sheep deficient in vitamin e preferentially select for a ... · sheep deficient in vitamin e...
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Sheep deficient in vitamin E preferentially select for a feed rich
in vitamin E
This thesis is submitted to fulfill the requirements for a Master of Science
(Animal Science) by way of Thesis & Coursework
Faculty of Natural and Agricultural Sciences
The University of Western Australia
July 2013
DORAID ESHO AMANOEL
BSc (Animal Production) (UOD) Iraq
BSc (Animal Science) (Hons) (UWA) Aust
Supervisors:
Dr Dean Thomas (CSIRO Livestock Industries)
Dr Hayley Norman (CSIRO Livestock Industries)
Associate Professor Dominique Blache (School of Animal Biology, UWA)
Journal Formatting: Animal Behaviour
i
DECLARATION
I certify that the substance of this thesis is original, has not already been submitted
for any degree and is not currently being submitted for any other degree or qualification
at any university or other institution. The experiment and the writing of the thesis were
designed and carried out by myself in consultation with my supervisors Dr Dean
Thomas, Dr Hayley Norman and Associate Professor Dominique Blache. I certify that
all the sources used have been duly acknowledged in this thesis.
Doraid Esho Amanoel
July 2013
ii
ABSTRACT
In Mediterranean environments, vitamin E deficiency is common in young weaner
sheep during summer and autumn due to shortages of green feed. Vitamin E deficiency
can cause diseases and death in severe cases. To rebalance vitamin E levels, it is
possible to offer food rich in vitamin E such as saltbush. However, it is not known if the
deficient animals will actively select the vitamin E rich food amongst other feed
sources. It was hypothesised that sheep experiencing a vitamin E deficiency would
voluntarily select more of vitamin E enriched feed compared with non-deficient sheep.
Fifty six Dohne-Merino ewe lambs aged eight months, with an average live body
weight of 37.5 kg, were selected. Two groups (n=28 per group) were constituted after a
depletion/enrichment phase (40 days), one group with high concentrations of vitamin E
(α-tocopherol) in the plasma and the other with low concentrations. In preparation for a
15 days preference testing phase, each group was randomly sub-divided into two sub-
groups (n=14 per sub-group). Animals in the four sub-groups were presented a choice
between pairs of vitamin E enriched and deficient feeds that were offered ad libitum.
Each feed type was flavoured with either strawberry or orange so that the animals were
able to learn to associate the vitamin E status of the feed with a particular flavour. The
experimental design was balanced so that two sub-groups (high and low plasma vitamin
E) were offered vitamin E enriched feed flavoured with strawberry and deficient feed
flavoured with orange and in the other two sub-groups the flavourings were reversed.
There was a significant three way interaction between the high and low vitamin E
treatment groups × vitamin E content and type of flavour in the feed × time (days)
suggesting that preference for vitamin E enriched feed with an orange flavour changed
with time differently to the strawberry flavoured vitamin E enriched feed. Sheep with a
vitamin E deficiency modified their relative intake and preferentially selected more of
the vitamin E rich feed compared to non-deficient sheep. Self-learning by the low
vitamin E group could explain that they overcame the aversive effect of the orange
flavour to consume more vitamin E to compensate for the deficiency. The results of this
study demonstrated that sheep deficient in vitamin E will voluntarily alter their
preference over time and select for vitamin E rich feed, presumably to remediate the
deficiency.
Key Words
Vitamin E deficiency, weaner sheep, preference, post-ingestive feedback, saltbush
iii
ACKNOWLEDGEMENTS
I am very appreciative of the support and contributions I have received from a
number of people and organisations that have enabled me to undertake this research.
From the outset, I would like to express my heartfelt thanks to my supervisors Dr
Dean Thomas (CSIRO), Dr Hayley Norman (CSIRO) and Associate Professor
Dominique Blache (UWA) for their rigorous engagement with my work. They have
been a constant source of wisdom and guidance throughout the progress of this study.
Their constant words of encouragement, belief that this study was valuable and their
collective abilities to keep me on track, motivated me to see it through to the end. I
thank you for your knowledge, feedback and valuable advice. I am truly grateful to all
three of you.
I have relied on many people around me throughout this study. In particular, I have
had the pleasure and good fortune of benefitting from the assistance of Matt Wilmot
(CSIRO). The practical help with the animals, his generosity, good humour and
friendship throughout the experiment will always be treasured. Special thanks go to
Miranda Taafe (CSIRO), Nathan Phillips (CSIRO) and Andrew Toovey (CSIRO) for
their welcome assistance in the animal house. Also, I am grateful to Paul Young
(CSIRO) for conducting the laboratory analyses of the blood samples.
No scientific study can be fully realised without the knowledge, wisdom and
generosity of other great minds. I have been more than fortunate in having the help of
supportive scientists who contributed to my research. I am indebted to Associate
Professor John Milton (UWA) for his invaluable assistance. His expertise in ruminant
nutrition enabled me to have the dietary pellets manufactured to meet the specific
requirements of this study. In addition, I was able to draw on Dr Dean Revell’s
(CSIRO) vast research knowledge of animal nutrition and preferences and I extend my
appreciation to him for his insights, rigor and wise words.
I have been fortunate to have been supported in my studies by a number of
organisations. I wish to thank the Commonwealth Scientific and Industrial Research
Organisation (CSIRO) for the opportunity to undertake this study within the
organisation. In particular, I thank the staff from the CSIRO Livestock Industries
iv
Division located at the Centre for Environment and Life Sciences (CELS) in Floreat,
Perth, Western Australia for their continued help and support. Also, I warmly
acknowledge the staff members of the UWA School of Animal Biology who were
always there to assist me when I needed them. Finally, I thank my sponsors, AusAID,
for having made my study at UWA possible through the provision my Australia – Iraq
Agricultural scholarship (AIAS).
I also wish to acknowledge Harry Williams who supplied the sheep for this study
and staff from the Department of Agriculture and Food Western Australia, Malcolm
McGrath and Gerard Smith for their assistance and cordiality.
I am immensely thankful to Noel Chamberlain for his continued wise counsel,
support, guidance and friendship. His enthusiasm and attention to detail inspired me to
strive for excellence in my work.
Last, but not least, I want to thank my wonderful family. I started this journey four
and a half years ago with your love, hopes and dreams for my success. Although you
have been half a world away, you have always been there for me. To my mother Shirini,
father Esho and my lovely sisters Dalya, Dalal, Dana and Diana, I thank you for your
unwavering support and encouragement.
v
TABLE OF CONTENTS
DECLARATION ................................................................................................................. i
ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGEMENTS ............................................................................................... iii
TABLE OF CONTENTS .................................................................................................... v
LIST OF ABBREVIATIONS ........................................................................................... vii
LIST OF FIGURES ......................................................................................................... viii
LIST OF TABLES ............................................................................................................. ix
CHAPTER 1: INTRODUCTION .................................................................................... 1
Hypothesis ........................................................................................................................... 2
Aims and Objectives ............................................................................................................ 2
CHAPTER 2: REVIEW OF THE LITERATURE ........................................................ 3
Introduction ......................................................................................................................... 3
Vitamin E and its Source in the Body.................................................................................. 3
The Role of Vitamin E in Animal Health, Meat Quality and Production ........................... 4
Vitamin E Requirements for Sheep ..................................................................................... 5
Vitamin E Deficiency and its Occurrence ........................................................................... 5
The Consequences of Vitamin E Deficiency ....................................................................... 6
The Effects of Vitamin E Deficiency on the Voluntary Food Intake in Ruminants ............. 7
Native Australian Perennial Shrubs as a Source of Vitamin E for Livestock .................... 8
Dietary Learning and Feedback Effects on Preference ...................................................... 9
Conclusion ........................................................................................................................ 12
CHAPTER 3: MATERIALS AND METHODS .......................................................... 13
Animals .............................................................................................................................. 13
vi
Experimental Design ......................................................................................................... 13
Preparation of Experimental Feeds .................................................................................. 15
Preparation of Experimental Animals .............................................................................. 16
Conditioning the Animals to the Experimental Feeds....................................................... 17
Acclimatising the Animals to the Animal House ............................................................... 17
Vitamin E Depletion/Enrichment Phase ........................................................................... 17
Definition of Vitamin E Deficiency and the Diagnosis of Nutritional Myopathy ............. 18
Preference Testing Phase .................................................................................................. 19
Feed Intake and Preference Measurements ...................................................................... 19
Animal Measurements ....................................................................................................... 20
Statistical Analysis ............................................................................................................ 21
CHAPTER 4: RESULTS ................................................................................................ 22
Plasma Vitamin E (a-tocopherol) Level in the Experimental Animals ............................. 22
The Effect of Vitamin E Deficiency on Feed Intake (as-fed basis) ................................... 23
The Relationship between Vitamin E Intake and Plasma Vitamin E Level....................... 26
The Effect of Vitamin E Deficiency on Growth Rate......................................................... 26
The Effect of Vitamin E Deficiency on the Feed Preference of Sheep .............................. 27
The Selection of Vitamin E Enriched Feed by Individual Animals ................................... 29
CHAPTER 5: DISCUSSION ......................................................................................... 31
Conclusion ........................................................................................................................ 35
REFERENCES ................................................................................................................ 37
vii
LIST OF ABBREVIATIONS
These abbreviations were created specifically for this study.
HIGHVE
Animals with high concentrations of vitamin E in the plasma
LOWVE
Animals with low concentrations of vitamin E in the plasma
+VEOR
Vitamin E enriched feed flavoured with orange
-VEOR
Vitamin E deficient feed flavoured with orange
+VEST
Vitamin E enriched feed flavoured with strawberry
-VEST
Vitamin E deficient feed flavoured with strawberry
viii
LIST OF FIGURES
Figure 1. Factors that can influence the selection and preference of diets by
animals (Arnold 1964).
10
Figure 2. Diagrammatic representation of the experimental design indicating
the vitamin E depletion/enrichment and preference testing phases of
the experiment, phase duration and feed provided in each phase.
14
Figure 3. Mean values of vitamin E concentrations in the plasma (α-
tocopherol measured in mg/L) in the HIGHVE
(black columns) and
LOWVE
(grey columns) treatment groups (n = 28) before and after
the preference testing phase (mean ± SE). The asterisk (*) on the
columns indicates significant differences between the groups
(P<0.001).
22
Figure 4. Total daily feed intake (mean ± SE in kg/animal/day) presented as-
fed basis for the HIGHVE
(black column) and LOWVE
(grey
column) groups (n = 28) during the preference testing phase (15
days). Error bars are standard errors of the means.
23
Figure 5. Mean values of the daily feed intake (kg/animal) of the feed
combinations (enriched or deficient feeds) with either flavour
(strawberry or orange) by the sub-treatment groups (HIGHVE
and
LOWVE
). The asterisk (*) indicates significant differences between
the intake of the vitamin E enriched and deficient feed, with either
flavours, within each sub-group (P<0.001).
25
Figure 6. The preference index of the HIGHVE
(black columns) and LOWVE
(grey columns) sub-groups across the preference testing period (15
days; mean ± SE). Preference index highlights animal preference
toward the +VEOR
feed (A) and +VEST
feed (B). The index ranges
from 0-1 indicating the extent to which a particular feed is preferred
by animals. The asterisk (*) indicates significant differences
between HIGHVE
and LOWVE
groups in their preference toward
+VEOR
and +VEST
feeds (P<0.05).
28
Figure 7. Daily frequency or proportion (%) of individual sheep in the
HIGHVE
(black columns) and LOWVE
(grey columns) sub-groups
selecting for the vitamin E enriched orange flavoured feed (A) and
vitamin E enriched strawberry flavoured feed (B) with high
preference (PI >0.5).
30
ix
LIST OF TABLES
Table 1
A summary of the deficient and adequate ranges plus the critical
value of α-tocopherol, used as an indicator in sheep, to determine the
level of vitamin E in the plasma
6
Table 2
Daily feed intake (mean values measured in kg) of the four sub-
groups within each treatment group (HIGHVE
and LOWVE
) during
the preference testing phase.
23
Table 3
Total vitamin E intake (mean in mg/kg with SE in brackets) of the
four sub-groups within each treatment group (HIGHVE
and LOWVE
)
during the preference testing phase and plasma concentrations of
vitamin E (mg/L) before and after preference testing.
26
Table 4
Average growth rate measured in kg/animal/day of sheep in the
HIGHVE
and LOWVE
groups during the depletion/enrichment and
preference testing phases.
26
1
CHAPTER ONE
INTRODUCTION
In areas with Mediterranean type climates, during summer and autumn when green
vegetation rich in vitamin E is scarce, sheep become deficient in vitamin E (Gabbedy et
al. 1977; Masters & White 1996; Pearce et al. 2005; Steele et al. 1980; White et al.
1992). Vitamin E deficiency is a major problem for fast growing weaner sheep 6-12
months old (Steele et al. 1980), which have an increased demand of vitamin E
(Gabbedy et al. 1977). These young animals graze for up to six months on dry senesced
pastures, cereal grains and stubbles, that have low vitamin E content which results in
insufficient liver reserves (Kumagai & White 1995; Smith et al. 1994; White & Rewell
2007). Consequently, weaner sheep can develop nutritional myopathy, or White Muscle
Disease (WMD), which can cause damage to body organs (such as heart) and skeletal
muscles, and in severe cases, it can lead to animals’ death (Smith et al. 1994; White &
Rewell 2007).
About 58% of weaner sheep flocks are deficient in vitamin E in Western Australia
(White & Rewell 2007). Farmers, who have become more aware of the importance of
vitamin E for their livestock, have increased the use of synthetic sources of vitamin E to
overcome the problem. In 2003-04, for instance, more than one million sheep in
Western Australia (≈ 5%) were supplemented with this vitamin during summer and
autumn as a means to reduce the incidence of nutritional myopathy (Pearce et al. 2005;
White & Rewell 2007). However, administering ‘off the shelf’ products (vitamin E
drenches, injections and feed additives) increase production cost due to the added
expense of the products and labour (Pearce et al. 2005). Thus, there is a significant
opportunity to explore other novel, sustainable alternatives to improve vitamin E
nutrition and to minimise, or even eliminate, the use of synthetic vitamin E in the
livestock industry. One possible low-cost and sustainable alternative is the provision of
vitamin E in autumn and summer from green native perennial forages. For example, old
man saltbush (Atriplex nummularia) has the potential to reduce the incidence of vitamin
E deficiency in sheep because it contains high levels of vitamin E, up to 139 mg/kg DM
(Pearce et al. 2005), and it remains green throughout summer and autumn (Fancote et al.
2013; Pearce et al. 2010). However, saltbush can contain high levels of oxalates,
nitrates, sodium (Na), chloride (Cl), potassium (K), sulphur (S) which can have anti-
2
nutritional properties that limit the voluntary food intake (VFI) and reduce preference
(Ben Salem et al. 2010; Norman et al. 2004). Nevertheless, if ruminants can learn to
prefer particular feeds because they raise fitness (Howery et al. 2010; Villalba et al.
2006b), they may also learn to overcome the aversive effects of the unfavourable
charecteristics of the feeds and consume more of it to remediate a vitamin E deficiency.
Ruminants learn to associate what they eat with the metabolic consequences of
eating it and they change food preference according to their experience and nutritional
status (Burritt & Provenza 1996). When ruminants experience deficiencies, they alter
their diet preference accordingly to include different and sometimes unusual feeds in
their diet such as soil, bones or manure to rectify imbalances and meet their specific
needs (Blair-West et al. 1992; Provenza 1995; Villalba et al. 2008). It is known that
ruminants exhibit preference, when offered choice, toward some essential nutrients such
as energy, protein and minerals (phosphorus, calcium and sodium) to remediate the
deficiencies (Bach et al. 2012; Villalba & Provenza 1997a; Villalba & Provenza 1997b;
Villalba et al. 2008). However, it is not known if sheep deficient in vitamin E will
modify their preference and increase the intake of a vitamin E enriched diet to remediate
the deficiency. Thus, the extent to which sheep experiencing a vitamin E deficiency will
alter their preference and preferentially select a vitamin E enriched feed is to be
investigated.
Hypothesis
Sheep experiencing a vitamin E deficiency would voluntarily select more of a
vitamin E enriched feed compared with non-deficient sheep.
Aims and Objectives
The aim of this project was to investigate whether sheep deficient in vitamin E alter
their preference and voluntarily consume more of vitamin E rich feeds to alleviate the
deficiency. To test the hypothesis, a vitamin E deficiency was induced in a group of fast
growing weaner sheep. Preference for feeds enriched or deficient of vitamin E was
compared with another group not experiencing the deficiency.
3
CHAPTER TWO
REVIEW OF THE LITERATURE
Introduction
Vitamin E deficiency in livestock can cause health issues to emerge. If ruminants are
able to alter their food preference according to experience and the nutritional status of
the body (Burritt & Provenza 1996), they might preferentially utilise vitamin E rich
forages when experiencing a vitamin E deficiency. This literature review explains the
biological role of vitamin E in the body of a living organism summarises the vitamin E
dietary requirements for sheep and highlights the times when sheep are at greatest risk
of developing a vitamin E deficiency. The consequences of vitamin E deficiency in
sheep are highlighted and the effects of the deficiency on the voluntary food intake are
explained. Ruminants’ capability to possess nutritional wisdom toward particular
nutrients is explored and factors influencing the selection and preference of diets are
summarised. The possibility of the inclusion of native Australian perennial shrubs (such
as saltbush) as sources of dietary vitamin E in livestock farming systems is considered.
Vitamin E and its Sources in the Body
Vitamin E was first identified in the early twenties in studies related to reproduction
in rodents (Bramley et al. 2000). Vitamin E is naturally available in plant material in
eight different forms: α, β, γ, δ -tocopherol and α, β, γ, δ -tocotrienol (Bramley et al.
2000). Alpha-tocopherol is the most important form of vitamin E because it is
biologically considered as a tissue active form of vitamin E (Hidiroglou & Charmley
1990). Alpha-tocopherol represents about 90% of all tocopherols in animal tissue thus it
is often used as a reliable indicator for the determination of vitamin E levels in the body
(Wolf et al. 1998). Vitamin E is absorbed through the lymphatic pathway, transported
with chylomicrons (lipoprotein particles) and stored as α-tocopherol in the liver
(Bjorneboe et al. 1990), an organ that regulates the provision of vitamin E to other
tissues (Fry 1993). Adipose tissue is another storage site of vitamin E in the body (Puls
1994). In livestock, vitamin E is not stored in sizeable amounts thus they easily become
deficient if they are not supplemented adequately (Puls 1994). It is estimated that over
one million sheep across WA are supplemented with synthetic vitamin E each year
4
during summer and autumn periods as a mean to reduce the incidence of nutritional
myopathy (Pearce et al. 2005).
Vitamin E is a potent antioxidant that works in conjunction with Selenium (Se) to
maintain the health of animals (Freer & Dove 2002; Yang et al. 2002). Vitamin E and
Se are positively correlated and have the ability to offset the deficiencies of each other
(Combs & Scott 1977; Hatfield et al. 2000). Therefore, studies investigating the effect
of vitamin E on the performance of animals (for example health and production) must
maintain Se at adequate levels in order to investigate the effect of vitamin E more
correctly.
The Role of Vitamin E in Animal Health, Meat Quality and Production
Vitamin E is a lipid-soluble antioxidant that is primarily involved in free radical
defence mechanisms and protects animal cells from oxidation damage (Huber 1988).
More specifically, vitamin E protects against peroxidative degradation of lipids in the
cell membrane (sourced from diets containing fat) and the consequent formation of free
radicals (Huber 1988). Free radicals are formed from normal metabolic processes in the
body when oxygen interacts with tissue molecules (Horton et al. 2005). Unlike the
normal paired electron structure of the oxygen atom, free radicals are oxygen atoms
with only a single electron (Chew 1996). The unpaired oxygen atom becomes unstable,
highly reactive and presents a strong oxidizing agent that can start a chain reaction with
other biomolecules (particularly proteins and lipids due to their strong oxidation ability)
which negatively affects their integrity and function (Chew 1996). If the oxygen free
radicals are not removed from the biological system by vitamin E, physiological
disorders can emerge (Chew 1996; Coehlo 1991).
In addition, vitamin E effectively interacts with the immune system and protects
against disease (Reffett et al. 1988), maximises immunocompetence through
maintaining the integrity of cells in the immune system (Coehlo 1991; Sheffy & Schultz
1979) and sustains humoral immunity (Reffett et al. 1988). It delays lipid and colour
oxidation of meat thus extending the shelf life of beef (Robbins et al. 2003; Stubbs et al.
2002) and lamb meat (Lauzurica et al. 2005; Pearce et al. 2005). Supplementing lambs
with vitamin E increases α-tocopherol concentrations in the muscles and improves the
moisture holding capacity of meat, reduces the production of off-flavours and odours,
5
and lowers the formation of peroxides and aldehydes which can be toxic to humans
(Morrissey et al. 1994). Vitamin E supplementation also increases α-tocopherol levels in
the colostrum and milk of ewes, boosts the neonatal birth weight, maintains the level of
vitamin E in the new born (Capper et al. 2005) and reduces the lamb mortality rate (Kott
et al. 1983; Kott et al. 1998).
Vitamin E Requirements for Sheep
The minimal and optimal vitamin E requirements for sheep are yet to be determined
(Freer et al. 2007). The recommended level of vitamin E in the diet for sheep is 10 to 20
mg/kg DM diet (Agricultural Research Council 1980). The National Research Council
(1985) recommends a 15 mg/kg DM vitamin E concentration for lambs and up to a 20
mg/kg DM diet for heavier animals including pregnant and lactating ewes. All these
values are based on an assumption that Se intake is adequate (Freer et al. 2007). If, for
example, the dietary Se is low, the concentration of vitamin E in the diet needs to be
increased. However, this does not apply when the nutritional conditions are inadequate;
when animals are only maintaining or losing weight (Agricultural Research Council
1980). As indicated, vitamin E contributes to sustaining the immune system of the
animal and this is most likely achieved at dietary levels of vitamin E higher than that
required for growth and maintenance (Puls 1994). Vitamin E concentrations in a diet
that are 6 to 20 times greater than National Research Council recommendations would
boost the immune system in animals to become more responsive (Nockels 1986). It is
important to indicate that several factors such as type of breed, stocking rate, climatic
conditions, proportional dietary amount of vitamin E as opposed to Se and the
physiological state of animals (pregnant and lactating) have a major role in the
determination of vitamin E dietary requirements for ruminants (Freer et al. 2007).
Vitamin E Deficiency and its Occurrence
Vitamin E deficiency is defined as a nutritional condition that occurs because of the
lack of α-tocopherol supply from the body reserves (liver and adipose tissue) due to an
inadequate vitamin E supply in the diet. Plasma α-tocopherol is often used as an
indicator to determine vitamin E deficiency in animals (White & Rewell 2007). A
summary of the deficient and adequate ranges, as well as the critical value of α-
tocopherol in the plasma of sheep, are summarised in Table 1.
6
Table 1
A summary of the deficient and adequate ranges plus the critical value of α-tocopherol, used as
an indicator in sheep, to determine the level of vitamin E in the plasma
Indicator Units Range Critical
value Reference
Deficient Adequate
Plasma α-
tocopherol mg/L ≤ 1.0
b,c 1.0 – 4.0
a 0. 7
a
a(White & Rewell 2007)
b(Smith et al. 1994)
c (Njeru et al. 1994)
Vitamin E deficiency generally occurs in young weaner sheep flocks. It was
estimated in 2007 that about 58% of weaner flocks were deficient in WA (White &
Rewell 2007). The occurrence of vitamin E deficiency is common in summer and
autumn when forage quality and quantity are at a minimum and less frequent during
winter and spring when fast-growing green pastures are generally available. The
concentrations of α-tocopherol in green pasture species during winter and spring range
from 50 to 200 mg/kg DM (Beeckman et al. 2010; Tramontano et al. 1993). In contrast,
in dry senesced pasture available during summer and autumn, vitamin E levels range
from 2 to 20 mg/kg DM (Tramontano et al. 1993) which potentially can result in a
vitamin E deficiency in livestock flocks (Hatfield et al. 2000; White & Rewell 2007).
Thus, herbivores grazing on spring pasture species tend to have higher vitamin E levels
in the body as opposed to those feeding on dry and stored feeds (Hatfield et al. 2000)
suggesting that supplementation of vitamin E during dry seasons is essential to maintain
health and productivity (Pearce et al. 2005).
The Consequences of Vitamin E Deficiency
Nutritional myopathy, also called nutritional muscular dystrophy or White Muscle
Disease (WMD), is one of the consequences of vitamin E deficiency in a combined
effect with Se deficiency (National Research Council 1985; White & Rewell 2007). The
cause of the disease is mainly due to a chronic imbalance between pro-oxidants
(polyunsaturated fatty acids; PUFAs) and antioxidants (vitamin E and Se) (Freer &
Dove 2002; Lykkesfeldt & Svendsen 2007). High uptakes of PUFAs with low
antioxidant concentrations in the body make an animal prone to a suppressed immune
response (Kelley & Bendich 1996) and tissue damage (Lykkesfeldt & Svendsen 2007),
leading to nutritional myopathy. It often occurs in vitamin E and Se deficient calves and
weaner sheep when they are turned out onto spring green pastures, where PUFAs are
7
abundant (Combs & Scott 1977). In WA, about 6% of weaner sheep show severe
muscle damage (White & Rewell 2007) mainly during two distinct time periods in
which the death rate is at its maximum; the first during summer and autumn when
vitamin E deficiency is predominant and the second during winter due to the incidence
of Se deficiency (Gabbedy et al. 1977; Turner et al. 2002; White & Rewell 2007). The
myopathy primarily damages the skeletal muscles and the affected animals have stiff
movements, an arched back and may become recumbent (National Research Council
1985). Severe cases of WMD can lead to sudden death within 2 to 3 days of birth due to
heart and muscle failure (National Research Council 1985). If sheep are not adequately
supplemented with vitamin E and Se, WMD can significantly impact on the
productivity and profitability of livestock farming systems.
The Effects of Vitamin E Deficiency on the Voluntary Food Intake in Ruminants
The direct effects of vitamin E deficiency on the voluntary food intake (VFI) of
ruminants have not been fully investigated. One of the signs of vitamin E deficiency in
sheep is reduced growth rate (National Research Council 1985) but that is not directly
correlated to reduced food intake. Sheep experiencing WMD exhibit normal appetite
toward foods but their live body weight gain is reduced due to the consequences of the
disease (Andrews 1992; Suttle 1992). For instance, skeletal muscle damage might cause
sheep to be unable to stand for long periods to consume food sufficiently or are unable
to swallow foods properly because of the damage to tongue muscles (National Research
Council 1996). In contrast, in rodents and broiler chicks, there is a direct correlation
between food intake and vitamin E availability in the diet. For example, supplementing
vitamin E improves food intake and boosts nutrient utilisation by rats (Ainsah 1999) and
chickens (Nwaigwe et al. 2010). Broiler chicks fed diets deficient in vitamin E
decreased their body weight because vitamin E decreased their food intake (Swain et al.
2000). Nevertheless, it appears that there is no current research available that is directly
related to the practical importance of vitamin E on food intake in ruminants, therefore,
future investigation in this area is required.
8
Native Australian Perennial Shrubs as a Source of Vitamin E for Livestock
Native Australian shrubs, such as old man saltbush (Atriplex nummularia), are used
to feed ruminants in a range of Mediterranean environments (Ben Salem et al. 2010).
These shrubs are tolerant to drought and salinity, perform well in various types of
nutrient poor soils and provide green biomass that is readily eaten by ruminants during
the summer and autumn feed gap (Masters et al. 2007; Norman et al. 2010a). Saltbush,
for example, has low organic matter digestibility (48-63% Organic Matter Digestibility,
OMD), moderate to high crude protein content (10-25% edible dry matter, EDM) and
up to 30% ash, predominately sodium (Na), potassium (K) and chloride (Cl) (Masters et
al. 2009; Masters et al. 2005; Norman et al. 2010a). More importantly, it has elevated
levels of antioxidant vitamin E (139 mg/kg EDM in old man saltbush) which has been
identified to have a beneficial role in enhancing animal health and improving meat
quality (Fancote et al. 2013; Pearce et al. 2005). Nevertheless, there are variations in the
nutritive value in saltbush, and possibly other forage shrubs, due to environmental
factors such as soil fertility and water content (Atiq-ur-Rehman et al. 1999; Masters et
al. 2009).
In addition to the nutritive value, Atriplex species (such as old man saltbush) contain
an array of compounds that have anti-nutritional effects such as oxalates and nitrates
(Masters et al. 2001; Norman et al. 2004). Elevated levels of salt (NaCl; 220 g/kg DM)
and sulphur (S; 4.6 g/kg Dry Matter; DM) in the leaves can limit VFI and create
aversions toward the plant (Ben Salem et al. 2010; Norman et al. 2004). If salty diets
were to be offered with other low salt alternatives such as a grass or legume understory
and fresh water ad libitum, the intake of both high and low salt diets would be
dependent on the quality (OMD) of the understory (Norman et al. 2010b). For instance,
the intake of saltbush by sheep increases from about 13% DM during spring to
approximately 54% DM during autumn due to a decline in the quality of the understory
from about 68% OMD in spring to 45% OMD in autumn (Norman et al. 2010b). Thus,
if saltbush is grazed concurrently with other plant material (dry stubble) during summer
and autumn periods, then there are potential opportunities for grazing systems to
become more sustainable, diverse and profitable (Monjardino et al. 2010). One of these
opportunities can be increased uptake of less preferred perennial forage shrubs
(saltbush) by vitamin E deficient animals to remediate the deficiency during the summer
and autumn feed gap.
9
Dietary Learning and Feedback Effects on Preference
Sheep consume a wide array of plant species and possess, to some extent, a
nutritional wisdom based on their experience and nutritional status (Burritt & Provenza
1996; Provenza 1995). This enables them to select for foods that are nutritionally
beneficial (Naim et al. 1991; Swithers & Davidson 2008), contain certain types of
substances (medications) to ameliorate malaises (Phy & Provenza 1998; Villalba et al.
2006b) and avoid others that are nutritionally deficient or contain elevated amount of
toxins and plant secondary compounds that are harmful (Provenza 1995; Provenza
1996; Provenza et al. 1990; Villalba & Provenza 1997b). Sheep do not instinctively
detect the needed nutrients or medicines available in the feed stuff through sensory
means (taste, smell, touch and sight) but they learn about their feeds across time using
post-ingestive feedbacks (Provenza & Balph 1990; Provenza & Villalba 2006). Sheep
attain information about their feed using the characteristics of the feed, such as flavours,
as cues to make associations with the post-ingestive feedbacks; positive or negative
gastrointestinal feedback stimulated in the body (Favreau et al. 2010b; Provenza 1995;
Provenza 1996). It is important to note that food preference is a complex design that is
influenced by a multitude of factors such as the physiological state, nutritional status
and experience of animals, as well as the chemical characteristics of feed (Fig.
1)(Arnold 1964).
10
ANIMAL
Motor outflow
Reflexes of attention, approach, examination and consumption or rejection
Sense of sight, smell, touch and taste
PLANTS
Plant species present and their chemical and physical characteristics
and relative availability.
Modified by
PLANT ENVIRONMENT
Soil type, soil fertility and Plant community,
rainfall, soil moisture
Modified by
PHYSICAL ENVIRONMENT
Topography (i.e. slope, aspect and site of plant)
Distance plant is from water
Distance plant is from tracks or shade
Modified by
ANIMAL FACTORS
Animal species
Animal individuality
Physiological condition (food demand)
Grazing behaviour
Social behaviour
Modified by
PREVIOUS EXPERIENCE
DIET COMPOSITION
Figure 1. Factors that can influence the selection and preference of diets by animals (Arnold
1964).
11
Studies have established that sheep develop preferences toward nutritious feeds
(crude protein, sodium Na, Phosphorus P, and energy) that meet their nutritional status
for the achievement of homeostasis (Bach et al. 2012; Villalba & Provenza 1997a;
Villalba & Provenza 1997b). For example, lambs provided deficient diets in crude
protein adjusted for the deficiency when an appropriate level of protein in an alternative
diet was offered (Bach et al. 2012). Sheep deficient in Na preferentially increased the
intake of water containing Na compared to normal water to compensate for the
deficiency (Beilharz et al. 1962; Denton & Sabine 1963). Similarly, sheep deficient in
Na had higher intake of plant species containing high levels of Na in preference to other
plants with low Na levels (Arnold 1964). Additionally, sheep deficient in P
preferentially increased the intake of subclover containing high levels of P compared to
low levels of P, suggesting that the selection for subclover containing high P
concentrations was beneficial for sheep to compensate for P degraded during the dry
season (Ozanne et al. 1976; Ozanne & Howes 1971; Villalba et al. 2006a). Lambs
consumed more flavoured (onion or oregano) straw in preference to unflavoured straw
when low doses of starch or propionate were infused into their rumen, after the
flavoured straw was consumed (Villalba & Provenza 1997a; Villalba & Provenza
1997b), indicating that sheep attributed the availability of energy to the flavour (via
positive post-ingestive feedback) and developed a preference towards that flavour.
In addition to the feeds that are nutritious, sheep voluntarily ingest low and
moderate quality shrubs for the content of some compounds (medicines) to self-
medicate against metabolic disorders such as acidosis (Phy & Provenza 1998),
gastrointestinal parasites (Lisonbee et al. 2009; Osoro et al. 2007; Villalba et al. 2010)
and metabolic disorders caused by excess amounts of secondary compounds such as
tannins and oxalates (Villalba et al. 2006b). Lambs were able to overcome the
consequences of acidosis by preferentially consuming water containing a sodium
bicarbonate compound (alkaline) while other lambs with no signs of acidosis consumed
plain water (Provenza et al. 1994), suggesting that the need to rectify acidosis
stimulated the lambs to consume the alkaline water.
Further, ruminants develop aversions toward some forages due to elevated levels of
plant toxins and other undesirable compounds existent in the plants (Provenza et al.
1994). Ruminants exhibit a variety of strategies to avoid the toxic effects of some
12
plants. Initially, they tend to sample small amounts of various plant species to buffer
any potential toxic effect (Voth 2010), then, based on the post-ingestive feedback, they
determine whether to increase or decrease the intake of these feeds (Provenza 1995).
Lambs experiencing negative post-ingestive feedback, stimulated by a toxin dose after
ingesting a rice diet flavoured with cinnamon, generated a similar aversion toward a
wheat diet mixed with cinnamon, suggesting that lambs were able to relate the
consequence to the flavour and formulated an aversion to any diet offered with the same
flavour (Launchbaugh & Provenza 1993). It can be concluded that the intensity of the
aversive post-ingestive signals, positive or negative, triggered from the gut and
translated in the brain (central nervous system) determine the extent to which foods are
preferred or avoided.
Conclusion
This review highlighted the biological importance of the antioxidant vitamin E and the
consequences associated with vitamin E deficiency in sheep, particularly in the autumn
and summer feed-gap when green pastures are lacking. The mechanisms at work in diet
selection and preference were examined. It was established that ruminants associate
between the sensory characteristics of the feed and post-ingestive feedback to sense the
consequences of food ingestion and alter feed selection and preference based on their
experience and the nutritional status of the body. However, as it is not known whether
sheep experiencing vitamin E deficiency alter their preference and select for a vitamin E
rich feed to remediate the deficiency, it was decided that this would be the ultimate
object of this research. If the proposed concept is proven to be right, then this will
provide worthwhile evidence for further investigation to see if vitamin E deficiency
would drive sheep to change feed preference and use perennial forage shrubs as sources
of vitamin E.
13
CHAPTER THREE
MATERIALS AND METHODS
This study was conducted at the Commonwealth Scientific and Industrial Research
Organisation (CSIRO) - Centre for Environment and Life Sciences (CELS) in Floreat,
Western Australia. The experimental protocol was approved as conforming to the
Australian Code of Practice for the Care and Use of Animals for Scientific Purposes,
and the welfare of the animals was closely monitored by the CSIRO Animal, Food and
Health Sciences, Floreat Animal Ethics Committee (AEC approval number: 1203).
Animals
Fifty six Dohne Merino ewe lambs (a dual purpose Merino resultant from a cross
between Peppin style Merino ewes and German Mutton Merino sires) aged eight
months, with an average live body weight of 37.5 kg were selected from a flock on a
commercial farm located near Pingelly, Western Australia. The lambs were selected
based on body weight, condition score and visual health status. The lambs were de-
stressed and acclimatised to the presence of humans and handling in a small yard
adjacent to the animal house so that the lambs became calmer, less reactive to human
activity and easier to handle throughout the study. The lambs had ad libitum access to
water and they were exercised on a weekly basis by allowing them to walk freely in a
large yard.
Experimental Design
The 56 ewe lambs were stratified according to the following parameters: plasma
vitamin E level (α-tocopherol concentration in the plasma), live body weight, condition
score, and genetics (animals were either second or third cross) to ensure there were no
differences in the mean of the indicated parameters. After stratification, the animals
were randomly allocated to individual pens in the animal house. All animals underwent
a vitamin E depletion/enrichment phase for 40 days to form two treatment groups: low
(LOWVE
) and high (HIGHVE
) concentrations of vitamin E in the plasma (n=28
animals/treatment group; Fig. 2). Following the depletion/enrichment phase, animals in
the HIGHVE
and LOWVE
groups underwent a preference testing phase for 15 days in
which each group was randomly sub-divided into two sub-groups (n=14 animals/ sub-
14
group; Fig. 2). The four sub-groups were offered a choice between vitamin E enriched
and deficient feeds ad libitum, each feed type flavoured with either strawberry or
orange, depending on the sub-group. One of the HIGHVE
sub-groups received a feed
combination of vitamin E enriched orange flavoured feed (+VEOR
) and vitamin E deficient
strawberry flavoured feed (-VEST
). The other HIGHVE
sub-group received vitamin E
enriched strawberry flavoured feed (+VEST
) and vitamin E deficient orange flavoured feed
(-VEOR
) (Fig. 2). The same procedure for the feed offerings and flavouring regime was
used for the LOWVE
sub-groups. Swapping the two flavours between sub-groups
determined whether feed preference was due to the vitamin E content of the feed or the
flavour.
Figure 2: Diagrammatic representation of the experimental design indicating the vitamin E
depletion/enrichment and preference testing phases of the experiment, phase duration and feed
provided in each phase.
15
Preparation of Experimental Feeds
The type of feed used in this study was based on commercially available pellets
composed of wheat, lupins and straw (70% dry matter digestibility, 16% crude protein
and 12.1% metabolisable energy). The pellets were specifically formulated for young
lambs to allow them to grow at a rate of about 200 g/day when offered 1.1
kg/animal/day (as-fed basis). These pellets were specially manufactured without the
addition of vitamin E so the pellets only contained the natural source of the vitamin (α-
tocopherol), which was equivalent to 6.9 mg/kg fresh matter (FM). Half of the
manufactured pellets were used as a ‘vitamin E deficient feed’ with no added vitamin E,
whereas the remaining half was sprayed with a synthetic type of vitamin E
(Nanodispersed Natural-Source Vitamin E by Kentucky Equine Research, Victoria,
Australia) to form a ‘vitamin E enriched feed’.
Liquid vitamin E was sprayed on the pellets while being rotated in a cement mixer.
The concentrated solution of the vitamin (250 IU d-α-tocopherol per 1 ml of the
vitamin, which is equivalent to 168 mg/ml) was diluted (1 part to 34 parts of tap water)
and the resultant solution applied using a manual trigger sprayer at a rate of 75 mg of
vitamin E per kg FM of feed. The pellets were examined to check that the liquid vitamin
was distributed evenly and the structure of the pellets was not affected (no visual and
textural changes). The adherence of the vitamin E on the internal surface of the mixer
was taken into account by an initial light application of the solution to the surface.
The vitamin E enriched and deficient feeds were alternately mixed with two
commercially available flavours: strawberry and orange. The flavours were synthetic
water soluble human food-flavouring agents (Queen Fine Foods Pty, Ltd., Queensland,
Australia). The flavours were used as ‘dietary cues’ to allow the sheep to differentiate
between the vitamin E enriched and deficient feeds and to associate the flavour in the
feed with post-ingestive feedbacks (positive feedback associated with a recovery from
vitamin E deficiency and negative feedback due to the aggravation of the deficiency).
The two flavours were diluted in tap water (1 part flavour to 1 part tap water) and
applied to the pellets at a 1% rate (Early & Provenza 1998) using a manual trigger
sprayer in two cement mixers (one cement mixer per flavour) to avoid cross
contamination between the two flavours. After flavouring the pellets, humans were able
16
to identify the orange and strawberry treatments by their smell. The addition of the
flavours did not affect the nutritional value of the pellets.
Preparation of Experimental Animals
Prior to transferring the animals to CSIRO, health records and vaccinations
administered to the animals on the farm were checked and a follow-up health
assessment was conducted to ensure that the selected animals were suitable for this
study. Health records indicated that the lambs were previously vaccinated
with Websters Low Volume 3 in 1 Vaccine with Selenium and Vitamin B12 (Virbac
Animal Health Pty, Ltd., New South Wales, Australia) for the prevention of cheesy
gland caused by Corynbacterium pseudotuberculosis, pulpy kidney (enterotoxaemia)
caused by Clostridium perfringens type D, tetanus caused by Clostridium tetani,
selenium responsive conditions, white muscle disease, vitamin B12 deficiency and
unthriftiness. Additionally, the lambs had been given Cydectin Weanerguard SE B12 6
in 1 Vaccine and Wormer (Virbac Animal Health Pty, Ltd., NSW, Australia) for the
prevention of five clostridial diseases, cheesy gland, internal parasites, nasal bot, itch
mite, and for the supplementation of vitamin B12 and selenium. The animals were shorn
on the farm two weeks prior to transportation to CSIRO Floreat.
Upon arrival at the CSIRO animal house facility, and prior to entering the animal
house, all lambs were held outside for 14 days in a small yard adjacent to the animal
house for the final on-site health check and quarantine purposes. The lambs were
weighed and their feet inspected and clipped as required. The lambs were also examined
for scabby mouth disease, checked for external parasites, vaccinated against lice and
blowfly strike using Vanquish Long Wool Spray (Alpha-cypermethrin 50 g/L, Coopers
Animal Health - A division of Schering Plough Pty Ltd, NSW Australia) and
supplemented with 200 mg of vitamin B1 (Thiamine hydrochloride 125 mg/mL, Nature
Vet Pty Limited, NSW Australia) subcutaneously.
17
Conditioning the Animals to the Experimental Feeds
All animals were initially offered a familiar feed comprised of oaten hay (1
kg/animal/day) whole lupins (250 g/animal/day) and Siromin (ad libitum), a complete
mineral supplement developed by CSIRO for sheep fed on dry herbage (White et al.
1992), outside in a small yard adjacent to the animal house. Then, the familiar feed was
gradually replaced by the vitamin E deficient feed to allow the microbial populations in
the rumen to adjust and adapt to the new feed and to ensure the lambs were given
adequate time for a vitamin E deficiency to occur. Over five consecutive days, 20% of
the vitamin E deficient feed was added daily to the familiar feed, with a concomitant
reduction of the familiar feed, until the familiar feed was fully replaced by the deficient
feed.
Acclimatising the Animals to the Animal House
The lambs were acclimatised to the animal house over three consecutive days. On
the first day, the sheep were fed the vitamin E deficient feed in the morning (at 0900
hours) outside the animal house in the adjacent yard and moved into the animal house in
the afternoon (at 1200 hours). At this stage, the sheep were restricted to walking
through the aisles (three aisles) for one hour but they were not allowed to walk into the
pens to ensure that the animals were not stressed due to separation or the novelty of the
new environment. On Day two, the sheep were fed outside in the morning (at 0900
hours) and moved into the animal house in the afternoon (at 1200 hours) and kept in
individual pens for an hour, then released back into the yard. On the third day, the sheep
were permanently moved into the animal house in the morning (at 0900 hours), housed
in individual pens and offered the vitamin E deficient feed in individual feeding bins
attached to the pens.
Vitamin E Depletion/Enrichment Phase
The vitamin E depletion/enrichment phase was conducted for 40 consecutive days in
which the animals in the LOWVE
and HIGHVE
groups were offered 1.1 kg/animal/day of
the vitamin E enriched or deficient feeds, depending on the treatment group, as one bout
in the morning (at 0900 hours; Fig. 2). In the last week of the depletion/enrichment
phase, the same quantity (1.1 kg/animal/day) of either feeds (vitamin E enriched or
18
deficient feeds) was offered simultaneously in the morning (at 0900 hours) in two
separate buckets attached to each other (each bucket containing 550 g) with random
left-right positions to familiarise the animals with the ‘two buckets’ presentation
procedure conducted in the preference testing phase. The depletion and enrichment
phase elapsed when two distinct vitamin E concentrations in the plasma were formed in
the HIGHVE
and LOWVE
groups.
Definition of Vitamin E Deficiency and the Diagnosis of Nutritional Myopathy
In the current study, vitamin E deficiency was defined as any plasma α-tocopherol
reading between 0.5 - 0.7 mg/L, as discussed by White and Rewell (2007). The
sufficient level of vitamin E in the plasma was defined as plasma α-tocopherol that is ≥
2 mg/L. The animals that exhibited plasma concentrations of vitamin E below the
critical level (below 0.5 mg/L) during the depletion/enrichment phase were immediately
drenched with 4 ml of vitamin E (Nanodispersed Natural-Source Vitamin E by
Kentucky Equine Research, Victoria, Australia) to ensure that the vitamin E level
remained within the defined deficiency range and safeguard the welfare of the animals
against the emergence of the subclinical nutritional myopathy.
In addition to monitoring the vitamin E concentration in the plasma, evidence of
sub-clinical nutritional myopathy was monitored using commercial kits (Roche
Diagnostics, F Hoffman-La Roche Ltd., Basel, Switzerland) at the Department of
Agriculture and Food Western Australia for the diagnosis of creatine kinase (CK) and
alanine aminotransferase (ALT) enzymes. The two tests have a sensitivity of 98% for
the diagnosis of subclinical nutritional myopathy as described by Fry et al. (1994). The
reference ranges for CK, that indicates the severity of muscle damage, was defined as
follows: CK value between 400 and 1200 U/L indicates mild muscle damage and >1200
U/L indicates severe muscle damage. For ALT, values between 30 and 80 U/L indicates
mild muscle damage and >80 U/L indicates severe muscle damage (Fry et al. 1994).
Throughout the study, the ALT and CK analysis revealed no evidence of sub-clinical
nutritional myopathy in the experimental sheep.
19
Preference Testing Phase
In the context of this study, the term ‘preference’ implies a behavioural
characteristic of an animal by which it voluntarily and preferentially selects one feed
over another. Thus, when offered a choice of feeds, one feed is indicated as ‘preferred’
if the animal more often selects it rather than the other feed on offer.
The preference testing phase was conducted for 15 successive days during which all
animals in the four sub-groups were offered 2 kg/animal/day (our established at ad
libitum rate) of either vitamin E enriched or deficient feed (flavoured with strawberry or
orange) in the morning (0900 hours). The feeds were offered simultaneously in two
separate buckets of identical size (25 cm diameter) and colour. The buckets were placed in
the feeding bins and supported from the base to prevent spillage of feeds caused by the
animals. The buckets were allocated and labelled according to the feed type and flavour for
the duration of the experiment to avoid any possible cross contamination of vitamin E or
flavour. The relative position of the flavoured feeds for each animal was alternated from
left to right daily to account for any positional effect. Every morning (0900 hours)
throughout the preference testing phase, the refusals of the vitamin E enriched and
deficient feeds of the previous day were weighed.
Feed Intake and Preference Measurements
Feed intake (as-fed basis) was quantified and relative preference for the vitamin E
enriched feed when fed in combination with the vitamin E deficient feed was calculated
using the following Preference Index (PI) equation. The calculation, as described by
(Bell 1959) is commonly used for determining relative preference (Colebrook et al.
1990; Dunlop 1986; Kenney & Black 1984):
In order to manage the occasions where the intake of one or both feeds were equal to
zero, a small constant amount of 5 g of either feed was added to the numerator and
denominator of the PI equation. The value 5 g was chosen to be less than the smallest
feed intake unit measured throughout the experiment (11 g). Thus, the equation used to
20
calculate PI in this study is:
The index has a scale from 0 to 1 indicating relative preference between vitamin E
enriched and deficient feeds. An equal preference corresponds to a PI value of 0.5
whereas PI values below 0.5 correspond to low preference and values higher than 0.5
indicate high preference.
Animal Measurements
Live body weight and condition scores were measured weekly in the morning (0800
hours) prior to feeding. Condition was scored on a scale of 1-5 according to the methods
described by Suiter (1994) and the procedure was conducted by the same trained person
to avoid any possible sources of variation. Throughout the depletion/enrichment and
preference testing phases blood samples were taken periodically (10 day intervals) by
means of direct jugular venipuncture from all animals before feeding at 0800 hours to
monitor the vitamin E level in the plasma. A volume of 10 ml of blood was collected
from the jugular vein using a 10 ml syringe and injected into Lithium heparinised tubes,
placed immediately into an ice bath and protected from the light because vitamin E is
light and temperature sensitive. Plasma was separated by centrifugation at 3000 rpm for
twenty minutes and stored at -20 °C until analysed. The plasma samples were analysed
for the measurement of α-tocopherol using a high-performance liquid chromatography
with a fluorometric detection technique at the CSIRO, CELS laboratory according to the
method described by McMurray & Blanchflower (1979). The intra-assay and inter-
assay coefficient of variation were equal to 1% and 2%, respectively.
21
Statistical Analysis
All statistical analyses were conducted using the Genstat statistical package with a
significance level of 95% (Genstat 2012). A Repeated Measures Analysis of Variance
(ANOVA) with mixed models was fitted using a Residual Maximum Likelihood
(REML) method. The analysis determined the effect of vitamin E deficiency status
(HIGHVE
and LOWVE
groups), feed combination (vitamin E deficient and enriched),
flavours (strawberry and orange), and the interaction effects on animal diet selection
and preference (expressed as feed intake and preference index) across the preference
testing period (15 days).
A preliminary repeated measures ANOVA model with mixed models REML was
fitted for the 15 days of the preference testing phase. In this model, high and low plasma
vitamin E treatment groups, vitamin E enriched and deficient feeds (each flavoured with
either strawberry or orange flavours) were used as fixed factors, and individual animals
(n=56) and time (n=15 days) were used as random factors. A time effect with variance
was detected, thus, a second model (repeated measures ANOVA with mixed models
REML) was fitted.
In the second model, the period of the preference testing (15 days) was divided into
two time periods: the training period (the first nine days) and the post training period
(the last six days) in order to determine in which period the treatment (high/low vitamin
E level) had an effect on animal diet selection and preference. The HIGHVE
and LOWVE
groups, feed combination (vitamin E enriched and deficient feeds each with either
flavour), training and post training periods were used as fixed factors, with individual
animals and the total time period (15 days) used as random factors. Examination of the
distribution of the residual values was conducted for both models using repeated
measures ANOVA (mixed models REML) and the results indicated that the
assumptions concerning homogeneity of variance and normality of data were adequately
met.
22
CHAPTER FOUR
RESULTS
Plasma Vitamin E (α-tocopherol) Level in the Experimental Animals
After the depletion/enrichment phase, prior to the commencement of the preference
testing, the average plasma concentrations of vitamin E differed between the HIGHVE
(1.92 ± 0.16 mg/L) and LOWVE
(0.60 ± 0.04 mg/L) groups (P <0.001; Fig. 3 A). The
concentrations of vitamin E in the plasma of the two groups become statistically similar
(1.98 ± 0.14 for HIGHVE
vs. 1.69 ± 0.11 for LOWVE
) at the end of the preference testing
phase (P=0.122, Fig 3 B).
Figure 3. Mean values of vitamin E concentrations in the plasma (α-tocopherol measured in
mg/L) in the HIGHVE
(black columns) and LOWVE
(grey columns) treatment groups (n = 28)
before and after the preference testing phase (mean ± SE). The asterisk (*) on the columns
indicates significant differences between the groups (P<0.001).
23
The Effect of Vitamin E Deficiency on Feed Intake (as-fed basis)
Total feed intake per day (mean ± SE kg/animal/day), averaged across the entire
preference testing period (15 days), did not differ between the HIGHVE
and LOWVE
groups (0.92 ± 0.03 vs. 0.93 ± 0.03; P=0.962; Fig. 4).
Figure 4. Total daily feed intake (mean ± SE in kg/animal/day) presented as-fed basis for the
HIGHVE
(black column) and LOWVE
(grey column) groups (n = 28) during the preference
testing phase (15 days). Error bars are standard errors of the means.
On average, animals in both the LOWVE
and HIGHVE
groups preferred strawberry
flavoured feed as evidenced by greater intake of strawberry pellets over orange
flavoured pellets (P<0.001; Table 2).
Table 2
Daily feed intake (mean values measured in kg) of the four sub-groups within each treatment
group (HIGHVE
and LOWVE
) during the preference testing phase.
Vitamin E
concentration
in the plasma
Feed intake (kg/animal/day) of the sub- groups
S.E.D P value
-VEOR
+VEOR
-VEST
+VEST
HIGHVE
0.531 a 0.658
b 1.199
c 1.298
c 0.053 P<0.001
LOWVE
0.737 a 0.751
a 1.051
b 1.181
c 0.053 P<0.001
Different letters within rows indicate significant difference between means (P<0.001).
24
However, when the time of the preference testing phase (15 days) was taken into
account, there was a three-way interaction (P<0.001) between the treatment groups
(HIGHVE
and LOWVE
) × feed combination (vitamin E + flavour content) × days. The
feed intake of vitamin E enriched and deficient feeds (flavoured with either orange or
strawberry) differed between the HIGHVE
and LOWVE
sub-groups over time (Fig. 5).
The LOWVE
sub-group offered a choice between the -VEST
feed and +VEOR
feed had a
higher intake of the -VEST
feed than for the +VEOR
feed during the first nine days
(P<0.001; Fig. 5 A). Then, their intake of the +VEOR
feed was higher from Day 10 to 12
followed by a reduction from Day 13 onwards. By contrast, the HIGHVE
sub-group
offered the same feeds maintained higher intakes for -VEST
compared with +VEOR
feed
across the 15 days of the preference testing phase (P<0.001; Fig. 5 B).
The other HIGHVE
and LOWVE
sub-groups offered the +VEST
and -VEOR
feed
combination (Fig. 5 C and D) consumed more of the +VEST
compared to the -VEOR
feed
across the whole preference testing period, except on Day 3, when the LOWVE
sub-
group had a higher intake of the -VEOR
feed as opposed to the +VEST
(P<0.001; Fig. 5
C). Feed intake of the +VEST
feed by the LOWVE
sub-group stabilised from Day 8
onwards (Fig. 5 C).
25
Figure 5. Mean values of the daily feed intake (kg/animal) of the feed combinations (enriched
or deficient feeds) with either flavour (strawberry or orange) by the sub-treatment groups
(HIGHVE
and LOWVE
). The asterisk (*) indicates significant differences between the intake of
the vitamin E enriched and deficient feed, with either flavours, within each sub-group
(P<0.001).
26
The Relationship between Vitamin E Intake and Plasma Vitamin E Level
During preference testing, vitamin E intake (mean ± SE mg α-tocopherol/kg FM) of
the LOWVE
sub-group from the +VEST
feed was greater than the amount of the vitamin
E ingested by the same sub-group from the +VEOR
(1328 vs. 844 mg/kg; P<0.001;
Table 3). By the end of the preference testing phase, the difference in vitamin E plasma
concentrations between the HIGHVE
and LOWVE
groups was eliminated (P=0.122). The
vitamin E intake from the diet and plasma concentrations of vitamin E were positively
correlated in the LOWVE
group (y = 0.234 + 0.0013x, R² = 0.63, P<0.001).
Table 3
Total vitamin E intake (mean in mg/kg with SE in brackets) of the four sub-groups within each
treatment group (HIGHVE
and LOWVE
) during the preference testing phase and plasma
concentrations of vitamin E (mg/L) before and after preference testing.
Treatment
groups
Sub-
groups
Total vitamin
E intake
(mg/kg)
Vitamin E
concentration in
the plasma before
preference testing
phase (mg/L)
Vitamin E
concentration in the
plasma after
preference testing
phase (mg/L)
∆
plasma
vitamin
E
(mg/L)
LOWVE
+VEOR
844.27
a
(59.33)
0.62 a
(0.06)
1.35 a
(0.12) 0.73
+VEST
1328.79
b
(64.56)
0.58 a
(0.04)
2.04 b
(0.12) 1.46
HIGHVE
+VEOR
740.06
a
(83.03)
1.78 b
(0.2)
1.57 a
(0.19) -0.21
+VEST
1459.92
b
(71.68)
2.06 b
(0.25)
2.38 b
(0.16) 0.32
Different letters within columns indicate significant difference between means (P<0.001)
The Effect of Vitamin E Deficiency on Growth Rate
On average, the growth rate (kg/animal/day) of animals in the HIGHVE and LOW
VE
groups did not differ during the depletion/enrichment phase (P=0.859) and preference
testing phase (P=0.598; Table 4).
27
Table 4
Average growth rate measured in kg/animal/day of sheep in the HIGHVE
and LOWVE
groups
during the depletion/enrichment and preference testing phases.
Phase Growth rate (kg/animal/day)
S.E.D P value HIGH
VE LOW
VE
Depletion/enrichment phase 0.108 0.107 0.009 P=0.859
Preference testing phase 0.205 0.214 0.017 P=0.598
The Effect of Vitamin E Deficiency on the Feed Preference of Sheep
Throughout the 15 days of preference testing, preference for the strawberry
flavoured feed was greater than the orange flavoured feed in the HIGHVE
and LOWVE
groups (P<0.001; Fig. 6). However, when the time of the preference testing phase was
included as a factor in the analysis, there was a significant three-way interaction effect
(P=0.002) between the treatment groups, feed combination (vitamin E in the feed +
flavours), and time (days).
Feed preferences of the HIGHVE and LOW
VE groups also differed between the first
nine days and the last six days of the preference testing phase (P=0.001). The HIGHVE
and LOWVE
sub-groups offered the combination of +VEOR
and -VEST
feeds had similar
low preferences (P=0.241) toward the +VEOR
feed during the first nine days of
preference testing (Fig. 6 A). However, during the last six days of the preference testing
period, the LOWVE
sub-group exhibited higher preferences toward the +VEOR
feed as
opposed to the other corresponding HIGHVE
sub-group offered the same feed
combination (P=0.008; Fig. 6 A). By contrast, the HIGHVE
and LOWVE
sub-groups
offered +VEST
and -VEOR
feeds had a higher preference pattern toward the +VEST
feed
across all 15 days of the preference testing phase (P > 0.05; Fig. 6 B). However, the
LOWVE
sub-group tended to have a more consistent and higher preference toward
+VEST
from Day 8 onwards (Fig. 6 B).
28
Figure 6. The preference index of the HIGHVE
(black columns) and LOWVE
(grey columns)
sub-groups across the preference testing period (15 days; mean ± SE). Preference index
highlights animal preference toward the +VEOR
feed (A) and +VEST
feed (B). The index ranges
from 0-1 indicating the extent to which a particular feed is preferred by animals. The asterisk (*)
indicates significant differences between HIGHVE
and LOWVE
groups in their preference toward
+VEOR
and +VEST
feeds (P<0.05).
29
The Selection of Vitamin E Enriched Feed by Individual Animals
Across the 15 days of the preference testing period, individual variability existed
between animals in both the HIGHVE
and LOWVE
groups in their tendency to exhibit
higher preference (preference index > 0.5) for the +VEOR
and +VEST
feeds (Fig. 7).
During the first nine days of the preference testing phase, on average, 15% of animals in
the LOWVE
sub-group exhibited a higher preference for the +VEOR
feed, which
increased to 80% in the last six days of testing (Fig. 7 A). By contrast, in the HIGHVE
sub-group receiving the same feed combination and flavouring pattern, the proportion
of the animals showing high preferences for +VEOR
was 29% in the first nine days as
opposed to 27% in the last six days of the preference testing phase (Fig. 7 A).
In the LOWVE
sub-group, offered the +VEST
feed, the average proportion of the
animals selecting for the +VEST
feed with higher preference was 67% during the first
nine days of preference testing which increased to 93% in the last six days of testing
(Fig. 7 B). In the corresponding HIGHVE
sub-group offered the same feed combination
and flavouring pattern, 84% of the animals selected for the +VEST
feed in the first nine
days and this increased to 88% in the last six days of preference testing (Fig. 7 B).
30
Figure 7. Daily frequency or proportion (%) of individual sheep in the HIGH
VE (black columns)
and LOWVE
(grey columns) sub-groups selecting for the vitamin E enriched orange flavoured
feed (A) and vitamin E enriched strawberry flavoured feed (B) with high preference (PI >0.5).
31
CHAPTER FIVE
DISCUSSION
The hypothesis that sheep experiencing a vitamin E deficiency would voluntarily
select more of a vitamin E enriched feed compared with non-deficient sheep was
supported. The results of this study showed that all sheep in the LOWVE
and HIGHVE
groups preferred the vitamin E enriched feed when it was flavoured with strawberry, but
only the LOWVE
group preferred the vitamin E enriched feed when flavoured with
orange. This is the first demonstration of the ability of sheep, experiencing a vitamin E
deficiency, to voluntarily alter their preference and select for vitamin E rich feed,
presumably to remediate the deficiency. It is evident that the deficient animals were able
to associate the flavours of food, using sensory means, with the positive post-ingestive
feedback stimulated from the deficiency-alleviating effects of the vitamin, which
encouraged them to further consume the vitamin E enriched feed. As a response to the
vitamin E intake, the LOWVE
group remediated the deficiency evidenced by the
increased α-tocopherol concentrations in the plasma reaching adequate concentrations
(> 1 mg/L) (Njeru et al. 1994; Smith et al. 1994) by the end of preference testing.
Several experiments indicated that sheep associated the pre-ingestive sensory
characteristics of food with the post-ingestive consequences (positive or negative
feedbacks) occurring at the gut level (du Toit et al. 1991; Villalba & Provenza 1997b)
and on that basis the animals adjusted their feed choices accordingly (Forbes &
Provenza 2000; Provenza 1995). However, the finding of this study suggests that post-
ingestive feedback can occur, not only at the gut level, but also at the tissue level, which
can influence the feeding behaviour of animals. It also suggests that the post-ingestive
feedback mechanisms arising due to a vitamin E deficiency are sensitive to the deficits
of the vitamin and operate even before animals show clinical signs of the deficiency or
reduce their feed intake.
The review of the literature did not find any other published studies where ruminants
or monogastrics have selected vitamin E rich diets as a response to their low vitamin E
status. However, findings of the current study are consistent with other studies where
animals have changed preference and selected for other nutrients that were limited in
their diets. For example, lambs provided deficient diets in crude protein adjusted for the
deficiency when an appropriate level of protein in an alternative diet was offered (Bach
32
et al. 2012). Lambs preferred to consume more of straw flavoured with either onion or
oregano in preference to unflavoured straw when low doses of starch or propionate were
infused into their rumen, after the flavoured straw was consumed (Villalba & Provenza
1997a; Villalba & Provenza 1997b). Also, sheep experiencing mineral deficiencies,
such as P and Ca, were able to identify the diets containing the needed mineral, using
sensory cues (flavours), associated the flavour of the required mineral with the recovery
from a deficit of that mineral and exhibited a strong preference toward the feed
supplying the mineral (Villalba et al. 2008; Villalba et al. 2006a). Similarly, sheep
deficient in Na had higher intake of plant species containing high levels of Na in
preference to other plants deficient in Na (Arnold 1964). In other circumstances, sheep
voluntarily ingested soil, bones, urine and manure and this has been linked to the
deficits of P and Ca (Blair-West et al. 1992; Villalba et al. 2008).
The regulation of protein, energy, P and Ca appears to be exercised at the level of
the digestive system in which by-products of microbial fermentation, such as volatile
fatty acids, interact to cause satiety and affect food preference and selection (Bennink et
al. 1978; Farningham et al. 1993). Thus, satiety seems to be influenced by the post-
ingestive feedbacks triggered from the chemo-, osmo, and mechano- receptors in the gut
and signalled to the central nervous system (Anil et al. 1993; Denton et al. 1996;
Mbanya et al. 1993). However, the physiological and/or molecular mechanisms
underlying the regulation of vitamin E intake in living organisms are not clearly defined
in the literature and it is not known how animals deficient in vitamin E physiologically
detect the deficiency and select for the feed containing vitamin E.
The rate of learning seemed to be influenced by the flavour of the feed. The LOWVE
group in this study took nine days to exhibit preferences for the vitamin E enriched feed
paired with the flavour they originally disliked (orange). This was evidenced by the
higher preferences (Preference Index >0.5) and greater proportion (80%) of individuals
preferring the +VEOR
feed during the last six days as opposed to the first nine days of
preference testing. However, when the vitamin E enriched feed was flavoured with
strawberry, which they initially liked, the learning took 8 days to establish a strong and
consistent preference that continued to Day 15 of preference testing. These results
support the notion that ruminants do not instinctively detect the needed nutrients (or
medicines) available in the feeds but they take time to learn about their feeds by
33
associating the sensory properties of feeds with post-ingestive feedbacks (Provenza &
Balph 1990; Provenza & Villalba 2006). The association, by animals, of post-ingestive
effects with food flavours has been identified as a means through which herbivores
learn about the consequences of feeds (Duncan & Young 2002). Learning about the
feeds on offer is an important tool herbivores use to modify foraging behaviour (either
to prefer or to avoid feeds) in order to cope and adapt quickly to the changing internal
and external environments for the achievement of nutritional homeostasis (Howery et al.
2010; Villalba et al. 2006b).
It has been experimentally established that foraging behaviour is formed by two
learning mechanisms: self-learning from experience and trial and error by relying on
post-ingestive feedbacks and learning from peers or from the mother (Provenza & Balph
1990; Provenza & Cincotta 1993). In this study, where sheep were confined in
individual pens and social interactions between animals were restricted, the sheep had to
learn about their feeds by themselves and this may have delayed their learning process.
Additionally, recent research has demonstrated that the learning process about diets is
delayed when experimental procedures become complex (Duncan & Young 2002) by
offering several feeds simultaneously (Duncan et al. 2007) and by increasing the
number of consequences associated with the ingestion of feeds (Ginane et al. 2005). In
this study, where sheep were not familiarised with the flavours prior to preference
testing, the food offering procedure of the two flavoured feeds and the two associated
post-ingestive consequences (positive feedback due to vitamin E intake vs. negative
feedback because of the aggravation of the deficiency) was perceived as complex
(Duncan & Young 2002; Favreau et al. 2010a), evidenced by the reduced self-learning
efficiency in the LOWVE
group during the first nine days of the preference testing
phase. The complexity of the feeding procedure in this study and the novelty of flavours
to the sheep might have potentially contributed to the delayed association of the
flavours with the post-ingestive signals. It is also possible that the delay in the
stimulation of post-ingestive feedbacks after the ingestion of vitamin E might have been
associated with the delay in responses of the receptors to vitamin E located at the tissue
level. However, the responsible receptors and the mechanisms related to how these
receptors operate as a response to vitamin E intake are yet to be determined.
34
Throughout the preference testing phase, a strong flavour effect was observed. It
was apparent that sheep in HIGHVE
and LOWVE
groups preferred strawberry flavour as
opposed to the orange flavour, explained by high feed intakes of feeds flavoured with
strawberry as opposed to those flavoured with orange. Higher intakes of strawberry
flavoured feeds could have been due to the sweet taste of the flavour which might have
stimulated feed intake (Burritt et al. 2005; McMeniman et al. 2006) compared to the
orange citric flavour that had a strong associated flavour that potentially depressed
intake, presumably due to low palatability (Bampidis & Robinson 2006). Nevertheless,
our results demonstrated that, from Day 10 onwards, the LOWVE
group was able to
overcome the aversive effect of orange flavour to compensate for vitamin E deficiency
evidenced by higher preferences for the +VEOR
feed as opposed to the -VEST
feed.
The ability of the vitamin E deficient sheep in our study to consume feeds paired
with a less preferred flavour is consistent with a number of studies where ruminants
preferred to consume less preferred feeds or substances (only when a positive post-
ingestive feedback was associated with the consumption of these feeds or substances) to
rectify illnesses or internal burdens and increase fitness (Janzen 1978; Lisonbee et al.
2009; Phy & Provenza 1998; Provenza et al. 2000; Villalba & Provenza 2001; Villalba
et al. 2010). For example, sheep experiencing acidosis, caused by the consumption of a
grain based diet, preferentially selected sodium bicarbonate solutions, a substance
normally not consumed by sheep under favourable conditions, to attenuate the acidosis
effects (Phy & Provenza 1998). Similarly, sheep increased the intake of polyethylene
glycol, a non-preferred substance by sheep that binds with tannins, to remediate the
illness caused by tannins (Provenza et al. 2000; Villalba & Provenza 2001).
Furthermore, lambs experiencing a malaise caused by gastrointestinal parasites were
able to ‘feel’ the presence of the internal parasites (or associated the symptoms), modify
their feed choices and selected for feeds containing tannins (anti-nutritional compounds
in plants that are normally avoided by ruminants) to attenuate the malaise (Lisonbee et
al. 2009; Villalba et al. 2010).
As the sheep in this study were able to overcome the aversive effect of the orange
flavour to select for the vitamin E nutrient available in the feed, it is highly likely that
sheep (and possibly other ruminant species) will even overcome the unusual (and
potentially off-putting) plant characteristics in perennial forages to actively select the
35
vitamin E rich dietary source. The inclusion of these less preferred perennial forage
shrubs in farm systems can provide a valuable source of the naturally occurring vitamin.
This gives farmers a practical strategy to tackle vitamin E deficiency and reduce the
incidence of nutritional myopathy, particularly during summer and autumn when the
risks of both are high (Fancote et al. 2013). Ultimately, the diversity of the feed supply
base for livestock through the inclusion of preferred perennial forage shrubs will
improve and the productivity of marginal lands will increase bringing environmental
benefits. These benefits include a reduction of dryland salinity, a decrease in soil
erosion and the provision of shade and shelter to livestock which will potentially boost
livestock production within the context of clean, green, and ethical approaches.
Conclusion
It is believed that this experiment provides the first demonstration of the ability of
sheep experiencing a vitamin E deficiency to identify a benefit from consuming vitamin
E enriched feed and exhibit a preference towards that feed. The sensory cues (flavours)
used in the experimental feed facilitated the association processes of the sheep between
a particular flavour (orange or strawberry) used in the vitamin E enriched feed and the
post-ingestive signals. It was evident that the positive post-ingestive feedback, due to
the relief effect from the vitamin deficiency, was stimulated at the tissue level after the
ingestion of the vitamin E enriched feed which expedited the strong preference shown
towards the enriched feed with the associated flavour. The rate of learning seemed to be
influenced by the flavour available in the feeds. The LOWVE
group offered the +VEST
feed tended to learn faster than the other LOWVE
group offered +VEOR
feed. After nine
days of experience and self-learning, the LOWVE
group was able to overcome the
aversive effect of the orange flavour used in the vitamin E enriched feed and actively
selected, with higher preference, for the vitamin E enriched orange flavoured feed.
The findings of this study have the potential to provide low-cost management
opportunities for the livestock industry in areas with Mediterranean climates where the
incidence of vitamin E deficiency and the associated nutritional myopathy are prevalent
due to a lack of green pastures during dry seasons. It is anticipated that these findings
will provide science based evidence for livestock producers that, if their livestock
become nutritionally deficient in vitamin E, the animals will actively increase the
utilisation of forage shrubs that are rich in vitamin E even though these forage shrubs
36
are often less preferred by the animals under more favourable conditions. Our results
suggest it is likely that sheep (and possibly other ruminant species) will, to some extent,
overcome unusual (and potentially off-putting) plant characteristics within forage
shrubs to actively select the vitamin E rich dietary source if the animals are given
sufficient time to learn about their feeds. The inclusion of perennial forage shrubs will
help to rectify seasonal vitamin E deficiencies in livestock and possibly replace the
application of synthetic supplements that are currently widely adopted by farmers. It
will also give livestock producers the confidence that the expense of planting native
forage shrubs, such as saltbush, may be mitigated by sheep supplementing themselves
with vitamin E at critical times during the year.
37
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