in the induction o f TI between 22 and 36 days o f age. The
means o f induction o f TI were 2.6 times on day 10, and 2.1 and 2.2 times on days 22
and 36, respectively. The duration o f TI was significantly different at 10 days o f age
between the continuous and increasing photoperiod programs; the birds on the
increasing photoperiod program had a shorter duration o f TI (Table 4.5). The birds had
a similar duration o f TI under continuous and increasing photoperiod programs at 22
days o f age (Table 4.5). On day 36, the birds on the increasing photoperiod program
showed a longer duration o f TI than the birds under the continuous daylength (Table
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92
4.5). Neither the microwave toe-treatment nor sex had an effect on the duration o f TI at
10, 22, and 36 days o f age (Table 4.5).
Table 4. 5 Effect of lighting, microwave toe-treatment, and sex on the
duration of tonic immobility of broiler chickens at 10, 22, and
36 days of age
Treatment
Duration o f tonic immobility (s)
Day 22
Day 36
Day 10
Continuous-light
94a
122
126d
Increasing-light
61b
145
184c
Intact-toe
Treated-toe
85
68
121
147
152
151
Female
81
133
169
Male
71
134
137
a-b, c-a jy f e a n s j n c o l u m n s
within a treatment are significantly different in
Repeated Measures Analysis o f Proc Mixed (p<0.05).
The duration o f tonic immobility was different between day 10 and days 22
and 36, there was no difference in the duration o f tonic immobility between
day 22 and day 36.
Data were transformed by Log, and means were transformed back and
reported.
TI is an unlearned state o f reduced responsiveness to external stimulation and is
induced by physical restraint (Beuving et al., 1989). As a logical extension o f the fear
hypothesis, it was suggested that TI might be a terminal reaction o f the anti-predator
response, which was proposed to include freezing, flight, fight and immobility stages
(Jones, 1986). Some characteristics o f TI include temporary suppression o f the righting
response, reduced vocalization, intermittent eye closure, rigidity, Parkinsonian-like
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93
muscle tremors in the extremities, altered electroencephalographic patterns and changes
in heart rate, respiration and core temperature (Wallnau, 1981).
The effect o f age on the induction o f TI in our study is consistent w ith findings o f
Heiblum et al. (1998), who proposed that the TI response was strongly affected by age.
These authors investigated the ontogeny o f TI using White Leghorn male chicks and
they found that TI was poorly developed during the first 3 days o f life, when the median
TI duration in chicks was 10 s and the mean number o f induction trials 2.3±0.3. After
the third day o f life, TI duration increased by up to 15 times and susceptibility by about
two.
The toe-treated birds and the birds with intact-toes had a similar duration o f TI at
10, 22, and 36 days o f age indicating they had a similar level o f fear at those ages. To
the author’s knowledge, there has been no publication on the effect o f toe-treatment on
TI response. Honaker and Ruszler (2004) evaluated the level o f fearfulness o f
microwave toe-treated L eghom hens by subjective observation. The descriptive scale
from 1 to 10 was used in their study ( liv e r y calm; 2=mild chirp; 3=normal movement;
4=loud chirp; 5=excited movement; 6=fearful chirp; 7=flighty; 8=squak; 9=wild
movement; 10=panic). They found that from 6 to 8 wk, fearfulness score peaked at 8 to
10 for intact-toe birds and 3 to 4 for toe-treated birds; by 16 to 18 wk, fearfulness score
subsided to 2 to 3 for toe-treated birds and 6-8 for intact-toe birds. Compton et al.
(1981) also reported that toe-treated cage reared Leghorn hens were less active and
fearful. Another study indicated that there were less fighting, less flying, fewer skin
tears and bruises for toe-treated turkeys (Owings et al., 1972). The fact that toetreatment significantly reduced carcass scratches in broilers (Hargis et al., 1989)
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94
suggests that toe-treated birds may experience less discomfort in the growing period,
and therefore may show a lower level o f fearfulness. Another possible explanation for
the lower level o f fear o f the toe-treated birds is that the contact with humans early in
life due to the toe-treatment procedure could decrease fearfulness to humans in the later
life. The different conclusions between our study and the reports cited above could be
due to the different measurements used to evaluate fearfulness and the different types o f
birds employed in the experiments.
The birds on the continuous lighting (CL) had a longer duration o f TI on day 10
indicating that birds on CL had a higher level o f fear at this time. There were no lighting
effects on the duration o f TI at 22 days o f age suggesting that there was a similar level
o f fear between CL birds and birds on the increasing lighting (IL). The trend reversed
on day 36, the birds on IL were found to have a longer duration o f TI. This may be a
sign that birds on IL could not easily adjust to the change o f daylengths later in life,
resulting in a higher level o f fear. A few studies have demonstrated that continuous light
causes a higher level o f fearfulness as indicated by a longer duration o f TI in growing
broilers. Sanotra et al. (2002) placed broilers under two light-dark programs and one
continuous lighting program and found that both light-dark programs reduced the
duration o f TI on days 21 and 35 compared with the continuous lighting. Campo and
Davila (2002) measured the duration o f TI in 36-wk hens o f a synthetic breed exposed
to lighting programs o f 23:1 LD or 14:10 LD and reported that the duration o f TI o f
hens housed under 23:1 LD was longer than that o f hens housed under 14:10 LD
(236±32 vs. 137±32 s, respectively).
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95
Sex did not affect TI response in the current experiment. The relationship between
gender and the TI response in birds is unclear. Differences between sexes have been
observed in the responses o f domestic chicks and adolescent birds to a variety o f
potentially frightening situations and this dichotomy has been attributed to greater
fearfulness in males (Banks et al., 1979; Jones, 1985). However, no sex differences in
the duration o f TI were found in Japanese quail and domestic chicks (Gallup, 1974;
Benoff and Siegel, 1976).
M any factors can influence the duration o f TI. Ferrante et al. (2001) examined the
fear reaction o f two strains o f chickens (an egg-type strain and a meat-type strain) and
found that meat-type birds showed a significantly lower duration o f TI. Individuals vary
in their susceptibility to as well as in the duration o f TI (Erhard et al., 1999). Chicks
showed longer duration o f TI when they could see the experimenter and response was
prolonged if the latter maintained direct eye contact with the bird rather than averting
his gaze (Gallup et al., 1972). Evidence o f diurnal periodicity in the TI response o f 1wk-old W hite Leghorn chicks was presented by Rovee et al. (1976). Chicks reared on a
12:12 LD regime with light onset at 8:00 AM showed the longest duration o f TI from
13:00 to 14:00 h and the briefest from 06:00 to 11:00 h. Jones (1986) in a review
summarized some general factors affecting TI, including age effect, influence o f the
experimenter, regular handling, genetic factors, social factors and influence o f housing
conditions.
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96
4.5 Conclusions
The microwave toe-treatment did not affect either the activity o f plasma creatine
kinase or the heterophil to lymphocyte ratio at both 21 and 35 days o f age indicating
that the microwave toe-treatment did not influence the stress level o f the broilers at
these ages. The microwave toe-treatment did not influence the duration and induction o f
tonic immobility reaction on days 10, 22, and 36 suggesting that the microwave toetreatment did not influence the level o f fear o f the birds. The birds on the continuous
lighting program had increased activity o f plasma CK, but similar H/L ratios compared
to the birds on the increasing lighting program. The sensitivity o f these two methods for
measuring stress may be different or other factors such as skeletal and heart muscle
myopathy or rapid growth that elicit a response for CK may not influence H/L ratio.
The increased plasma CK activity in this study m ay suggest that the continuous lighting
program could have detrimental effects on meat quality o f the broilers. The increasing
lighting program decreased the duration o f tonic immobility on day 10, had no effects
on day 22, and increased the duration o f tonic immobility at 36 days o f age, compared
to the continuous lighting. This confirms that short or moderate daylengths reduce the
level o f fear in broilers. This may also indicate that birds on IL could not easily adjust to
the change o f daylengths in later life. The lower activity o f plasma creatine kinase and
heterophil to lymphocyte ratios in the males suggest that the males experienced a lower
level o f stress in this study. The males and females had a similar level o f fearfulness as
indicated by the duration o f tonic immobility.
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97
CHAPTER 5
CARCASS QUALITY AND SKIN INTEGRITY OF
MICROWAVE TOE-TREATED BROILER CHICKENS GROWN
WITH TWO PHOTOPERIOD PROGRAMS
5.1 Abstract
Carcass scratches and bruises to broiler chickens result in carcass downgrading at
the processing plant. The primary cause for carcass scratches is injury inflicted by
toenails o f other broilers. Increasing photoperiod programs reduce the incidence o f heart
disease and leg problems. However, birds given this type o f program may have an
increased incidence o f carcass scratches due to increased activity. Microwave toetreatment is a potential solution for reducing skin scratching damage. Rearing broilers
under lighting programs with long darkness reduced skin tearing at the processing plant,
and collagen is proposed to be a major determinant o f skin tears. Matrix
metalloproteinases (MMPs) are responsible for extracellular collagen degradation and
remodeling. The objective o f this study was to investigate the influence o f microwave
toe-treatment a nd p hotoperiod o n e arcass s cratches a nd b raises, a nd s kin i ntegrity o f
broiler chickens. T wo r eplicate trials w ere conducted w ith 728 fem ale an d 728 m ale
broilers in each trial. H alf o f the birds from each sex were toe-treated with microwave
energy upon delivery from the hatchery. The birds in each trial were randomly assigned
to four sections o f two rooms with a total o f 32 floor pens. Two sections were given the
continuous p hotoperiod from s tart t o finish a nd the o ther t wo w ere o n a n i ncreasing
lighting program with short photoperiod after the first three days, which increased to 23
h by day 30. The activity o f plasma MMPs was measured using zymography at 21 and
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98
35 days of age. Skin puncture strength was examined on day 38 with a texture analyzer.
Skin type I and III collagen content was determined using immunohistochemistry.
Carcass scratches and bruises were monitored at the end o f the trial (day 38). The
microwave toe-treatment significantly reduced the incidence o f carcass scratches
(p<0.01), and tended to decrease carcass bruises (p=0.061). The increasing photoperiod
program increased the incidence o f carcass scratches (p=0.05), whereas it did not affect
the incidence o f carcass bruises. Both sexes had a similar incidence o f carcass scratches
and bruises. The males had stronger skin strength than the females (p<0.05). The toetreated birds had higher skin strength than the intact-toe birds (p<0.05). The increasing
photoperiod program tended to increase skin type I collagen content compared to the
continuous lighting (p=0.08). The birds with the increasing lighting and the continuous
lighting had similar type III collagen content in the skin. Neither the microwave toetreatment nor sex had significant influences on type I and III collagen content in the
skin.
5.2 Introduction
Carcass scratches and bruises to broiler chickens result in carcass downgrading at
the processing plant. During the broiler’s short life, scratches may develop into an
inflammation o f the skin or dermatitis known as scabby hip syndrome (Proudfoot and
Hulan, 1985), a severely afflicted flock o f which m ay have the number o f Grade A
carcasses reduced by as much as 50% (Proudfoot and Hulan, 1985). Scratches also can
lead to the development o f cellulitis, the most common cause for carcass condemnation
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99
in Canada (Kumor et al., 1998). The prim ary cause for scratches is injury inflicted by
toenails o f other broilers (Hargis et al., 1989). In recent years, there is interest in
increasing p hotoperiod programs f or growing b roilers b ecause t his 1ighting p attem i s
associated with a reduction in the incidence o f heart disease and leg problems (Classen
et al., 1991; Rozenboim et al., 1999). However, birds given this type o f program m ay
have an increased incidence o f carcass scratches due to increased activity (Blair et al.,
1993). One potential solution for reducing skin scratching damage is to treat the toes o f
day-old broilers with microwave energy. Turkeys are routinely subjected to toe-clipping
at the hatchery to reduce downgrading due to scratching (Moran, 1985; M cEwen and
Barbut, 1992). Currently, the turkey industry uses microwave technology to restrict
claw growth and reduce carcass scratches. However, this procedure has not been
generally practiced in broiler chickens due to the cost per bird and uncertain effects
(Proudfoot et al., 1979; Hargis et al., 1989).
Rearing broilers under lighting programs with long darkness has been shown to
reduce skin tearing at the processing plant compared to continuous lighting programs
(Hammershoj, 1997). Downgrading problems associated with skin integrity such as cuts
and tears cause substantial economic losses to the broiler industry (Bilgili et al., 1993).
Christensen et al. (1994) reported that birds with lower skin strength exhibited an
increased incidence o f skin tears during slaughter in the processing plant. Olkowski et
al. (2001) demonstrated that broilers raised at cool temperture increased the incidence
o f heart disease and those birds with heart disease had elevated levels o f matrix
metalloproteinase-2
(MMP-2)
circulating
in
their
blood
plasma.
Matrix
metalloproteinases (MMPs), an enzyme system, are responsible for extracellular
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100
collagen degradation an d rem odeling (Spinale e ta l., 1999). Collagen is th e principal
protein in the skin and is a major determinant o f skin strength (Granot et al., 1991a).
Collagen is also proposed to be a major determinant o f skin tears (Granot et al., 1991b).
Continuous lighting increased the incidence o f heart disease compared to increasing
photoperiod programs, so if continuous lighting also increases activities o f plasma
MMPs or influences the synthesis o f collagen, this may reduce the structural integrity o f
collagen and increase susceptibility o f broilers’ skin to bruises and tears. The present
study was conducted to investigate the influence o f microwave toe-treatment and
photoperiod on carcass scratches and bruises, and skin integrity in broiler chickens.
Skin puncture strength, activities o f plasma MMPs, and skin type I and III collagen
content were measured to evaluate skin integrity.
5.3 Materials and Methods
5.3.1 Birds, Diets and Housing
Two replicate trials were conducted with 728 female and 728 male broilers in
each trial. The first trial was performed in the fall and the other in the winter. Sexed,
day-old broilers (Ross 308) were obtained from a commercial hatchery and were placed
in sex-separated pens in a confinement house with untreated wood shavings as litter.
Animal care was provided according to the standards o f the Canadian Council on
Animal Care (1993) and the protocol was approved by Nova Scotia Agricultural
College Animal Care and Use Committee. Two room s were used in each trial; each
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101
room was separated by a black plastic divider into two sections for two photoperiods. In
each trial, the birds were randomly assigned to four sections o f the two rooms with 8
floor pens per section, giving 4 replications o f each lighting program, toe-treatment and
sex treatm ent combination. T h e stocking density was 0.07 m 2 b ird ’1. Feed and w ater
were available ad libitum with one suspended tube feeder per pen and a nipple drinker
system in each room. There were ten nipples per pen. Box feeders were also used to
provide feed in the first week. All the birds were given the same diets that consisted o f
starter, grower, and finisher rations. The starter diets contained 23% crude protein and
3050 kcal ME kg’1 and were fed as mash from day 0 to 13 days o f age. The grower diets
contained 20% crude protein and 3150 kcal ME k g '1 and were fed as mash from day 14
to 23 days o f age. The finisher diets contained 18% crude protein and 3200 kcal ME kg’
1 and were fed as pellets from days 24 to 38. The ingredients used in the diets and the
calculated a nalyses a re s hown i n T able 5.1. T he b rooding t emperature w as 3 0-32°C
from day 0 to day 7 after which it was reduced by 3°C per week until it reached 21°C
where it remained for the rest o f the trial.
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102
Table 5.1 Ingredient composition and calculated analyses of diets used in the
experiment
Ingredients (%)
Com
Wheat
Soybean meal
Poultry grease
Limestone
Dicalcium phosphate
Iodized salt
DL-Methionine
Amprolium11
BMDV
Pel-Stikw
Vit/M in Premixxy
Calculated content
Crude protein, %
Metabolizable energy, kcal kg"1
Calcium, %
Available phosphoms, %
Lysine, %
Methionine + Cystine, %
Starter
(0-13 d)
Grower
(14-23 d)
Finisher
(24-38 d)
42.90
10.00
38.69
3.86
1.97
1.10
0.32
0.103
0.05
0.0044
0.50
0.50
50.08
10.00
31.29
4.47
1.85
0.95
0.30
0.002
0.05
0.0044
0.50
0.50
55.74
10.00
26.23
3.98
1.84
0.91
0.30
23.00
3050
1.00
0.45
1.37
0.95
20.00
3150
0.92
0.40
1.15
0.76
18.00
3200
0.90
0.38
1.00
0.70
0.50
0.50
u Amprolium: Merck & Co., Inc., Whitehouse station, NJ, USA.
VBMD: a dried precipitated germentation product obtained by culturing Bacillus subtilis
tracy on media adapted for microbiological production o f bacitracin;calcuim carbonate.
Alpharma, Fort Lee, NJ. USA.
w Pel-Stik: Uniscope, Inc., Johnstown, CO, USA.
x Supplied per kg starter diet: vitamin A, 19,500 IU; vitamin D 3, 4000 IU; vitamin E, 2.97
mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.046; niacin, 29.7; folic
acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg; thiamine, 5.8 mg;
manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50 mg; wheat
middlings, 1432 mg; ground limestone, 500 mg.
y Supplied per kg grower & finisher diet: vitamin A, 19,500 IU; vitamin D3, 4000 IU;
vitamin E, 2.97 mg; riboflavin, 7.6 mg; DL Ca-pantothenate, 13.5; vitamin B, 0.024;
niacin, 29.7; folic acid, 4.0 mg; choline, 801 mg; biotin, 0.3 mg; pyridoxine, 5.9 mg;
thiamine, 5.8 mg; manganese, 70.2 mg; zinc, 80.0 mg; selenium, 0.30 mg; ethoxyquin, 50
mg; wheat middlings, 1543 mg; ground limestone, 500 mg.
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103
5.3.2 Microwave Toe-treatment
H alf o f the birds from each sex were toe-treated using a Microwave Claw
Processor (MCP, Nova-Tech Engineering Inc., Willmar, MN) upon delivery from the
hatchery. To conduct the microwave toe-treatment, the chicks were turned upside down
and their legs were inserted into shackles o f the MCP between the knee and ankle. The
ventral side o f the bird was toward the operator. Each o f the three front toes were pulled
into place by a vacuum and treated with microwave energy for 0.8 s.
5.3.3 Experimental Lighting Programs
Light was provided by incandescent bulbs mounted at approximately 2.0 m above
the floor level. All birds were subjected to the same light intensity. Light intensity was
measured at the bird head level with a light meter (Cal-Light 400, The Cooke
Corporation) and adjusted by rheostat. All broilers were given 24 h light for the first
three days. Then in two sections, the lighting schedule was 23 h per day until the end o f
the trial. The other two sections were on an increasing photoperiod program. The two
lighting programs used in the experiment are in Table 5.2.
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104
Table 5. 2 Two lighting programs used in the experiment
Age (d)
Lighting
Continuous
Increasing
Light intensity
(lux)
0-3
24L1:0D2
24L:0D
20
4-6
23L:1D
10L:14D
20
7-9
23L:1D
10L:14D
5
10-16
23L:1D
12L:12D
5
17-22
23L:1D
14L:10D
5
23-29
23L:1D
18L:6D
5
30-38
23L:1D
23L.TD
5
lL: light
2D: darkness
5.3.4 Blood Sampling
At 21 and 35 days o f age, blood samples were collected from 3 randomly selected
birds from each o f the 32 pens in each trial. The blood-sampled birds were wing-banded
on day 21 and the same birds were used for blood sampling on day 35. Approximately
3.5 ml blood was collected via venipuncture from the brachial vein into a 7 ml
Vacutainer® tube containing sodium heparin as an anticoagulant. The blood samples
were immediately placed on ice until centrifuged at 1600 x g at 4°C to separate plasma.
The p lasma sa mples w ere t ransferred i nto m icrocentrifuge t ubes and frozen a t - 80°C
until analyzed.
5.3.5 Skin Sampling
At the age o f 38 days, 2 birds per pen were selected randomly and sacrificed by
cervical dislocation. The feathers from the caudal-dorsal pelvic area and thigh were dry-
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105
picked. Skin samples o f approximately 5 x 5 cm were removed with surgical scissors
from the right pelvic back area and immediately placed on ice until tested for skin
puncture strength within 24 hr. Approximately 2 x 3 cm samples were removed from
the same area for skin collagen content analysis, rolled and frozen in liquid nitrogen and
stored at -80°C until analyzed.
5.3.6 Carcass Scratches and Bruises
Five randomly selected birds per pen, for a total o f 160 in each trial, were wingbanded at 37 days o f age. These birds were shipped for custom processing on day 38,
and the incidence o f scratches and bruises were evaluated separately for each carcass.
5.3.7 Laboratory Analysis
5.3.7.1
Skin Puncture Strength
Fresh skin samples were mounted between specially constructed aluminum plates
(9.8 x 10.4 cm) with etched surfaces to prevent slippage. The plates contained a center
hole (1.6 cm diameter) and a simple clamping mechanism to tightly secure the sample.
Special care was taken to tighten the samples consistently. Secured samples were placed
with the epidermal surface facing the probe, in the stand o f a Lloyd® texture analyzer
(Lloyd® Instrument Ltd, Fareham, Hants). The punch test was performed by passing a
blunt-tipped probe (1.0 cm diameter) through the sample at a constant crosshead speed
o f 100 mm min' 1. Skin puncture strength variables consisted o f load at th e breaking
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106
point ( the f orce r equired t o p uncture t he s ample; N ) a nd s kin d isplacement ( distance
probe travels to the breaking point; mm).
5.3.7.2 Zymography
The activity o f plasma MMPs was measured using gelatin zymography. Briefly,
5pi diluted samples (5pi plasma samples diluted with 500pl sample buffer: 62.5mM
Tris-HCI, 25% glycerol, 4% SDS, 0.01% bromophenol blue, pH 6.8) were loaded on
8% SDS-PAGE in which the separating gels were co-polymerized with 2 mg/ml o f
gelatin (Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA). Gels were cast
between the glass plates o f a Bio-Rad Mini Protean® 3 Cell electrophoresis apparatus
(Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, California) and were
electrophoresed at 90 V in running buffer (10 x running buffer: 25 mM Tris, 192 mM
glycine, 0.1% SDS, pH 8.3) until the bromophenol blue marker dye reached the bottom
o f gels. After electrophoresis, the gels were washed with 2.5% Triton X-100 solution
twice for 20 min each to remove SDS and then incubated in incubation buffer (0.15M
NaCl, 5mM CaCl2, 50mM Tris-HCI, 0.05% NaN3, pH 7.5) at 37°C for 18 h. After
incubation, the gels were stained with 0.05% Coomassie® brilliant blue G-250 (BioRad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA) in a mixture o f methanol:
acetic acid: w ater (2.5: 1 : 6.5) f or 9 0 m in an d destained in 4 % m ethanol w ith 8%
acetic acid until the bands became clear.
The activity o f MMPs was detected as
transparent bands against the blue background o f Coomassie® brilliant blue. To evaluate
the activity o f the detected enzymes, the size o f the zones o f clearing on zymograms
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107
were analyzed using Gene genius bio imaging system (SynGene, a division o f
Synoptics, Ltd., Beacon house, Nuffield road, Cambridge, UK).
5.3.7.3 Immunohistochemistry
Skin type I and III collagen content was analyzed using immunohistochemistry.
Cryostat cross-sections (5 pm) were prepared from the frozen chicken skin samples
using a Kryostat® (Leica CM 1850-kryostat, D-69226 Nussloch, Germany) and
mounted on super frost microscopy slides. The slides were air dried for 30 min before
cold acetone (4°C) fixation for 10 min. After rehydration in phosphate buffer saline
(PBS) (1 liter for 10 x PBS: 80g NaCl, 2g KC1, 14.4g Na2H P 0 4, 2.4g KH2P 0 4, pH 7.2),
the slides were treated in 0.3% H20 2 in methanol for 10 min, and then nonspecific
binding sites were blocked with 2% bovine serum albumin (BSA) (Pierce, Rockford,
IL) in PBS for 1 h in a humid chamber at room temperature. Rabbit anti-chicken
collagen type I and III polyclonal antibodies (Concentration, 1 mg/ml, Chemicon
international, Temecula, CA) and the negative control (rabbit IgG, concentration, 25
mg/ml, Chemicon International, Temecula, CA) were diluted in 2% BSA in PBS
(collagen type I antibody: 1:1000 dilution, collagen type III antibody: 1:800 dilution;
final IgG concentration, 1 and 1.2 pg/ml) and applied to the sections overnight at room
temperature. After the sections were washed in PBS, they were incubated with
secondary antibody (goat anti-rabbit I gG bio tin conjugated affinity purified antibody,
Chemicon international, Temecula, CA) for 1 h at RT. The slides were washed in PBS
and Avidin and Biotinylated horseradish peroxidase macromolecular Complex (ABC)
was applied to the sections for 30 min to amplify antibody reaction. This was followed
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108
by a wash in PBS. Antibody binding was detected with 3, 3 ’-diaminobenzidine (DAB).
Then aqueous hematoxylin was used to counterstain the nuclei for 1 min, followed by
the addition o f aqueous mounting media (Aquaperm; Fisher Scientific, Ottawa, ON,
CA). Staining intensity was scored on a 5-point scale (i.e., positive but very weak, 1;
positive but weak, 2; moderate, 3; strong, 4; very strong, 5).
5.3.8 Statistical Analysis
The experiment was a split-plot factorial design. Four room by trial combinations
(2 rooms in each trial and 2 trials) were the blocks, two sections o f a room were the
whole plots and individual pens within each section were subplots. Two photoperiods
(continuous and increasing) were whole plot treatments and a factorial o f toe-treatment
(treated-toe and intact-toe) and sex (male and female) were subplot treatments. The
response variables including skin strength variables and scores o f skin type I and III
collagen were analyzed using Proc M ixed procedure in SAS® software (SAS Institute,
1999); the activity o f plasma MMPs was analyzed with Repeated Measures Analysis
using Proc Mixed procedure in SAS® software (SAS Institute, 1999). The statistical
significance was determined using a (level o f significance) o f 5%. The following model
was employed for statistical analysis o f data for a given time point:
y mim = M+ a t + Pj + (afi)y + r k + (ay)ik + (Pr)Jk + (« ^ r)P + ^ + (acT)n + (/?<?)/V
+ {ap(7)ijl + (yS)kl + (ayS)ik, +(fiyS)ijk +(apSr ) ij!d + s iJk!m
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109
Where y ijklm is the response o f interest; // is the overall mean; a t is the main
effect o f blocking factor; ft. is the effect o f the / h photoperiod factor (/= 1,2); y k is the
effect o f the klh toe-treatment (k= 1,2); Sl is the effect o f Ith sex factor (/= 1,2); (f i y) jk is
the effect o f the interaction between photoperiod and toe-treatment; {/3S) jt is the effect
o f the interaction between photoperiod and sex; ( jS ) u is the effect o f the interaction
between toe-treatment and sex; (/3yS)jU is the effect o f the interaction between
photoperiod, toe-treatment and sex; s iJklm is the random error component assumed to be
normal, independent and have constant variance. Note that the interactions w ith the
blocking factor are used as errors for testing the effects o f photoperiod, toe-treatment
and sex. For Repeated Measures Analysis, the repeated statement in Proc M ixed was
used, and time was added as another factor.
The assumptions o f above model were tested in SAS®. Transformation was
conducted when the distribution o f the error terms was not normal. When significant
difference was found among main effects or their interactions, Least Squares Means
(LSmeans) comparison tests in SAS® were used to compare the means.
The data o f carcass scratches and bruises were analyzed by Chi-square test in
Minitab®. The statistical significance was determined using a (level o f significance) o f
5%.
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110
5.4
Results and Discussion
5.4.1 Carcass Scratches and Bruises
The microwave toe-treatment reduced the incidence o f carcass scratches
significantly (p<0.001) (Table 5.3). Percentage scratched birds with treated-toes was
only 4.40%, compared to 81.76% scratched birds with intact toes (Table 5.3). The birds
on the increasing photoperiod program had a higher incidence o f scratches than those
with the continuous lighting (p=0.05) (Table 5.3). There was no sex effect on carcass
scratches (Table 5.3). The incidence o f carcass bruises was not influenced by
photoperiod, or the sex o f the bird, but the toe-treatment had a marginal effect
(p=0.061) (Table 5.3).
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Ill
Table 5. 3 Effect of lighting, microwave toe-treatment and sex on carcass
scratches and bruises at 38 days of age
Treatment
Lighting
Toe
Sex
Continuous Increasing
Intact Treated
Female Male
Scratched birds
59
76
130
7
70
66
No scratched birds
100
83
29
152
89
93
Total birds
159
159
159
159
159
159
37.11
47.80
81.76
4.40
Scratched birds (%)
44.03 41.51
X2
3.720
194.016
0.206
Probability
0.050
<0.001
0.650
Bruised birds
41
33
43
29
39
34
No bruised birds
118
126
116
130
120
125
Total birds
159
159
159
159
159
159
25.79
20.75
27.04
18.24
Bruised birds (%)
24.53 21.38
X2
1.127
3.519
0.445
Probability
0.288
0.061
0.505
Carcass scratches and bruises data were analyzed by Chi-square.
It i s evident th at th e m icrow ave toe-treatment reduced th e incidence o f c arcass
scratches significantly confirming that the primary cause for carcass scratches is injury
inflicted by toenails o f other broilers (Hargis et al., 1989).
Our findings agree with
Hargis et al. (1989), who reported that toe-trimming broiler chickens with a hot blade
resulted in a 3.7 to 4.8-fold reduction in subjective lesion scores and a 7 to 10-fold
increase in percentage o f USDA Grade A carcasses at a commercial processing plant.
Proudfoot et al. (1979), using large experimental pens, however, did not observe any
benefit o f toenail removal on carcass quality in broiler turkeys. Owings e t al. (1972)
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112
found only a small improvement with 1.2 to 5% more toms classified as Grade A. The
difference in response to toe-treatment between broiler chickens and turkeys or the
difference in experimental conditions could be an explanation for the different findings.
In this experiment, the incidence o f carcass scratches was more than 80% for the birds
with intact toes, much greater than commercial situation. The greater incidence o f
scratches for the intact-toe birds in this study may be due to the influence o f increased
handling for research.
The increased incidence o f carcass scratches with the increasing lighting is due to
increased bird activity with increasing lighting programs (Blair et al., 1993). Blair et al.
(1993) reported that broilers on an increasing photoperiod were more mobile than those
on the continuous lighting program, as judged by their higher intake o f feed over a 2-h
period from feeders accessible only by jumping onto a 30-cm-high platform. Hester et
al. (1985) found that tom turkeys were more active (i.e., spent less time lying down)
when reared on a treatment in which the photoperiod was increased in length from 9 to
15 h between 56 and 126 days than on a treatment in which the photoperiod remained at
15 h from 12 to 133 days. Sanotra et al. (2002) observed the behavior o f broilers with
different lighting treatments at 11, 18, 25, 32, and 40 days o f age and found that broilers
from c ontrol h ouses w ith c ontinuous 1ight w ere s itting m ost o f t he t ime a nd s ho wed
very little activity, compared to the birds with light-dark lighting programs.
According to our results, the photoperiod program or sex had no effects on
carcass bruises. A higher incidence o f carcass bruises with continuous lighting than
increasing lighting was observed in a previous study with broilers (Rathgeber and
Maclsaac, 2003). Taylor and Helbacka (1968) have shown that female broilers bruise
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113
more easily and more severely than males. They found a positive correlation between
the ether extractable component o f the carcass and the bruising score, an d suggested
that this explained w hy females which are fatter, and why highly finished birds bruised
more easily. Mayes (1980) reported that the incidence o f bruising was greater in female
flocks than in male, but the bruising in the females tended to be less severe. Mayes
(1980) also found that the birds that were heavy for their age bruised more easily and
more severely; and the birds bruised less easily on cold days. The toe-treated birds
tended to have less carcass bruises than the birds with intact-toes (p=0.061) in this
study. This may be due to the stronger skin o f the toe-treated birds.
5.4.2 Skin Puncture Strength
Skin puncture strength variables consisted o f load at the breaking point and skin
displacement. The load at the breaking point was higher for the males than the females
(Table 5.4). Larger forces were needed to break the skin o f toe-treated birds than that o f
intact-toe birds (Table 5.4). The skin displacement was different between the toe-treated
birds and the controls, with the skin from toe-treated birds being more extendable
(Table 5.4). The male and female skin was not different for extendibility (Table 5.4).
The lighting programs did not influence either measurement on skin samples (Table
5.4).
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114
Table 5. 4 Effect of lighting, toe-treatment and sex on skin puncture strength in
broiler chickens at 38 days of age
Treatment
Lighting
Sex
Toe
Skin strength
variables
Continous Increasing
Intact
Treated
Female Male
Load(N )
87.2
84.2
83.4b
88.0a
82.2b
89.3a
Extension (mm) 22.5
22.8
21.8d
23.6C
22.6
22.8
a"b’c"d Means in rows within a treatment are significantly different (p<0.05).
Data were transformed by square root, and means were transformed back and reported.
The significant difference in the load at the breaking point between two sexes
indicates that the males had stronger skin than the females. This observation is
consistent with previous reports (Weinberg et al., 1986; Bilgili et al., 1993; Christensen
et ah, 1994; Yalqin et ah, 1998). Smith et ah (1977) postulated that high levels o f fat,
accompanied by a reduction in total collagen concentration, made the skin o f females
weaker. Pines et ah (1996) suggested that the higher tensile strength o f the male skin
may be due to the elevated skin collagen content that resulted from increased expression
in collagen type I genes and from the higher amounts o f various collagen cross-links.
Yalfin et ah (1998) found that female broiler’s skin was higher for skin dry matter and
fat content. Collagen is the principal protein in the skin and is a major determinant o f
skin strength (Granot et ah, 1991a). However, the differences in breaking strength were
not consistently associated with collagen content o f the skin (Kafri et ah, 1985). The
rate o f cross-linking (Granot et ah, 1991b) and the state o f maturation o f the collagen
(Crosley et ah, 1992) and the ratio o f type I and type III may also play a role (Burgeson
and Nimni, 1992). Granot et ah (1991b) postulated that variations in skin strength could
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115
be affected by biochem ical components such as total protein, fat, or the specific matrix
constituents (collagen or proteoglycans). Christensen et al. (1994) found that males
have a thicker dermal layer than females even though the total skin thickness is less in
males. This supports the conclusion o f Ramshaw et al. (1986), who proposed that skin
tensile strength is a direct function o f the collagenous dermal layer o f the skin.
Skin strength o f broilers increases linearly with age (Bilgili et al., 1993;
Christensen et al., 1994). An explanation for this is that skin collagen content increases
with age (Pines et al., 1996). Dietary energy, protein, and fat have been shown to be
contributors to skin strength (Kafri et al., 1985; Granot et al., 1991b). Christensen et al.
(1994), however, found that neither added dietary fat nor ambient temperature had an
effect on skin strength o f broilers. There was a genetic effect on skin strength, for
example, low-weight female chicken strains having weaker skins when compared to
high-weight strains (Kafri et al., 1984).
According t o o ur r esults, t he 1oad a 11 he b reaking p oint w as 1arger f or t he t oetreated birds suggesting that the toe-treated birds had stronger skin than the intact-toe
birds. On the other hand, differences in skin displacement, an indirect measure o f skin
elasticity, between the toe-treated birds and the birds with intact toes indicates that the
skin o f the toe-treated birds was more elastic. These results may suggest that microwave
toe-treatment could benefit wellbeing o f the birds due to decreased levels o f stress and
fearfulness ( Compton e ta l., 1981; Honaker an d Ruszler, 2 004). Stress a n d fe a rm a y
affect energy and mineral metabolism and interact with the immune system (Siegel,
1995). The toe-treated birds might have improved use o f nutrients such as amino acids,
minerals and vitam ins which are directly o r indirectly involved i n c ollagen synthesis
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116
(Pinion et al., 1995). The higher incidence o f scratches o f intact-toe birds may have
been contributed to the weaker skin o f these birds. The stronger skin o f toe-treated birds
was not expectation. A following-up study focusing on the influence o f microwave toetreatment on skin integrity m ay be beneficial.
5.4.3 Zymography and Immunohistochemistry
The microwave toe-treatment did not affect the activity o f plasma pro-MMP-2
either at 21 days or 35 days o f age. The intact-toe birds had a higher activity o f plasma
active-MMP-2 on day 35 (Table 5.5). There was no significant difference in the activity
o f plasma pro-MMP-2 and active-MMP-2 between the continuous lighting and the
increasing lighting groups on days 21 and 35 (Table 5.5). Sex did not affect the activity
o f the plasma pro-MMP-2 and active-MMP-2 on days 21 and 35 (Table 5.5). The
activity o f the plasma pro-MMP-2 was significantly higher on day 35 than on day 21
(Table 5.5).
Type 1 collagen content in the broiler skin tended to be higher with the increasing
photoperiod program than on the continuous lighting program (p=0.08). Type III
collagen content in the skin was not affected by the lighting treatments (Table 5.6).
There was no microwave toe-treatment or sex effect on the content o f type I and type III
collagen in the skin (Table 5.6).
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117
Table 5. 5 Effect of lighting, microwave toe-treatment, and sex on the activity of
plasma pro-MMP-2 and active MMP-2 in broiler chickens at 21 and 35
days of age
Treatment
Age (d)
21
35
Continuous-light
Increasing-light
Activity o f plasma MMP-2* (pixel intensity)
Pro-MMP-2^
65229
73566
A c tiv e-M M P ^
2424
2194
Intact-toe
Treated-toe
69343
69169
2498
2124
Female
Male
68503
69337
2352
2263
Continuous-light
Increasing-light
57552
58014
2581
2070
Intact-toe
Treated-toe
59643
55616
1978°
2686a
Female
Male
57111
57614
2595
2057
* MMP-2: matrix metalloproteinases-2, named as gelatinase as well.
^ Pro-MMP-2: 72-kDa gelatinase *Active-MMP-2: 69-kDa gelatinase.
ab
Means m columns with no common superscript are significantly different m
Repeated Measures Analysis o f Proc M ixed (p<0.05).
The activity o f plasma MMP-2 was obtained by the background color minus the
color o f the band, the lower number indicates higher activity o f plasma MMP-2.
The activity of plasma pro-MMP-2 was significantly higher on day 35 than on day 21.
The data were transformed by cube root, and means were transformed back and
reported.
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118
Table 5. 6 Means of the scores of skin type I and III collagen staining
intensity
Score o f staining intensity
Treatment
Type I collagen
Type in collagen
Continuous-light
3.02±0.22
3.33±0.24
Increasing-light
3.50±0.22
3.53±0.25
Intact-toe
3.18±0.22
3.40±0.25
Treated-toe
3.34±0.22
3.46±0.24
Female
3.26±0.22
3.29±0.25
Male
3.26±0.22
3.57±0.25
Staining intensity by immunohistochemistry was scored on a 5-point scale
(i.e., positive but very weak, 1; positive but weak, 2; moderate, 3; strong,
4; very strong, 5).
Presented are mean±standard error.
No significant treatment effects were found on the scores o f staining
intensity o f type I and type III collagen in the skin o f briolers.
Matrix metalloproteinases
(MMPs),
an
endogenous
enzyme
system,
are
responsible for extracellular collagen degradation and remodeling (Spinale et al., 1999).
Collagen degradation in connective tissue is mediated mainly by MMPs, with the 72kDa gelatinase (pro-MMP-2) playing a key role in this process (Creemers et al., 1998).
It has been found that both human and chicken MMP-2 are capable o f cleaving type I,
IV and V collagen (Aimes and Quigley, 1995). In the current study, the activity o f proMMP-2 was significantly higher on day 35 than on day 21. This may be due to more
active collagen remodeling in the older birds. Olkowski et al. (2001) demonstrated that
the activity o f this enzyme appears to be considerably higher in preparations from
broilers, particularly in the left ventriculum o f fast growing birds, in comparison to
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119
Leghorns or slow growing broilers. They proposed that the pathogenesis o f heart failure
and ascites in fast growing broilers m ay be associated with the increased activity o f
MMPs.
The skin o f th e m odem broiler chickens contains close to 5 0% fat and around
3.5% collagen (Cliche et al., 2003), approximately 75% o f the collagen is type I and
15% type III (Abedin and Riemschneider, 1984). It has been shown that skin collagen is
significantly influenced by genetics and sex o f the birds (Granot et al., 1991b). In our
study, sex did not affect the activity o f pro-MM P-2 and active-MMP-2, or the skin type
I and III collagen content. In previous studies, it has been reported that males have
higher skin collagen content than females (Smith et al., 1977; Pines et al., 1996). Our
results show that the toe-treated birds and the birds with intact toes had similar skin type
I and III collagen content. However, the skin strength was different between them. This
might be explained by that the differences in breaking strength are not consistently
associated with collagen content o f the skin (Kafri et al., 1985). The toe-treated birds
also had a lower activity o f active-MMP-2 on day 35, which may indicate that the toetreated birds may have higher skin collagen content at this age. These results suggest
that the association between activity o f MMPs, collagen content, and skin strength is
not simple.
In the current study, the increasing lighting tended to increase skin type I collagen
content score (p=0.08). We used a split-plot design, and two photoperiods were whole
plot treatments. This design could have reduced the lighting effects in the statistical
analysis due to few degrees o f freedom o f the lighting factor (3 d f o f lighting in this
experiment). The semi-quantitative method o f immunohistochemistry used for the skin
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120
collagen content analysis may also make treatment effects less distinguishable. We
suggest that further attention should be paid to the effect o f photoperiod on skin
collagen content.
5.5
Conclusions
The microwave toe-treatment reduced the incidence o f carcass scratches
significantly, whereas the increasing photoperiod program increased the incidence.
There was no significant difference in carcass scratches between the males and females.
The lighting treatment or sex did not influence the incidence o f carcass bruises. The toetreated birds tended to have a lower incidence o f carcass bruises compared to the birds
with intact toes.
The toe-treated birds had increased skin strength and reduced activities o f activeMMP-2 on day 35 compared to the birds with intact toes. This indicates that the
microwave toe-treatment benefits skin integrity in broiler chickens. The male broilers
had stronger skin than the females but there was no difference in type I and III collagen
content between the sexes. The photoperiod programs did not affect skin strength,
activities o f plasma MMP-2 and type III collagen content. The increasing lighting
tended to increase type I c ollagen c ontent suggesting that further attention should be
paid to the effect o f photoperiod on skin collagen content o f broilers.
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121
CHAPTER 6
OVERALL DISSCUSSION AND CONCLUSION
The microwave toe-treatment reduced body weight o f broilers early in life, but
did not have a detrimental effect on bird weight by market age. The microwave toetreatment reduced feed intake but did not affect feed conversion ratio. The lower early
feed intake m ay have been contributed to the reduction in early growth o f toe-treated
birds. The reduction in early growth caused by the toe-treatment may suggest that the
toe-treatment could pose an initial stress in the broilers. The toe-treated group had
higher mortality during the 38-day growing period due to the higher mortality o f toetreated birds in the first week. The major cause for mortality o f the toe-treated birds was
found to be dehydration, indicating the-treatment procedure could influence bird ability
to access to feed and water. In order for microwave toe-treatment to become a viable
commercial procedure, the level o f exposure to microwave energy may need to be
reduced in effort to reduce related mortality. Additional projects to determine the
optimum level for broiler chickens is recommended. Increased availability o f feed and
water for toe-treated birds may also help reduce the mortality.
The microwave toe-treatment did not affect either the activity o f plasm a creatine
kinase or the heterophil to lymphocyte ratios suggesting that the microwave toetreatment did not influence the stress level in birds at the ages tested. Despite the
findings by previous studies which found toe-treatment reduce fear in Leghorn
chickens, there was no difference in the duration and induction o f tonic immobility
between the toe-treated birds and the birds with intact toes in this study indicating that
the microwave toe-treatment did not influence the level o f fear in broilers.
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122
This project provided evidence that microwave toe-treatment could benefit the
skin integrity in broiler chickens. The microwave toe-treatment reduced the incidence o f
carcass scratches significantly, and tended to decrease the incidence o f carcass bruises
(p=0.061). The toe-treated birds had increased skin puncture strength and reduced
activities o f the active-MMP-2 compared to the birds with intact toes.
The birds on the increasing photoperiod program were significantly smaller
during most o f the growout phase, but had a similar final body weight compared to the
birds under the continuous lighting program. The reduction in early growth rate on the
increasing lighting benefited bird health and resulted in decreased mortality in later life.
The birds on the increasing lighting consumed less feed, especially starter and grower
feed which are more expensive due to higher protein content, but had the same overall
feed conversion as the birds with the continuous light. These results suggest that
increasing lighting programs would be more economic than continuous photoperiod for
growing broilers.
The birds on the continuous photoperiod program had a higher activity o f plasma
creatine kinase, but did not show elevated heterophil to lymphocyte ratios. The
difference b etween t he r esults f or t hese t wo m ethods f or m easuring t he stress w ould
seem to be most easily explained in terms o f sensitivity o f each method. The elevated
activity o f plasma creatine kinase with the continuous lighting may indicate lower meat
quality o f birds with the continuous lighting. Increasing lighting significantly decreased
the duration o f tonic immobility at 10 days o f age, had no effect on day 22, wheares
increased the duration at 36 days o f age. These findings suggest that short daylengths
could reduce the level o f fear in broilers and may also indicate that birds on the
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123
increasing lighting could not easily adjust to the change o f daylengths in later life,
resulting in a higher level o f fear at this age.
Increasing lighting increased the incidence o f carcass scratches, but did not have
an e ffect o n t he i ncidence o f carcass b raises. T he 1ighting t reatments d id n ot h ave a
significant effect on skin strength or the activity o f plasma MMP-2. There was no
difference in the type III collagen content in the skin between the two photoperiod
programs. The increasing lighting tended to increase the type I collagen content in the
skin (p=0.08). A split-plot design used in the study could have reduced the lighting
effects in the statistical analysis due to few degrees o f freedom for the lighting factor.
Further research is recommended to examine the effects o f photoperiod on skin collagen
o f broilers.
M ale broilers had a higher feed intake and heavier body weight after 3 wk o f age.
The males had lower mortality than the females in this study. The males showed larger
body weight differences between the continuous and increasing lighting programs. The
males also showed larger body weight differences than the females between the intacttoe and treated-toe groups. Lower activity o f the plasma creatine kinase and the
heterophil to lymphocyte ratios in the males suggest that they had a lower level o f
stress; this finding is consistent with the lower mortality o f the males in this study. The
evidence suggests that the male chicks could tolerate the negative effect caused by toetreatment better than the females. There was no significant difference in the level o f fear
between the males and the females. The males and females had a similar incidence o f
carcass scratches and braises. The male broilers had stronger skin than the females,
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124
while t here w as n o d ifference i n t ype I and III c ollagen c ontent i n t he s kin b etween
sexes.
Collectively, this study demonstrated the use o f microwave toe-treatment in
combination with the increasing photoperiod benefited broiler chickens by improving
carcass quality without reducing overall performance and had little influence on the
level o f stress and fear o f the broilers. Additional projects to determine the optimum
level o f exposure to microwave energy for broiler chickens in effort to reduce related
mortality is recommended.
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125
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