AN ABSTRACT OF THE THESIS OF
Bruno Ieda Cappellozza for the degree of Master of Science in Animal Science presented
on February 17, 2012.
Title: Effects of Camelina Meal Supplementation on Ruminal Forage Degradability,
Performance, and Physiological Responses of Beef Cattle.
Abstract approved:
Reinaldo Fernandes Cooke
Three experiments compared ruminal, physiological, and performance responses
of beef steers consuming hay ad libitum and receiving grain-based supplements with
(CAM) or without (CO) inclusion of camelina meal. In Exp. 1, 9 steers fitted with
ruminal cannulas received CAM (2.04 kg of DM/d) or CO (2.20 kg of DM/d). Steers
receiving CAM had reduced (P = 0.01) total DMI and tended to have reduced (P = 0.10)
forage DMI compared to CO. No treatment effects were detected (P ≥ 0.35) for ruminal
hay degradability parameters. In Exp. 2, 14 steers receiving CAM (1.52 kg of DM/d) or
CO (1.65 kg of DM/d) were assigned to corticotropin-releasing hormone (CRH; 0.1
µg/kg of BW) and thyrotropin-releasing hormone (TRH; 0.33 µg/kg of BW) challenges.
Steers receiving CAM had greater (P < 0.05) serum concentrations of PUFA compared to
CO prior to challenges. Upon CRH infusion, mean plasma ceruloplasmin concentrations
increased at a lesser rate in CAM compared with CO (P < 0.01). Upon TRH infusion, no
treatment effects were detected (P ≥ 0.55) for serum TSH, T 3 , and T 4 . In Exp. 3, 60
steers were allocated to 20 drylot pens. Pens were randomly assigned to receive CAM
(2.04 kg of DM/steer daily) or CO (2.20 kg of DM/steer daily) during preconditioning
(PC; d -28 to 0). On the morning of d 0, steers were transported for 24 h. Upon arrival
from transport on d 1, pens were randomly assigned to receive, in a 2 x 2 factorial
arrangement, CAM or CO during feedlot receiving (FR; d 1 to 29). During PC, CAM had
reduced (P < 0.01) forage and total DMI, and tended to have reduced (P = 0.10) ADG
compared to CO. Plasma linolenic acid concentrations increased during PC for CAM, but
not for CO (P = 0.02). Steers that received CAM during FR had greater (P < 0.05) mean
plasma concentrations of PUFA, and reduced mean rectal temperature and concentrations
of haptoglobin and ceruloplasmin during FR compared to CO. Therefore, camelina
supplementation reduced forage and total DMI, did not alter thyroid gland function,
increased PUFA concentrations in blood, and attenuated the acute-phase protein reaction
elicited by neuroendocrine stress responses. In conclusion, camelina meal is a feasible
ingredient to reduce stress-induced inflammatory reactions and potentially promote cattle
welfare and productivity in beef operations.
© Copyright by Bruno Ieda Cappellozza
February 17, 2012
All Rights Reserved
EFFECTS OF CAMELINA MEAL SUPPLEMENTATION ON RUMINAL FORAGE
DEGRADABILITY, PERFORMANCE, AND PHYSIOLOGICAL RESPONSES OF
BEEF CATTLE
by
BRUNO IEDA CAPPELLOZZA
A THESIS
submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Master of Science
Presented February 17, 2012
Commencement June 2012
Master of Science thesis of Bruno Ieda Cappellozza presented on February 17, 2012.
Approved:
Major Professor, representing Animal Science
Head of the Department of Animal Sciences
Dean of the Graduate School
I understand that my thesis will become part of the collection of Oregon State University
libraries. My signature below authorizes release of my thesis to any reader upon request.
Bruno Ieda Cappellozza, Author
ACKNOWLEDGEMENTS
First of all, I would like to thank God for giving me health, protection, and
patiency. Second, I would like to thank my whole family for the support, patience, and
faith on me and that I’ve always needed. A special thank goes to my father Eduardo
Cappellozza, my mother Zaira Ieda Cappellozza, my grandfather José Ieda, and my
grandmother Lucy Ieda Cardinali for the best education and love provided to me. Also, I
would like to thank my girlfriend Maria Reis for her patience, love, and help during this
period.
I would like to thank Dr. Reinaldo F. Cooke for the opportunity, guidance during
my entire program, the excellent mentorship, for trusting in my work, and showing me
that everything happens the way we want no matter the difficulties found. I also would
like to thank Reinaldo and Flávia Cooke for their friendship and the happy moments.
I would like to thank Dr. David Bohnert for the opportunity and his guidance
when needed. I also would like to thank Flávia Cooke for the help and teaching during
my laboratory work. I would like to thank the interns and all the staff who assisted me
with my research projects, particularly César Trevisanuto, Victor Tabacow, Skip Nyman,
and Tony Runnels. I also would like to thank my former advisor, Dr. José Luiz Moraes
Vasconcelos, who introduced me to the scientific world, and always helped me and gave
me great opportunities during my academic formation. Lastly, I would like to thank my
friends Philipe Moriel and Fernanda Victor for their friendship and always helping me,
when needed.
TABLE OF CONTENTS
CHAPTER
Page
I. Introduction………………………………………………………..…………….1
II. Literature Review………………………………………………..………….….. 2
Immune System………………………………………………………… 2
Innate Immune System………………………….……………......... .2
Acute-phase Response…………………………………..……..…… 4
Stress and Hypothalamic-pituitary-adrenal axis………….…......……….8
Thyroid gland…………………………………………………..……….11
Camelina Meal…………………………………………………..………14
Fat Supplementation…………………………………………….………18
Ruminal Biohydrogenation……………………………………........ 21
PUFA and Immune System…………………………………........... 23
Development of strategies to alleviate the acute-phase
response in beef cattle…………………………..……………………… 26
III. Effects of camelina meal supplementation on ruminal forage
degradability, performance, and physiological responses of beef
cattle……………………………………………………………………………. 32
Abstract………………………………………………………………………… 32
Materials and Methods………………………………………………………….33
Results and Discussion………………………………………………………….45
IV. Literature Cited…………………………………………………………….. 71
LIST OF FIGURES
Figure
Page
2.1
Effects of proinflammatory cytokines on HPA axis, body tissues,
and organs…………………………………………………..………….29
2.2
Acute-phase reaction cascade event following activation by
macrophages, resulting in the production of acute-phase proteins…….30
2.3
Process of biodiesel production……………………………….………. 31
3.1
Plasma ceruloplasmin concentrations of beef steers offered
supplements containing (CAM) or not (CO) camelina meal and
receiving an i.v. corticotropin-releasing hormone (CRH) infusion
(0.1 µg/kg of BW) at h 0 in Exp. 2……………………………………69
3.2
Plasma linolenic acid concentrations during preconditioning (d -28
to d 0) of steers offered supplements containing (CAM) or not
(CO) camelina meal in Exp. 3……………………………………...….70
LIST OF TABLES
Table
Page
2.1
Fatty acid content of Camelina sativa oil from different locations
and cultivars……………………………………....……………………28
3.1
Ingredient composition, nutrient profile, and intake of treatments
offered during Exp. 1, 2, and 3……………………………………..…57
3.2
Fatty acid profile of feedstuffs offered to cattle during Exp. 1, 2,
and 3…………………………………………………………………...59
3.3
Plasma concentrations of CCK and ruminal in situ disappearance
parameters of mixed alfalfa-grass hay incubated in forage-fed
steers offered supplements containing (CAM) or not (CO) camelina
meal in Exp. 2………………………………………………………….60
3.4
Serum fatty acid concentrations (mg/ml of plasma) of steers offered
supplements containing (CAM) or not (CO) camelina meal during
Exp. 2…………………………………………………………………..61
3.5
Physiological responses of steers offered supplements containing
(CAM) or not (CO) camelina meal during Exp. 2…….……………….62
3.6
Performance and physiological responses during preconditioning
(d -28 to d 0) of steers offered supplements containing (CAM) or
not (CO) camelina meal during Exp. 2………………………………...63
3.7
Plasma fatty acid concentrations (mg/ml of plasma) during
preconditioning (d -28 to d 0) of steers offered supplements
containing (CAM) or not (CO) camelina meal in Exp. 3.......................64
3.8
Performance and physiological responses during feedlot receiving
(d 1 to d 29) of steers offered preconditioning supplements (from
d -28 to d 0) containing (CAM) or not (CO) camelina meal in
Exp. 3………………………………………………………………….65
3.9
Performance and physiological responses during feedlot receiving
(d 1 to d 29) of steers offered supplements containing (CAM) or
not (CO) camelina meal in Exp. 3…………………………………….66
LIST OF TABLES (Continued)
Table
Page
3.10
Plasma fatty acid concentrations (mg/ml of plasma) during feedlot
receiving (d 1 to d 29) of steers offered supplements containing
(CAM) or not (CO) camelina meal in Exp. 3………………………....67
3.11
Changes in plasma concentrations of acute-phase proteins in steers
after transport in Exp. 3………………………………………..……...68
CHAPTER 1
INTRODUCTION
Beef cattle production is the second largest commodity grown in the state of
Oregon, behind only of greenhouse and nursery products, being mainly composed of
cow-calf operations (OASS, 2010). In general, beef cattle producers in the state keep the
cow-calf pair on their property from birth to weaning. After weaning the producers have
several options, such as: 1) send the animals to the feedlot, retaining its ownership and
paying fees to the feedyard, and 2) selling the calves to the feedyard. The majority of
feedlots in the U.S. are located in the states of Texas, Kansas, and Nebraska (NASS,
2011), meaning that many newly weaned calves from the state of Oregon have to travel
long distances. Upon arrival in the feedlot, the animals go through several management
procedures, including vaccination, ear tagging, commingling with animals from different
locations, changes in the social hierarchy of the group, novel sources of feed and water,
castration, and dehorning (Loerch and Fluharty, 1999). All these management procedures
from weaning to feedlot arrival, which occur together or in a short period of time, are
very stressful to the animals, and expose them to viral and bacterial agents so that may
develop bovine respiratory disease (BRD) complex. Bovine respiratory disease is highly
correlated to morbidity and mortality rates in newly weaned and received cattle (Duff and
Galyean, 2007), presenting the most significant health problem in the U.S. beef cattle
industry. The calf death costs associated with BRD are estimated at almost $ 650,000
million yearly, and in the state of Oregon this cost is estimated to be in the order of $
10,971 million, representing 31.2% of the total calf loss within the state (NASS, 2011).
Therefore, strategies to alleviate the magnitude of these stressful events, thereby
enhancing survival rates and performance during the feedlot entry period have been
2
developed and are mainly focused on preweaning management, including creep feeding,
early weaning (Arthington et al., 2005; Carroll et al., 2009), and supplementation with
polyunsaturated fatty acids (PUFA; Araujo et al., 2010; Cooke et al., 2011). Additionally,
alleviating these stress-associated immune challenges may decrease costs associated with
labor and medication, which will result in greater overall profitability of beef cattle
operations.
CHAPTER 2
LITERATURE REVIEW
1. Immune System.
The immune system’s main role is to protect the body from infection, in order to
keep homeostasis. Several factors may impact the immune system’s functioning,
including age, genetics, gender, nutritional and hormonal status, and stress (Calder and
Kew, 2002). Generally, the immune system is divided into two branches: the innate and
acquired immunity. The acquired immune system is responsible for adapting and building
a immune response for each antigen it encounters in the body (Carroll and Forsberg,
2007), characterized by production of antibodies and creation of an immunological
memory. The main characteristic of the acquired immune system is its specificity,
meaning that for each antigen the body is challenged with, a specific response is
obtained. For the purpose of the studies reported herein, we will primarily review the
innate immune system.
1.1 Innate Immune System.
The innate immune system is considered the first-barrier against any pathogen in
the body, independently of the class (bacteria, protozoa, virus, or fungi). It includes
3
physical (skin, tears, mucosal secretion) and chemical (antimicrobial peptides, superoxide
anion, nitric oxide) barriers, as well as the complement system (Carroll and Forsberg,
2007). The innate system provides enough time for the acquired system to develop a
strong and effective response against a specific pathogen. The cellular components of the
innate immune system include phagocytic cells (neutrophils, monocytes in the
bloodstream, macrophages resident in the tissues, and dendritic cells), natural killer (NK)
cells, and cells that release inflammatory mediators (mast cells, basophils, and
eosinophils).
Unlike acquired immunity, the innate immune system does not recognize every
possible antigen infecting the body, nor does it generate a long-term protective
immunological memory (Murphy et al., 2008). Instead, the innate immune system
recognizes highly conserved structures present in many different microorganisms, known
as pathogen-associated molecular patterns (PAMPs). These structures are not found
typically in mammalian cells and thus, allow the recognition and attack of pathogens by
phagocytic cells (Carroll and Forsberg, 2007). Pathogen-associated molecular patterns
interact with a variety of different receptors in phagocytic cells, including the toll-like
receptors (TLR) located inside or on the surface of these cells. Upon the binding of
PAMPs to TLR, the cytokine response is initiated in neutrophils and macrophages, by
activation of the transcription factor nuclear factor kappa beta (NFĸB). The activation of
the NFĸB induces the gene expression of several important mediators of innate
immunity, such as cytokines, chemokines, and co-stimulatory molecules (Murphy et al.,
2008).
4
Cytokines are chemical messengers that can act to regulate the activity of cells
that produced the cytokine (autocrine action), cells that are in close proximity (paracrine
action), and cells that are in distant locations within the body (endocrine action; Calder
and Kew, 2002). Cytokines released by phagocytic cells during an immune response are
major mediators of intermediary metabolism in immunologically challenged animals
(Klasing, 1988). The primary cytokines that macrophages secrete are: interleukin-1 (IL1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which are collectively
referred to as proinflammatory cytokines because they stimulate an inflammatory
response within the body (Johnson, 1997). Additionally, the circulating concentrations of
these cytokines increase following an acute bacterial challenge (Lipopolysaccharide
(LPS) challenge; Carroll et al., 2009) and a neuroendocrine stress (corticotropin-releasing
hormone (CRH) infusion; Cooke and Bohnert, 2011). Proinflammatory cytokines present
two inherent properties: 1) they are extremely pleiotropic because they can serve different
functions in disparate organ systems (Kroemer et al., 1993), and 2) there is marked
redundancy in that different cytokines exhibit a similar biological function. Several
metabolic, neuroendocrine, and behavioral effects of proinflammatory cytokines in the
periphery and in the hypothalamic-pituitary-adrenal axis are shown in Figure 2.1. One of
the effects resulting from an inflammation is the activation of the acute-phase response
(APR), in order to restore the homeostasis in the damaged tissues within the infected
body.
1.2 Acute-phase response (APR).
Following an injury, trauma, or infection of a tissue, a complex series of reactions
are executed by the host in an effort to prevent further tissue damage, to destroy the
5
infective organism, and to activate the repair processes that are necessary to return to
homeostasis. All these actions are part of the inflammatory response. The early and
immediate set of are known as the APR (Baumann and Gauldie, 1994). The primary cell
types associated with the beginning of the APR are tissue macrophages and blood
monocytes (Gauldie, 1991). After activation, tissue macrophages and blood monocytes
release IL-1 and TNF-α and subsequently IL-6, which is considered the master regulator
of the APR in innate immune function. The schematic demonstration of the beginning of
this process is shown in Figure 2.2.
Several characteristic reactions are observed during an APR, including alterations
in plasma iron, zinc, and copper; increases in circulating white blood cells, and changes
in behavior such as lethargy, anorexia, decreased social, sexual, and aggressive behavior
(Carroll et al., 2009). In addition, the two main physiologic outcomes associated with
acute inflammation are: the febrile response and alterations in liver metabolism. Both
responses are regulated mainly by the proinflammatory cytokines previously mentioned.
The febrile response involves alteration of the temperature in the hypothalamus and in the
body. The febrile response is a defensive response involved in killing pathogens and
preventing the formation of bacterial protective coats and is mainly induced by PGE 2 .
The second reaction is the alteration in liver metabolism. Under homeostasis, hepatocytes
in the liver synthesize a range of acute-phase proteins (APP) at a relatively steady state;
however, during an inflammatory response, proinflammatory cytokines stimulate the
production and secretion of these proteins at a greater rate, whereas most of liver function
is dedicated to APP production (Carroll and Forsberg, 2007).
6
Acute-phase proteins are a group of blood proteins that change in concentration in
animals subjected to immune challenges (Murata et al., 2004). As previously mentioned,
APP are mainly synthesized and released by the hepatocytes and have several different
biological functions: protease inhibitors, tissue repair and remodeling, activation of
macrophages, enzymes, coagulation of proteins, metal-binding, and transport proteins for
products generated during the inflammatory process (Murata et al., 2004; Carroll and
Forsberg, 2007). The APP have been shown to be species-specific, which means that
some proteins that function as an APP in one species may not be a good indicator in
another species. In cattle, the most studied APP are ceruloplasmin, serum amyloid-A,
haptoglobin (Hp), and fibrinogen (Godson et al., 1995).
Bovine Hp is synthesized as a precursor polypeptide, which after oligomerization
and cleavage is split and then assembled to a tetramer of 2-α and 2-β chains of higher
polymerization, similar to Hp2-2 found in humans (Ametaj et al., 2011). The most
prominent function of Hp is to bind hemoglobin (Hb), thus preventing the binding of Fe
by bacteria inhibiting its growth, given that iron is required for bacterial metabolism.
Haptoglobin inhibits cyclooxygenase and lipooxygenase activities, enzymes catalyzing
reactions that may be harmful to body tissues (Saeed et al., 2007). In addition, Hp binds
high-density lipoprotein (HDL) apolipoprotein-A1 to promote adaptive immune response
and down-regulates the APR. Among all APP’s suggested as potential indicators of
inflammation in beef cattle, Hp seems to be the most reliable and consistent because it is
undetectable in healthy animals, whereas as increase is observed after disease, injury, or
stress responses (Carter et al., 2002; Petersen et al., 2004).
7
Ceruloplasmin is a copper-containing ferroxidase that oxidizes highly reactive
ferrous iron (Fe+2) to its non-toxic ferric iron form (Fe+3; Patel et al., 2002), allowing iron
binding to transferrin. Another function of ceruloplasmin is transport of copper in the
body (Eckerssal and Conner, 1988). As an APP, ceruloplasmin functions as a modulator
of the inflammatory response (Cousins, 1985) by creating an adaptive protection response
related to iron distribution in the host, thus limiting Fe for bacterial growth (Weinberg,
1984); working as a scavenger of reactive oxygen species secreted by phagocytes
(Eckerssal and Conner, 1988), and reducing the number of neutrophils attaching to the
endothelium. Ceruloplasmin concentrations are highly correlated with Cu status, and
during situations of Cu-deficiency, the circulating ceruloplasmin levels are reduced
(Mulhern and Koller, 1988) impairing the innate immune response. Arthington et al.
(1996) induced (Mo-supplemented) or not (control treatment) heifers to a Cu-depleting
period for 129 days, with a subsequent bovine herpesvirus-1 (BHV-1) inoculation to
induce an immune reaction. Heifers in the Mo-supplemented group (Cu-deficient) had
lower plasma Cu and subsequently lower plasma ceruloplasmin concentrations when
compared to control cohorts. According to The Merck Veterinary Manual (Merck, 2010),
the normal concentrations of ceruloplasmin in cattle range from 16.8 to 34.2 mg/dL. In
the study from Arthington et al. (1996), control animals had ceruloplasmin levels within
this range, whereas Mo-supplemented heifers had levels below the optimum range. These
results demonstrate that the mineral status, particularly Cu levels, have enormous impacts
in the ceruloplasmin concentrations.
The innate immune response has been shown to impact nutrient metabolism, by
shifting the partitioning of dietary nutrients away from skeletal muscle accretion, and
8
accelerating lipolysis and muscle protein degradation, resulting in an imbalance between
anabolic and catabolic processes that ultimately leads to impaired feed utilization and
animal growth (Johnson, 1997). In addition, some acute-phase proteins typically peak 3
days following a stress/disease stimuli, resulting in a decreased feed intake (Arthington et
al., 2008; Araujo et al., 2010; Cooke et al., 2011), and a subsequent impairment in animal
performance. Therefore, acute-phase proteins are negatively correlated to ADG after a
stressful event (Arthington et al., 2005; Qiu et al., 2007), and positively associated with
the incidence of morbidity and subsequent requirement for antimicrobial treatment
(Carter et al., 2002).
2. Stress and hypothalamic-pituitary-adrenal axis
Stress is known as the reaction of an animal to factors that potentially influence its
homeostasis, whereas animals that are unable to cope with these factors are classified as
stressed or distressed (Moberg, 2000). These factors (also known as stressors) elicit
physiologic responses within the body in an attempt to reestablish homeostasis, primarily
through the activation of hypothalamic-pituitary-adrenal (HPA) axis (Dobson and Smith,
2000). Stressors may be grouped in: 1) Psychological stress: such as commingling or
social mixing, novel environment, and restraint; 2) Physiological stress: such as
deviations in normal endocrine or neuroendocrine function caused by various conditions,
including human handling management procedures; and 3) Physical stress: such as
diseases, injury, fatigue, hunger, and dehydration (Carroll and Forsberg, 2007).
After a stress stimulus, CRH and vasopressin (VP) are secreted by their respective
neurons in the hypothalamus into the paraventricular nucleus (PVN; Carroll and
9
Forsberg, 2007). These hormones have been shown to be potent stimulators of
adrenocorticotropic hormone (ACTH) secretion from anterior pituitary gland, with CRH
being a more potent stimulator than VP in cattle (Carroll et al., 2007). Also,
administration of CRH has been related to inhibit processes related to maintenance of
life, such as feeding and reproduction. Upon the binding to their membrane receptors in
the anterior pituitary gland (de Souza et al., 1985), CRH activates protein kinase A,
which is coupled to adenylate cyclase to produce 3’,5’ cyclic AMP (cAMP). Stimulation
of corticotrophs with CRH causes a calcium influx of cytosolic origin, and it has been
speculated that the release of intracellular pools of calcium may be the main regulatory
signal that activates ACTH release in the pituitary gland (Link et al., 1993).
The pituitary gland, also known as hypophysis, is composed of three separate
endocrine organs (anterior lobe, intermediate lobe, and the posterior lobe). The anterior
pituitary gland is composed of five phenotypically distinct cell types: 1) Somatotrophs:
secretion of growth hormone (GH); 2) Mammotrophs: secretion of prolactin (PRL); 3)
Thyrotrophs: production of thyrotropin-stimulating hormone (TSH); 4) Gonadotrophs:
production of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and 5)
Corticotrophs: production of ACTH and related peptides. The primary action of ACTH is
to promote the uptake of cholesterol and subsequent synthesis and release of steroids by
the adrenal gland (Carroll and Forsberg, 2007).
The adrenal gland is divided into two regions: the adrenal cortex and the adrenal
medulla. The adrenal cortex is responsible for the synthesis and release of three major
classes of hormones: mineralocorticoids, androgens, and glucocorticoids. The primary
glucocorticoid in rodents is corticosterone, whereas in humans and most mammals is
10
cortisol (Carroll and Forsberg, 2007). Cortisol elicits several diverse biologic effects in
the body, including the breakdown of liver reserves (glycogen), muscle, and adipose
tissues to increase the availability of energy to the animal, APP production by the
hepatocytes, regulation of stress response, enhanced synthesis and secretion of
cathecolamines by the adrenal medulla, and suppression of the inflammatory and immune
systems to prevent autoimmune disorders (Munck et al., 1984). Hence, chronic exposure
to high concentrations of cortisol may result in severe physiologic problems, such as
immunosuppresion, excessive protein catabolism, reduced growth and reproduction rates,
and suppressed milk production (Friend, 1991). Similarly, inadequate supply of cortisol
may also lead to disorders such as fatigue, susceptibility to autoimmune disorders, and
inflammation (Chrousos and Gold, 1992).
Glucocorticoids also play a major role in regulating the balance between pro/antiinflammatory cytokines. Supporting this evidence, Blotta et al. (1997) observed that
glucocorticoid-treated macrophages produce significantly less proinflammatory cytokines
(IL-12 and TNF-α) resulting in an increased production of anti-inflammatory cytokines
(IL-4 and IL-10).
Several studies demonstrated that lipopolysaccharide (LPS) infusion may affect
the HPA axis, leading to an increase in cortisol concentrations and an acute-phase
reaction in cattle (Carroll et al., 2009). Lipopolysaccharide is a component of the cell
wall in gram-negative bacteria, that after binding to the TLR’s on the cells of the immune
system induces an immune reaction. However, this LPS-model for quantifying stress and
subsequent immune responses may not be as responsive to other stressful events, such as
transport, weaning, and feedlot entry, because these events are not associated with the
11
presence of a pathogen, and as mentioned earlier, the main cause of an immune reaction
is increased circulating cortisol (Crookshank, 1979; Arthington et al., 2003; Cooke et al.,
2011) and tissue mobilization. Therefore, in an effort to understand the mechanisms
underlying these effects, Cooke and Bohnert (2011) developed a stress-model using a
CRH challenge (non-pathogenic stress) in beef steers. These authors observed that after
CRH infusion at 0.1 µg/kg of body weight (BW), cortisol concentrations reached a peak
at 1 h, with a subsequent increase on plasma IL-6 and haptoglobin concentrations,
demonstrating that acute stress activates the innate immune system (Carroll and Forsberg,
2007), and a subsequent acute-phase response in healthy beef steers. In another study,
Cooke et al. (2012) demonstrated that the magnitude and duration of increasing
circulating cortisol concentrations alters the subsequent stress-induced immune reaction.
Increasing CRH-infusion dose from 0.1 to 0.5 µg of CRH/kg of BW did not result in
enhanced acute-phase reaction. These results may be attributed to the lack of increase on
proinflammatory cytokines and haptoglobin concentrations after the CRH infusion,
suggesting that prolonged CRH-induced increase in plasma cortisol did not stimulate
tissue degradation to activate the innate immune system, and was not sufficient to either
stimulate or suppress acute-phase and inflammatory reactions, respectively (Cooke et al.,
2012). Therefore, a balance on adrenal function must be reached in order to keep the
immune function in proper functioning without impairing the performance and health of
animals.
3. Thyroid gland
The thyroid gland secretes triiodothyronine (T 3 ) and thyroxine (T 4 ). The main
product synthesized by the thyroid gland, T 4 , is released upon hydrolysis of its precursor,
12
called
thyroglobulin
(Squires,
2003).
This
process
is
controlled
by
the
hypothalamus/anterior pituitary axis. First, the PVN of the hypothalamus produces the
thyrotropin-releasing hormone (TRH) that stimulates the release of TSH from the anterior
pituitary, which in turn, act on thyroid receptors to stimulate the synthesis and release of
T 4 and T 3 (Huszenicza et al., 2002). Thyroid-stimulating hormone interacts with
receptors in the thyroid follicular cells to activate adenylate cyclase to increase cAMP
production, resulting in increased synthesis and release of thyroid hormones. Thus,
thyroid hormones exert negative feedback on TSH secretion. In addition, TSH-response
following a TRH challenge is an extremely sensitive, safe, and reliable indicator of
thyroid function, being successfully used in dairy cattle (Laarveld et al., 1981). Thyroxine
is converted to the most biologically active form of thyroid hormone, T 3 , by the action of
the type I and II 5’-deiodinases in the thyroid gland and mainly in extrathyroidal tissues,
such as brain, liver, kidney, and skeletal muscle (Heuer and Visser, 2009). Both
hormones, T 4 and T 3 , are metabolized to the inactive diiodothyrosine (3,3’-T 2 ) by the
type III 5’-deiodinase enzyme (Squires, 2003), thus controlling local and systemic levels
of these hormones in the body (Gereben et al., 2008). Although all these thyroid
hormones are present in the circulation, the physiological effects of thyroid hormones,
such as control of metabolic rate, growth, and development, are attributed almost only to
T 3 (Flier et al., 2000).
Thyroid hormones are necessary for normal development, growth, differentiation,
and normal development of the central nervous system, but the most characteristic effect
of the thyroid hormones is control of basal metabolic rate (Paxton, 1986). Thrift et al.
(1999b) induced Brahman heifers to hypo- (HYPO) or hyperthyroidism (HYPER)
13
conditions and observed that HYPO heifers had reduced concentrations of T 4, T 3 , lower
rectal temperature (RT), greater BW, and body condition score (BCS) gains during the
treatment period (84 d). Hypothyroidism condition was accompanied by lethargy and a
palpable enlargement of the thyroid gland on d 84. Hypothyroidism is characterized by a
decreased rate of oxygen consumption per unit of body surface area and a decrease in
heat production (Loeb, 1996) resulting in a decline in basal metabolic rate. The lower RT,
increased BW, and BCS observed by Thrift et al. (1999b) are consequences of increased
energy availability associated with the reduced basal metabolic rate. I another study,
Thrift et al. (1999a) reported that HYPO cows had reduced milk production, luteal
progesterone (P 4 ) concentrations, and tended to have longer postpartum intervals and
longer intervals from calving to pregnancy. Bernal (1996) observed that cows treated
with propylthiouracil (PTU; thyroid gland antagonist) had a greater ratio of luteal:serum
P 4 , suggesting that P 4 is produced but not released into the circulation. Hypothyroidism
during early stages of development results in deficiencies in somatic, neural, and sexual
development (Squires, 2003). Moreover, inadequate thyroid hormone production leads to
high levels of TSH and hypertrophy of the thyroid gland, causing goiter (Ahlin et al.,
1994).
At physiological concentrations, thyroid hormones in conjunction with GH and
insulin stimulate protein synthesis and a reduction in N excretion. However, at excessive
concentrations (hyperthyroidism), thyroid hormones will increase glucose transport and
storage, glycogen and FA oxidation (decreasing adipose storage), and protein catabolism
(Paxton, 1986). Insulin increases the effects of T 3 , whereas glucagon is inhibitory
(Squires, 2003). Level of T 3 has been shown to decrease during fasting and increase
14
during refeeding. This happens due to changes in conversion of T 4 to T 3 , as well as
changes in TSH release from the pituitary (Squires, 2003).
4. Camelina meal
The production of biodiesel from oilseeds results in a large amount of byproducts
that may be useful supplements for livestock, and can be used in a wide variety of beef
cattle diets (Lardy, 2008), thus becoming a sustainable alternative for livestock
operations. The major oilseed crops that may be used for biodiesel production in the
Western of United States include mustard, soybeans, sunflowers, safflower, camelina,
and canola.
Camelina (Camelina sativa L.), a member of the Brassicaceae family, is usually
referred to as “false flax” or “gold of pleasure” (Fröhlich and Rice, 2005), because like
flax, its oil contains relatively high proportions of omega-3 fatty acids. Camelina used to
be an important oil crop during the Bronze and Iron Ages (Abramovič and Abram, 2005),
having desirable agronomical features including cold- and drought-tolerance (Lardy and
Anderson, 2009), low requirements for fertilizers, minimal needs of pesticide application
(Zubr, 1997), and an early maturation point (Fröhlich and Rice, 2005). During the
processing of camelina seeds for biodiesel production, two byproducts are obtained: the
camelina oil (glycerin) and the meal (Figure 2.3). Camelina meal contains approximately
13% fiber, 5% minerals (Bonjean and Le Goffic, 1999), 13% fat, 41% CP (Lardy, 2008),
and 10% oil with 28.5% 18:2n-6 and 41.3% 18:3n-3 (Hurtaud and Peyraud, 2007).
Linolenic acid (18:3n-3) is of a particular interest, due to its cardio protective effects,
including lowering cholesterol and blood pressure, and reducing the risk for coronary
15
heart disease (Nestel et al., 1997; Guallar and Jimenez, 1999). However, processing of
camelina seeds for biodiesel production is not a steady and well-defined activity,
resulting in variation between extractions that yields byproducts of different nutritional
composition (% oil, protein, fat, and fatty acid content). In addition, Abramovič and
Abram (2005) compared the fatty acid profile of camelina seeds from different locations
and cultivars, and observed that these two factors also affect the fatty acid composition in
the camelina oil (Table 2.1).
Camelina meal contains glucosinolates and 2 to 5% of erucic acid (22:1n-9;
Putnam et al., 1993), compounds that impair the activity of thyroid gland (Lardy and
Kerley, 1994) and promote myocardial lipidosis in rats (Kramer et al., 1990).
Additionally, camelina meal contains other anti-nutritional factors that may impair the
performance of animals, such as sinapines, saponins, and condensed tannins (Mawson et
al., 1993; Lardy, 2008).
Glucosinolates are a large group of sulphur-containing secondary plant
metabolites present in all the economically important varieties of Brassicaceae (rapeseed,
mustard, and camelina; Tripathi and Mishra, 2007). The ingestion of large amount of
glucosinolates may be deleterious to animal health and production, reducing feed intake
(Hill, 1991; Lardy and Kerley, 1994), stimulating iodine deficiency and hypertrophy of
thyroid gland (Burel et al., 2000), and increasing the mortality levels (CSWRI, 2002).
Glucosinolates are biologically inactive molecules, but upon ingestion, the derivative
products released (thiocyanate, vinyloxazolidinethiones, and isothiocyanate) are
biologically active and known for their diversified effects (Tripathi and Mishra, 2007).
These derivative products prevent the iodination of thyroid hormones resulting in inactive
16
hormones when released into the blood stream (Guyton, 1986). Ruminants species are
comparatively more tolerant to glucosinolates than monogastrics, but long-term, high
levels of low glucosinolate feeding have been shown to increase the levels of biologically
active products and depress T 4 in beef cattle (Vincent et al., 1988), as well as reducing
feed intake, milk production, and reproductive performance in dairy cows (Waldern,
1973; Laarveld et al., 1981; Ahlin, 1994). Lardy and Kerley (1994) supplemented
rapeseed meal to beef steers at different levels of inclusion in the diet, and observed that
steers fed higher levels of rapeseed meal had reduced DM and CP intake, seemed to
avoid consuming the rapeseed meal-containing supplements, and experienced decreased
serum T 4 concentrations. In addition, even when these authors treated the rapeseed meal
with ammonia, reducing the glucosinolate levels up to 75% (from 118 to 28 µmol/g),
there was no enhancement in terms of palatability or acceptability by steers compared to
those supplemented with soybean meal. These results may be explained by the fact that
the treatment of rapeseed meal yielded compounds with similar flavors and odors as
before the treatment (Fenwick et al., 1983) without an increase in consumption.
However, in a recent study, Moriel et al. (2011) supplemented camelina meal
(0.97 kg.heifer-1.d-1) for a 60-d period to beef heifers and reported no difference in DMI
or growth and reproductive performance between treatments. In addition, heifers
supplemented with camelina meal had greater T 3 concentrations compared to heifers
offered glycerin and control (corn/soybean meal) supplements. The differences in results
reported by Moriel et al. (2011) and Lardy and Kerley (1994) could be explained by the
lower glucosinolate content in camelina meal when compared with other plants from the
Brassicaceae family (22 µmol/g; Lange et al., 1995).
17
Erucic acid (22:1n-9) is a 22-carbon monounsaturated fatty acid with a single
double bond at the omega 9 position, and is found in the seeds of Brassicaceae, including
rapeseed, mustard, and camelina. It is digested, absorbed, and metabolized, for the most
part, like other fatty acids (Food Standards Australia New Zealand, 2003). The
Brassicaceae plants should have erucic acid content lower than 2% in order to prevent
any problems to human health. Two health concerns are associated with erucic acid:
Firstly, there is association between dietary erucic acid and myocardial lipidosis in nonruminants (Charlton et al., 1975; Kramer et al., 1990). Myocardial lipidosis reduces the
contractile force of heart muscle, and can be explained by the fact that erucic acid may
inhibit the mitochondrial oxidation of other fatty acids causing the accumulation of
triglyceride and cholesterol esters (Ray et al., 1979). Kramer et al. (1992) fed rats diets
with different proportions of erucic acid (2.5% or 9% of 22:1n-9 in oil) and reported that
the higher concentration of 22:1n-9 increased myocardial lipidosis when compared to the
lower level. Secondly, long-term (150 d) erucic acid intake is being associated with
higher levels of collagen, leading to heart lesions in rats (Ray et al., 1979). In addition,
Charlton et al. (1975) reported that rats offered diets with 20% rapeseed oil containing
two levels of erucic acid (4.3% and 22.3%) had a decreased amount of lipid in the
myocardium on d 28 and 112 after the beginning of supplementation, respectively. Even
though the lipid droplets were disappearing over time, animals with myocardial necrosis
and fibrosis were detected. Results analyzing the incidence of these diseases must be
conducted for each species separately. For example, pigs seem to have greater incidence
of myocardial lipidosis when 22:1n-9 levels exceed 5% of diet (Kramer et al., 1990),
18
whereas rats do not tolerate dietary levels higher than 3% of 22:1n-9 (Kramer et al.,
1979).
Besides these negative effects associated with camelina meal consumption, on
August 2010, the Food and Drug Administration (FDA) Division of Animal Feeds
regulated that camelina meal is allowed in feedlot diets at no more than 10% of the diet
(DM basis; Taasevigen, 2010).
5. Fat supplementation
The main reason for supplementing ruminant diets with fat is to increase the
energy density of the animal’s diet. Fats are essential components of every living cell,
having important functions for the integrity of bilipid structures of cell membranes, signal
transduction, sources of energy, and precursors for numerous biologically active
compounds (Squires, 2003). In addition, fat supplementation is a means to alter the fatty
acid composition of food products derived from ruminant animals (Hess et al., 2008),
preventing the occurrence of several diseases in humans. As an example, studies
conducted with poultry demonstrated that supplementation with sources rich in n-3 fatty
acids increase the content of these fatty acids in egg yolk (Juneja, 1997; Rokka et al.,
2002).
Polyunsaturated fatty acids (PUFA) are fatty acids that contain two or more
double bonds-chain of carbon atoms, and may be classified as n-3 or n-6 according to the
number and position of the first double bond in the fatty acid chain. Omega-3 (linolenic
acid) and omega-6 (linoleic acid) fatty acids are known as Essential Fatty Acids (EFA),
meaning that they cannot be synthesized by the body of mammals and need to be
19
obtained through the diet (Schmitz and Ecker, 2008). Linoleic acid is found in vegetable
oils such as corn and soybean meal, whereas linolenic acid is found in fish oils and
grasses. These EFA have important functions in several systems of our body, including
the reproductive (Funston, 2004; Hess et al., 2008) and immune (Calder, 2002) systems.
Although very important, fat supplementation must be adopted with caution. The
excessive and/or incorrect use of fat supplementation may have detrimental effects to
beef cattle, including alterations in the ruminal function and intake pattern, resulting in
reduced DMI, ADG, and subsequent performance. In a recent review, Hess et al. (2008)
suggested optimal levels of lipid inclusion in beef cattle diets. If the goal is to maximize
the use of forage-based diets, the lipid inclusion should be at less than 3% of dietary DM,
whereas it should be limited to 2% of DM or less if the goal is to prevent substitution
effects on forage consumption.
Several factors may be associated with reduced DMI and subsequent performance
following fat supplementation. Grummer et al. (1990) observed that when feeding fat to
cattle, an adaptation period and mixing with other ingredients (such as corn) may
improve the acceptability and consumption of the supplement by animals when compared
to fat sources offered alone. Other risk factors associated with fat supplementation are the
reduction in forage digestibility (Allen, 2000). These ruminal effects may include
modification of ruminal microbial population, physical coating of the fiber that prevents
microbial interaction, and inhibition of microbial activity in the forage cell membrane
(Devendra and Lewis, 1974). Brokaw et al. (2002) demonstrated that total tract OM
digestibility decreased when soybean oil (2.25% of dietary DM) was supplemented to
ruminants consuming high-forage diets. Free FA available in feedstuffs is highly
20
susceptible to ruminal biohydrogenation and can impair ruminal digestibility parameters
(Harfoot and Hazlewood, 1988). However, Cooke et al. (2011) offered a commercial
rumen-protected PUFA source at 3.6% of dietary DM and reported a reduction in DMI
but no difference in ruminal degradability parameters of forage, suggesting that other
factors such as reduced gut motility and enhanced cholecystokinin release may be related
to the reduction in performance when PUFA is fed (Drackley et al., 1992). It is important
to note that rumen-protected PUFA sources have a less degree of biohydrogenation when
compared to nonprotected sources, yielding less free FA for biohydrogenation and
leading to differences in results related to ruminal digestibility parameters. Additionally,
forage intake and diet digestibility were not affected in steers offered switchgrass hay and
canola seeds that provided approximately 4% of dietary DM as crude fat (Leupp et al.,
2006).
When
ruminants
consume
large
amounts
of
fat,
which
cannot
be
digested/absorbed, satiety signals may be generated (Palmquist, 1994). Satiety hormones,
including cholecystokinin (CCK) and pancreatic polypeptide, are integrated and
processed in the brain. Cholecystokinin is a peptide hormone produced by the duodenal
mucosa, existing in a number of forms, from a 33-amino-acid polypeptide in the
gastrointestinal tract to the 8-amino-acid (CCK-8) that is found in the central nervous
system. Both forms act as an important satiety signal to reduce feed intake (Squires,
2003). Immunizing pigs, but not sheep, against CCK-8 increased growth rate by about
10% and feed intake by 8%, with no changes in carcass composition (Squires, 2003).
Additionally, decreased production of CCK has been found in humans with bulimia,
whereas increased levels have been implicated with age-related anorexia (Squires, 2003).
21
Choi and Palmquist (1996) fed cows increasing amounts (0, 30, 60, and 90 g/kg of DM)
of a commercial calcium salt of fatty acid (CSFA, and observed that DMI, CP, ADF, and
NDF intakes decreased linearly in animals offered higher amounts of CSFA. Cows
supplemented with CSFA had greater CCK concentrations compared to control cohorts.
In addition, a linear increase in plasma CCK concentrations was correlated with
decreased DMI in response to increasing amounts of CSFA in the diet. These results
suggest that CCK is a potent satiety hormone and may be a factor mediating fat-induced
depression of feed intake in cattle (Choi and Palmquist, 1996). Other studies (Relling and
Reynolds, 2007; Bradford et al., 2008) have reported diets rich in PUFA appear to
influence CCK concentrations to a greater extent compared to diets rich in saturated fatty
acids (SFA).
5.1. Ruminal biohydrogenation
Concentrations of PUFA in the blood, meat, and milk are highly affected by
PUFA metabolism in the rumen (Jenkins et al., 2008) in a process known as
biohydrogenation that can greatly change the profile of FA absorbed and incorporated
into the tissues. Biohydrogenation is a process in which unsaturated fatty acids (UFA) are
converted to SFA through isomerization to trans FA intermediates accompanied by
hydrogenation of the double bonds (Harfoot and Hazlewood, 1988). After entering the
rumen, lipids are hydrolyzed by microbial lipases releasing free FA (Garton et al., 1961;
Dawson et al., 1977), whereas biohydrogenation subsequently occurs by action of
ruminal microbes (Jenkins et al., 2008). However, the rates of hydrolysis and
biohydrogenation are dependent on the type and amount of fat reaching the rumen (Beam
et al., 2000) as well as ruminal pH (Van Nevel and Demeyer, 1996). Unsaturated fatty
22
acids have more potent antimicrobial properties and promote greater inhibition of ruminal
fermentation than do SFA, because of its greater solubility and the permanent damage
caused in the cell membrane of gram positive bacteria (Jenkins, 1993).
The end product of complete biohydrogenation of dietary 18-carbon unsaturated
fatty acids is stearic acid (18:0), whereas incomplete biohydrogenation leads to an
increase in duodenal flow of unsaturated intermediate fatty acids (Kucuk et al., 2004).
Complete biohydrogenation of linoleic acid yields as an end product stearic acid, whereas
only 2 intermediates have been reported: cis-9, trans-11-conjugated linoleic acid (CLA)
and trans-11-18:1 (Garton, 1977). Recent studies have shown that this pathway is
consistent, identifying several isomers of these intermediates in digesta of cattle (Jenkins
et al., 2008). The CLA isomer is considered a health-promoting FA contributing to cancer
prevention, reduced body fat accretion, decreased atherosclerosis, improved immune
response (Whigam et al., 2000; Palmquist et al., 2005), and contributes to 80 to 90% of
the total CLA in milk fat (Parodi, 1977) and meat (Chin et al., 1992). The other
intermediate (trans-11-18:1) serves as precursor for endogenous synthesis of cis-9, trans11-CLA via Δ9-desaturase in the small intestine (Archibeque et al., 2005) and other
tissues (Griinari et al., 2000). Scholljegerdes et al. (2004) reported that ruminal
biohydrogenation of C18 UFA in heifers fed high-linoleate safflower seeds was higher
compared with heifers fed high-oleate or low-fat supplements. Also, linoleic acid can be
desaturated and elongated to form arachidonic acid (20:4n-6), which is a precursor of 2series prostaglandins. Moriel et al. (2011) observed that heifers supplemented with
camelina meal for a 60-d period had greater plasma concentrations of stearic, trans-11-
23
18:1, linoleic, arachidonic, and linolenic acids than heifers fed control and glycerol
supplements.
It is important to state that the amount and type of intermediates formed in the
rumen is variable according to the diet (concentrate vs. roughage) and the ruminal
bacterial population. Griinari and Bauman (1999) proposed that other intermediate, trans10, cis-12-CLA, may also be generated from linoleic acid, by bacteria strains frequently
present when high-concentrate diets are offered. This CLA isomer is known for its
positive effects on the immune function, enhancing the immune system, reducing the
catabolic effects of immune stimulation, and reduced production of pro-inflammatory
eicosanoids.
Much less is known about the biohydrogenation of linolenic acid. It is reported
that its biohydrogenation may lead to formation of a conjugated form of 18:2n-3 FA and
trans-11-18:1 acid as intermediates, and stearic acid as an end product. However, it has
not been reported the formation of any CLA isomers (Jenkins et al., 2008). In addition,
linolenic acid can be desaturated and elongated to form eicosapentaenoic (EPA; 20:5n-3)
and docosahexaenoic (DHA; 22:6n-3) acids, which are the precursors for the 3-series
prostaglandins (Staples et al., 1998; Yaqoob and Calder, 2007).
5.2. PUFA and immune system
The metabolism of linoleic and linolenic acids have different properties regarding
the immune function, competing for the same series of enzymes (i.e. cyclooxygenase-2)
and yielding very different compounds (eicosanoids), which may alter the immune
response within the body. Therefore, an excess of one type of EFA may cause a
24
significant decrease in the activity of the other (Schmitz and Ecker, 2008). Hence, the
total amount of fatty acids available and the n-6/n-3 ratio affects the formation of the
long-chain PUFAs. The enzymes have higher affinity for n-3 FA, and efficient
conversion of linolenic acid to long-chain n-3 PUFA can occur at a 4:1 ratio of
linoleic:linolenic acids in the diet (Squires, 2003).
The mechanisms involved in the modulation of the immune system by PUFA are
not yet understood, but evidences suggest that the reasons include modulation of
eicosanoid synthesis and cytokine profiles (Miles and Calder, 1998), and activation of the
transcription factor peroxisome proliferators-activated receptors (PPAR).
Cell membranes contain mainly arachidonic acid (AA) compared to omega-3 FA,
and this greater amount reflects in the class of eicosanoids produced. Arachidonic acid
may be converted to the prostaglandin-2 (PG 2 ), thromboxane-2 (TXB 2 ), and leukotriene4-series (LT 4 ) by the action of cyclooxygenases (COX) and lipoxygenases (LOX). The
eicosanoids produced in this case are pro-inflammatory (Yaqoob and Calder, 2007). For
example, prostaglandins (PGE 2 ) generated from AA have been shown to have proarrhythmic effects, potent stimulator of the APR and other inflammatory responses
(Schmitz and Ecker, 2008), including fever (Carroll and Forsberg, 2007). In addition,
leukotriene (LTB 4 ) has been shown to increase the production of inflammatory cytokines
such as TNF-α, IL-1, and IL-6 (Schmitz and Ecker, 2008).
Unlike omega-6 FA-derived, eicosanoids derived from omega-3 FA present antiinflammatory properties. Linolenic acid serves as precursor for its omega-3 derivatives,
EPA and DHA, which promote the synthesis of eicosanoids that do not stimulate the
25
acute-phase protein response, such as prostaglandin-3-series (PGE 3 ), thromboxane-3
(TXB 3 ), and leukotriene-B5 (LTB 5 ). Previous studies (Araujo et al., 2010; Cooke et al.,
2011) demonstrated that supplementation with rumen-protected PUFA prior to stressful
events, such as 24-hour transportation and feedlot entry, alleviated the acute-phase
protein response in beef cattle. The mechanisms underlying this response may involve a
shift in the production of pro-inflammatory (IL-1, IL-6, and TNF-α) to anti-inflammatory
(IL-4 and IL-10) cytokines, leading to a reduced acute-phase protein production (Cooke
et al., 2011). As the levels of EPA and DHA increase, the AA levels decrease in cell
membranes of platelets, erythrocytes, monocytes, neutrophils, and liver cells. It
subsequently reduces the levels of eicosanoids produced from AA that enhance a proinflammatory response and allergic reactions (Squires, 2003). Silvestre (2009) observed a
reduced n-6/n-3 FA ratio in neutrophils of dairy cows fed calcium salts (CS) of fish oil
compared with those fed CS of palm oil. In addition, mean concentration of TNF-α in
supernatants of isolated neutrophils were lower for cows fed CS of fish oil. Further, the
concentration of EPA and DHA in neutrophils was greater in cows fed CS of fish oil than
cows fed CS of palm oil. Farran et al. (2008), in an effort to evaluate the effects of FA on
immune responses, fed tallow, rolled full-fat soybeans, and flaxseed to beef cattle. The
authors observed that feeding flaxseed increased plasma concentrations of n-3 FA,
reduced rectal temperature, and plasma TNF-α concentration after a LPS challenge.
Peroxisome proliferator-activated receptors are found in inflammatory cells, and
appear to be regulated by PUFA and eicosanoids (Calder, 2002). Wang et al. (2001)
demonstrated that the activation of the PPAR may have anti-inflammatory actions, by
inhibiting the activation of inflammatory genes, including TNF-α, IL-1, IL-6, and COX.
26
Two mechanisms for the anti-inflammatory actions of PPAR have been proposed: 1)
stimulate the breakdown of inflammatory eicosanoids through induction of peroxisomal
β-oxidation, and 2) interfere with or antagonize the activation of other transcription
factors, including NFĸB (Calder, 2002). The mechanisms by which omega-3 FA affects
PPAR activities are not clear, but it may act by increasing the level of PPAR transcription
factors in inflammatory cells and inhibiting the activity of the inflammatory genes
mentioned previously.
In summary, the appropriate balance of n-6/n-3 ratio in the diet is important to
modulate an immune reaction following stress of pathogenic and neuroendocrine
challenges. Moreover, the requirements of beef cattle for these EFA are still unknown,
and this approach is needed to develop nutritional strategies in order to reduce or alleviate
the immune responses, leading to an enhancement on animal well-being and performance
(Cooke et al., 2011).
6. Development of strategies to alleviate the acute-phase response in beef cattle
Beef cattle production is the second largest commodity grown in the state of
Oregon, and the developments of strategies to enhance the profitability of these systems
are required. Newly weaned steers go through several stressful situations at the same time
or in a short period of time, including feedlot entry, commingling, and vaccination. These
stressful procedures result in an immune reaction, subsequently activating the immune
system.
When the immune system is challenged, several responses are created,
characterizing an inflammatory response. One of these responses is the activation of the
27
APR, which results decreases nutrient utilization and feed intake, and changes the
behavior and physiology. All these reactions impact the subsequent animal performance
and the profitability of the beef production system. Strategies have been developed in
order
to
alleviate
and/or
prevent
these
stress-induced
responses,
including
supplementation with PUFA. However, the exact mechanisms by which these stressful
events exert their actions are not totally understood.
Therefore, to address these mechanisms and possible to develop strategies to
alleviate and/or prevent these negative reactions, three experiments were conducted to
evaluate the effects of camelina meal supplementation on ruminal forage degradability,
animal performance, and physiological responses of beef cattle. Results from these
experiments are reported and discussed in the following chapter.
28
Table 2.1. Fatty acid content in Camelina sativa oil from different locations and cultivars
(Adapted from Abramovič and Abram, 2005).
Content of fatty acid, %
Fatty acid
Study 11
Palmitic acid (16:0)
Study 22
Study 33
Study 44
6.43
7.05
5.50
5.45
17.40
16.80
14.90
15.50
Stearic acid (18:0)
2.57
2.45
2.30
2.50
Linoleic acid (18:2)
16.90
21.50
15.80
15.00
α-Linolenic acid (18:3)
35.20
30.90
38.90
37.30
Oleic acid (18:1)
Erucic acid (22:1)
1.62
2.00
2.40
2.80
2
3
Abramovič and Abram (2005); Budin et al. (1995); Eidhin et al. (2003); 4Zubr
1
et al. (2002).
29
Figure 2.1. Effects of proinflammatory cytokines on the HPA axis, body tissues, and
organs (Adapted from Johnson, 1997).
30
Figure 2.2. Acute-phase reaction cascade event activated by macrophages, which results
in the production of acute-phase proteins (Adapted from Carroll and Forsberg, 2007). IL1 = interleukin-1; TNF-α = tumor necrosis factor-α; IL-6 = interleukin-6.
31
Figure 2.3. Process of biodiesel production (Adapted from Kerr et al., 2007).
32
CHAPTER 3
Effects of camelina meal supplementation on ruminal forage degradability,
performance, and physiological responses of beef cattle
ABSTRACT. Three experiments compared ruminal, physiological, and
performance responses of beef steers consuming hay ad libitum and receiving grainbased supplements without (CO) or with (CAM) the inclusion of camelina meal. In Exp.
1, 9 steers fitted with ruminal cannulas received CAM (2.04 kg of DM/d; n = 5) or CO
(2.20 kg of DM/d; n = 4). Steers receiving CAM had reduced (P = 0.01) total DMI and
tended to have reduced (P = 0.10) forage DMI compared to CO. No treatment effects
were detected (P ≥ 0.35) for ruminal hay degradability parameters. In Exp. 2, 14 steers
receiving CAM (1.52 kg of DM/d; n = 7) or CO (1.65 kg of DM/d; n = 7) were assigned
to a corticotropin-releasing hormone (CRH; 0.1 µg/kg of BW) and a thyrotropinreleasing hormone (TRH; 0.33 µg/kg of BW) challenge. Steers receiving CAM had
greater (P < 0.05) serum concentrations of PUFA compared to CO prior to challenges.
Upon CRH infusion, mean plasma haptoglobin concentrations tended (P = 0.10) to be
reduced and ceruloplasmin concentrations increased at a lesser rate in CAM compared
with CO (treatment x time; P < 0.01). Upon TRH infusion, no treatment effects were
detected (P ≥ 0.55) for serum thyrotropin-stimulating hormone, triiodothyronine, and
thyroxine. In Exp. 3, 60 steers were allocated to 20 drylot pens. Pens were randomly
assigned to receive CAM (2.04 kg of DM/steer daily; n = 10) or CO (2.20 kg of DM/steer
daily; n = 10) during preconditioning (PC; d -28 to 0). On the morning of d 0, steers were
transported for 24 h. Upon arrival, pens were randomly assigned to receive, in a 2 x 2
factorial arrangement, CAM or CO during feedlot receiving (FR; d 1 to 29). During PC,
33
CAM had reduced (P < 0.01) forage and total DMI, and tended to have reduced (P =
0.10) ADG compared to CO. Plasma linolenic acid concentrations increased during PC
for CAM, but not for CO (treatment x day; P = 0.02). During FR, steers that received
CAM during PC had reduced (P < 0.01) forage and total DMI, but tended (P = 0.10) to
have greater G:F compared to CO. Steers that received CAM during FR had greater (P <
0.05) mean plasma concentrations of PUFA, and reduced mean rectal temperature and
concentrations of haptoglobin and ceruloplasmin during FR compared to CO.
In
summary, camelina supplementation to beef steers impaired forage and total DMI, did
not alter thyroid gland function, increased circulating concentrations of PUFA, and
lessened the acute-phase protein reaction elicited by neuroendocrine stress responses.
Key Word: Beef cattle, camelina meal, inflammation, performance, thyroid gland,
transport
MATERIALS AND METHODS
All experiments were conducted at the Oregon State University – Eastern Oregon
Agricultural Research Center, Burns. All animals utilized were cared for in accordance
with acceptable practices and experimental protocols reviewed and approved by the
Oregon State University, Institutional Animal Care and Use Committee. Experiments 1
and 2 were conducted from August to November 2010. Experiment 3 was conducted
from September to November 2010, and divided into a preconditioning (PC; d -28 to 0)
and a feedlot receiving (FR; d 1 to 29) phase. Supplement ingredients provided during
Exp. 1, 2, and 3 were originated from the same batch. Similarly, hay provided during
Exp. 1, 2, and 3 was harvested from the same field during June 2010. All steers were
34
administered Clostrishield 7 and Virashield 6 + Somnus (Novartis Animal Health;
Bucyrus, KS) at approximately 30 d of age, and One Shot Ultra 7, Bovi-Shield Gold 5,
TSV-2 and Dectomax (Pfizer Animal Health; New York, NY) at weaning.
Experiment 1.
Animals.
Nine Angus x Hereford steers (initial BW 250 ± 9 kg; initial age 499 ± 5 d), housed
in individual drylot pens (11 x 21 m) and fitted with ruminal cannulas, were assigned on
d 0 of the study to receive a grain-based supplement with (CAM; n = 5) or without (CO;
n = 4) the inclusion of camelina meal (Table 3.1) for 21 d.
Diets.
Treatments were fed daily (0700 h) at a rate of 2.20 and 2.04 kg of DM per steer for
CO and CAM, respectively. Treatment intakes (DM basis) were formulated to be isocaloric, iso-nitrogenous, and corresponded to 0.89 and 0.80 % of initial shrunk BW for
CO and CAM supplemented steers, respectively. Mixed alfalfa-grass hay was offered
throughout the experiment (d 0 to 21) in amounts to ensure ad libitum consumption.
Treatments and hay were not mixed, and treatments were inserted directly into the rumen
of each steer via fistula to ensure complete treatment consumption. Samples of hay and
treatment ingredients were analyzed for nutrient content by a commercial laboratory
(Dairy One Forage Laboratory, Ithaca, NY). All samples were analyzed by wet chemistry
procedures for concentrations of ether extract (method 2003.05; AOAC 2006), CP
(method 984.13; AOAC 2006), ADF (method 973.18 modified for use in an Ankom 200
fiber analyzer, Ankom Technology Corp., Fairport, NY; AOAC 2006), and NDF (Van
Soest et al., 1991; method for use in an Ankom 200 fiber analyzer, Ankom Technology
35
Corp.). Calculations of TDN used the equation proposed by Weiss et al. (1992), whereas
NE m and NE g were calculated with the equations proposed by the NRC (1996). Further,
hay and supplement samples were analyzed for FA content using gas chromatography
(Model HP 6890, Hewlett-Packard Co., Wilmington, DE) according to the procedures
described by Folch et al. (1957) and Hayat et al. (2009). Composition and nutritional
profile of treatments are described in Tables 3.1 and 3.2, and were calculated from
samples collected at the beginning of the study. Hay quality was calculated from samples
collected weekly, and estimated at (DM basis) 54 % TDN, 60 % NDF, 40 % ADF, 1.15
Mcal/kg of NE m , 0.59 Mcal/kg of NE g , 12.8 % CP, and 2.1 % ether extract. Hay FA
profile is described in Table 3.2. A complete commercial mineral/vitamin mix
(Cattleman’s Choice; Performix Nutrition Systems, Nampa, ID) containing 14 % Ca, 10
% P, 16 % NaCl, 1.5 % Mg, 6000 ppm Zn, 3200 ppm Cu, 65 ppm I, 900 ppm Mn, 140
ppm Se, 136 IU/g of vitamin A, 13 IU/g of vitamin D 3 , and 0.05 IU/g of vitamin E was
included in the diet (Table 3.1), and water was available for ad libitum consumption
throughout the experiment.
Sampling.
Steer shrunk BW was recorded 7 d before the beginning of the experiment to
calculate treatment intake. Voluntary forage intake was evaluated daily from d 1 to 15,
whereas intake data from d 8 to 15 were used for treatment comparison. From d 1 to 15,
hay was offered in amounts to ensure ad libitum intake, and orts were collected and
weighed daily. Samples of the offered and the non-consumed hay were collected daily
from each pen and dried for 96 h at 50°C in forced-air ovens for DM calculation. Initial
shrunk BW was utilized for calculation of DMI as % of BW. From d 16 to d 19, steers
36
were restricted to receive 90 % of their voluntary forage DMI determined from d 1 to 15.
Immediately before treatment feeding on d 16, Dacron bags (nitrogen-free polyester; 10
cm x 20 cm; 50 ± 10 µm pore size; Ankom Technology Corp., Fairport, NY) containing 4
g (DM basis) of ground dietary hay (2-mm screen; Wiley Mill, Model 4; Arthur H.
Thomas, Philadelphia, PA) were suspended in the ventral rumen of each steer, and
incubated in triplicate for 0, 1, 3, 5, 8, 12, 24, 36, 48, 72, and 96 h. Prior to ruminal
incubation, all bags were soaked in warm water (39°C) for 15 min. After ruminal
incubation, bags were washed repeatedly with running water until the rinse water was
colorless, and subsequently dried for 96 h at 50°C in forced-air ovens. The 0-h bags were
not incubated in the rumen but were subjected to the same soaking, rising, and drying
procedures applied to the ruminally incubated bags. Dried samples were weighed for
residual DM determination, and then triplicates were combined and analyzed for NDF
(Robertson and Van Soest, 1981) using procedures modified for use in an Ankom 200
Fiber Analyzer (Ankom Technology Corp.). After removal of the last Dacron bags on d
19, steers were allowed to consume hay ad libitum until the end of the study. On d 21,
blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 9, and 12 h relative to treatment
feeding (0 h), for determination of plasma cholecystokinin (CCK) concentrations.
Blood Analysis.
Blood samples were collected via jugular venipuncture into commercial blood
collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing
sodium heparin, and were placed on ice immediately. Samples were centrifuged at 2,500
x g for 30 min for plasma harvest and stored at -80°C on the same day of collection.
Plasma concentrations of CCK were determined using a bovine-specific commercial
37
ELISA kit (KT-10170; Kamyia Biomedical Company, Seattle, WA). The intraassay CV
was 4.2 %.
Statistical analysis.
All data were analyzed using steer as the experimental unit. Forage and total DMI,
and plasma CCK concentrations, were analyzed using the PROC MIXED procedure of
SAS (SAS Inst., Inc., Cary, NC) and Satterthwaite approximation to determine the
denominator df for the tests of fixed effects. The model statements contained the effects
of treatment, day for DMI or time for CCK, and the resultant interaction. Data were
analyzed using steer(treatment) as the random variable. The specified term for the
repeated statement was day or time (for DMI or CCK, respectively), the subject was
steer(treatment), and the covariance structure utilized was autoregressive, which provided
the best fit for these analyses according to the Akaike information criterion. Kinetic
parameters of hay DM and NDF disappearance were estimated using nonlinear regression
procedures of SAS, as described by Vendramini et al. (2008). Treatment effects on
ruminal in situ forage degradability parameters were also analyzed using the PROC
MIXED procedure of SAS and Satterthwaite approximation. The model statement
contained the effect of treatment. Data were analyzed using steer(treatment) as random
variable. Results are reported as least square means and were separated using LSD.
Significance was set at P ≤ 0.05, and tendencies were determined if P > 0.05 and ≤ 0.10.
Results are reported according to treatment effects if no interactions were significant, or
according to the highest-order interaction detected.
Experiment 2.
Animals.
38
Fourteen weaned Angus steers (initial BW 191 ± 2 kg; initial age 178 ± 4 d) were
utilized in the study (d 0 to 36). All steers were weaned 30 d before the beginning of the
study and exposed daily to halter-training techniques to become acclimated to human
interaction; thus, minimizing confounding effects between human handling, weaning, and
hormones challenges measured herein (Arthington et al., 2005; Curley et al., 2008; Cooke
and Bohnert, 2011). Beginning 5 d prior to and during the study, steers were housed in
individual pens (2 × 5 m) contained within an enclosed barn. On d 0, steers were ranked
by initial BW and assigned to receive CO or CAM (7 steers/treatment).
Diets.
Treatments were fed daily (0800 h) at a rate of 1.65 and 1.52 kg of DM per steer for
CO and CAM, respectively. Treatment intakes (DM basis) were formulated to be isocaloric, iso-nitrogenous, and corresponded to 0.96 and 0.88 % of initial shrunk BW for
CO and CAM, respectively. Mixed alfalfa-grass hay was offered throughout the
experiment (d 0 to 36) in amounts to ensure ad libitum consumption. Treatments and hay
were not mixed, and treatments were readily consumed by steers. Samples of hay and
treatment ingredients were analyzed for nutrient content as in Exp. 1 (Tables 3.1 and 3.2).
Water was offered for ad libitum consumption throughout experiment.
Sampling.
From d 21 to 23 and d 31 to 33, steers were weighed daily. On d 24 and 34 of the
study, all steers were fitted with an indwelling jugular vein catheter for serial blood
collection according to the procedures described by Merrill et al. (2007), and an
indwelling RT monitoring device (d 24 only) as described by Reuter et al. (2010). On d
25, all steers received 0.1 µg of bovine corticotropin-releasing hormone (CRH)/kg of BW
39
to stimulate and evaluate treatment effects on a stress-induced acute-phase protein
response (Cooke and Bohnert, 2011). On d 35, all steers received 0.33 µg of bovine
thyrotropin-releasing hormone (TRH)/kg of BW to stimulate and evaluate treatment
effects on thyroid function (Wichtel et al., 1996). Bovine CRH (#34-3-11; American
Peptide Co., Inc., Sunnyvale, CA) and TRH (#52-0-80; American Peptide Co., Inc.) were
dissolved into 10 mL of physiological saline (0.9 %) immediately before challenge and
administered to steers via indwelling jugular catheters. Catheters were subsequently
flushed with 10 mL of physiological saline to ensure that the hormones reached the
circulation, followed by 5 mL of heparinized saline (20 IU of heparin/mL of saline) to
maintain catheter patency.
After CRH infusion (h 0; d 25), blood samples were collected hourly from -2 to 0 h
and 4 to 8 h, and every 30 min from 0 to 4 h via jugular catheters. Rectal temperatures
were recorded by the indwelling device (Reuter et al., 2010) every 30 min from −2 to 8 h
relative to infusion. Immediately after the collection at 8 h relative to infusion, catheters
and rectal probes were removed. Subsequent blood samples were collected via jugular
venipuncture and RT were assessed using a digital thermometer (GLA M750 digital
thermometer; GLA Agricultural Electronics, San Luis Obispo, CA) every 6 h from 12 to
72 h, and every 24 h from 96 to 168 h relative to treatment infusion. All samples were
analyzed for plasma concentrations of ceruloplasmin and haptoglobin. Samples collected
from -2 to 8 h were also analyzed for plasma concentrations of cortisol. Samples
collected at -2 h were analyzed for plasma FA content.
After TRH infusion (h 0; d 35), blood samples were collected hourly from -2 to 0 h
and 4 to 8 h, and every 30 min from 0 to 4 h via jugular catheters. Immediately after the
40
collection at 8 h relative to infusion, catheters were removed. Subsequent blood samples
were collected via jugular venipuncture every 4 h from 12 to 24 h relative to treatment
infusion. All samples were analyzed for serum concentrations of thyrotropin-stimulating
hormone (TSH), triiodothyronine (T 3 ), and thyroxine (T 4 ). Samples collected at -2 h
were also analyzed for plasma FA content.
Blood Analysis.
Blood samples that were collected via jugular catheters were immediately
transferred into commercial blood collection tubes (Vacutainer, 10 mL; Becton
Dickinson, Franklin Lakes, NJ) that did not contain additives for serum collection, or
contained sodium heparin for plasma collection. Samples were placed immediately on
ice, centrifuged within 30 min after collection at 2,500 × g for 30 min at 4ºC, and plasma
and serum were immediately stored at -80ºC. Blood samples obtained via jugular
venipuncture were collected and harvested for plasma serum as in Exp. 1.
Plasma concentrations of cortisol were determined using a bovine-specific
commercial ELISA kit (Endocrine Technologies Inc., Newark, CA). Plasma
concentrations of ceruloplasmin and haptoglobin were determined according to
procedures previously described (Demetriou et al., 1974; Makimura and Suzuki, 1982).
Serum concentrations of TSH, T 3 , and T 4 were determined using a bovine-specific
commercial ELISA kit (Endocrine Technologies Inc., Newark, CA, USA). The intra- and
interassay CV were, respectively, 9.5 and 8.4 % for T 3 , 11.2 and 8.1 % for T 4 , 4.5 and
8.12 % for TSH, 0.1 and 10.6 % for haptoglobin, 4.5 and 7.5 % for ceruloplasmin, and
3.4 and 9.2 % for cortisol. Serum samples were also analyzed for FA content according to
the procedures described by Folch et al. (1957) and Hayat et al. (2009).
41
Statistical Analysis.
All data were analyzed using steer as experimental unit, and the PROC MIXED
procedure of SAS with Satterthwaite approximation. Serum FA profile from samples
collected at -2 h relative to CRH (d 25) and TRH (d 35) challenges were analyzed jointly
given that steers received the same treatments throughout the experiment (d 0 to 36). This
model statement contained the effects of treatment, day, and the interaction. Data were
analyzed using steer(treatment) as the random variable. The specified term for the
repeated statement was day, and steer(treatment) was included as subject. The covariance
structure utilized was autoregressive, which provided the best fit for these analyses
according to the Akaike information criterion. The model statement for all other
physiological data contained the effects of treatment, time, and the interaction. Data were
analyzed using steer(treatment) as the random variable. The specified term for the
repeated statement was time, and steer(treatment) was included as subject. The
covariance structure utilized was autoregressive according to the Akaike information
criterion. Results are reported as least square means and separated using LSD.
Significance was set at P ≤ 0.05, and tendencies were determined if P > 0.05 and ≤ 0.10.
Results are reported according to treatment effects if no interactions were significant, or
according to the highest-order interaction detected.
Experiment 3.
Animals.
Sixty Angus x Hereford steers (BW = 221 ± 2 kg; initial age 182 ± 1 d), weaned 35
d prior to the beginning of the study, were ranked by initial shrunk BW (d -28 of the
study) and allocated to 20 drylot pens (3 steers/pen). Pens were randomly assigned to
42
receive CAM or CO during the preconditioning (PC) phase (d -28 to 0; Table 3.1). On the
morning of d 0, steers were combined into 1 group, loaded into a commercial livestock
trailer, transported for 24 h to simulate the stress challenge of a long-haul (approximately
1,500 km; Arthington et al., 2008), and returned to the research facility (d 1). Upon
arrival, pens receiving CAM or CO during PC were randomly assigned to receive, in a 2
x 2 factorial arrangement, CAM or CO during a 28-d feedlot receiving (FR) phase (10
pens/treatment). As a result, 4 treatment combinations were generated (5 pens/treatment
combination): 1) CO during PC and FR, 2) CAM during PC and FR, 3) CAM during PC
and CO during FR, and 4) CO during PC and CAM during FR. Steers received a booster
of Bovi-Shield Gold 5, UltraChoice 7, and TSV-2 (Pfizer Animal Health) at the
beginning of the study (d -28). No incidences of mortality or morbidity were observed
during the entire experiment.
Diets.
Treatments were fed daily (0700 h) at the same rate as in Exp. 1. Treatment intakes
(DM basis) were formulated to be iso-caloric, iso-nitrogenous, and corresponded to 0.99
and 0.92 % of initial shrunk BW for CO and CAM, respectively. Mixed alfalfa-grass hay
was offered throughout the experiment (from d -28 to d 29) in amounts to ensure ad
libitum consumption. Treatments and hay were not mixed, and treatments were readily
consumed by steers. Samples of hay and treatment ingredients were analyzed for nutrient
content as in Exp. 1 (Tables 3.1 and 3.2). Water was offered for ad libitum consumption
throughout experiment.
Sampling.
43
Steer shrunk BW was collected prior to the beginning (d -31) and at the end of the
study (d 30) after 16 h of feed and water restriction. Shrunk BW was also recorded on d 1
immediately after arrival unloading. Preconditioning ADG was determined using values
obtained on d -31 and 1, whereas ADG during FR was determined using values obtained
on d 1 and 30 of the study.
Throughout the experimental period, pen voluntary forage and total DMI were
recorded daily. Hay and treatment orts were collected from each pen daily, weighed, and
dried for 96 h at 50°C in forced-air ovens for DM calculation. Estimated duodenal flow
of linoleic and linolenic acids during the study were calculated based on treatment and
hay intake of each pen, feed nutritional analysis, and the CPM-Dairy model (CornellPenn-Miner Dairy; Version 3.08.01; Univ. of Pennsylvania, Kennett Square; Cornell
Univ., Ithaca, NY; and William H. Miner Agric. Res. Inst., Chazy, NY), which has been
shown to adequately estimate intestinal FA flow in cattle (Moate et al., 2004). Average
shrunk BW during PC (values obtained on d -31 and d 1) and FR phase (values obtained
on d 1 and d 30) were utilized for calculation of DMI as % of BW during each respective
period. Total BW gain achieved during PC and FR phases were divided by total DMI
consumed during each phase for G:F calculations. Blood samples were collected on d 28, -15, 0, 1, 4, 7, 10, 14, 21, and 29 of the study. Samples collected from d 0 to 29 were
analyzed for plasma concentrations of cortisol, haptoglobin, and ceruloplasmin. Blood
samples collected on d -28, -15, 0, 4, 14, and 29 were also analyzed for plasma
concentrations of FA. Steer RT was measured at 30-min intervals with an automatic
recording device (Reuter et al., 2010) during transport, whereas on d 4 and 7 RT was
44
measured with a digital thermometer (GLA M750 Digital Thermometer) concurrently
with blood collection.
Blood Analysis.
Blood samples obtained via jugular venipuncture were collected and harvested for
plasma as in Exp. 1. Plasma concentrations of cortisol, haptoglobin, ceruloplasmin, and
FA were determined using the same procedures as described in Exp. 2. The intra- and
interassay CV were, respectively, 1.8 and 9.2 % for haptoglobin, 3.7 and 8.4 % for
ceruloplasmin, and 4.3 and 15.4 % for cortisol.
Statistical analysis.
All data were analyzed using pen as the experimental unit with the PROC MIXED
procedure of SAS and Satterthwaite approximation. The model statement used for ADG
and G:F during PC contained the effects of PC treatment. Data were analyzed using
pen(PC treatment) as the random variable. The model statement for ADG and G:F during
FR contained the effects of PC treatment, FR treatment, and the resultant interaction.
Data were analyzed using pen(PC x FR treatment) as the random variable. The model
statements used for DMI analyses during PC and FR were similar to models used for
ADG, but also contained the effects of day, all resultant interactions, in addition to day as
the specified term for repeated statement, pen(treatment) as subject, and autoregressive as
covariance structure according to the Akaike information criterion. The model statement
used for FA data obtained on d -28, -15, and 0, as well as RT, cortisol, ceruloplasmin,
and haptoglobin data obtained on d 0 and 1 of the study contained the effects of PC
treatment, day, and the resultant interaction because steers were assigned to their FR
treatment after sampling on d 1. Data were analyzed using pen(PC treatment) and
45
steer(pen) as the random variables. The specified term for the repeated statement was
day, the subject was steer(pen), and the covariance structure utilized was autoregressive
according to the Akaike information criterion. The model statement used for RT, cortisol,
ceruloplasmin, FA, and haptoglobin data obtained from d 4 to 29 contained the effects of
PC treatment, FR treatment, day, and all the resultant the interactions. Data were
analyzed using pen(PC x FR treatment) and steer(pen) as random variables. The specified
term for the repeated statement was day, the subject was steer(pen), and the covariance
structure utilized was autoregressive according to the Akaike information criterion.
Results are reported as least square means and separated using LSD or PDIFF.
Significance was set at P ≤ 0.05, and tendencies were determined if P > 0.05 and ≤ 0.10.
Results are reported according to treatment effects if no interactions were significant, or
according to the highest-order interaction detected.
RESULTS AND DISCUSSION
Experiment 1
In experiment 1, our objective were to evaluate the effects of camelina meal
supplementation on forage and total DMI, in situ ruminal forage degradability
parameters, and plasma CCK concentrations.
Steers receiving CAM had reduced (P = 0.01; data not shown) total DMI and
tended to have reduced (P = 0.10; data not shown) voluntary forage DMI compared to
CO cohorts (3.72 vs. 4.04 and 2.91 vs. 3.14 % of BW, respectively; SEM = 0.08),
concurring with previous research (Araujo et al., 2010; Cooke et al., 2011) reporting that
PUFA supplementation impairs DMI in forage-fed cattle. Lardy and Kerley (1994)
reported that substitution of soybean meal for rapeseed meal, also a member of the
46
Brassicaceae family, reduced DMI in beef steers and attributed these outcomes to
reduced palatability of diets containing rapeseed meal. In the present study, treatments
were included directly into the rumen of each steer, which eliminated potential concerns
associated with diet palatability. One could speculate that reduced DMI in CAM steers
was due to impaired forage digestibility as well as increased CCK synthesis and release
(Schauff and Clark, 1989; Drackley et al., 1992; Allen et al., 2000). However, no
treatment effects were detected (P ≥ 0.35) for ruminal disappearance rate or effective
ruminal degradability of hay DM and NDF (Table 3.3). Further, no treatment effects were
detected (P = 0.35) on plasma CCK concentrations (Table 3.3).
These results support previous research from our group reporting that PUFA
supplementation at a rate of 3.6 % of diet DM did not impact ruminal in situ forage
degradability in beef cattle (Cooke et al., 2011). Hess et al. (2008) indicated that
inclusion of supplemental fat up to 3% of diet DM is recommended to maximize the use
of forage-based diets and prevent impaired forage digestibility and intake. Moreover,
Leupp et al. (2006) reported that ruminal forage digestibility was not affected in foragefed steers offered supplemental fat at 4 % of diet DM. In the present study, according to
DMI evaluation and feed nutritional analysis, supplemental fat was included at 2.1 and
1.1 % of diet DM for CAM and CO, respectively, which supports the lack of treatment
effects on ruminal in situ forage degradability parameters. Gut-peptide hormones, such as
CCK, are mediators of DMI in animals (Choi and Palmquist, 1996), whereas diet FA
profile appears to modulate circulating CCK concentrations and subsequent DMI in
cattle. In fact, diets rich in PUFA appear to influence CCK concentrations to a greater
extent compared to diets rich in SFA (Relling and Reynolds, 2007; Bradford et al., 2008).
47
Conversely, Benson and Reynolds (2001) and Litherland et al. (2005) reported that
abomasal infusion of PUFA to dairy cows reduced DMI but did not impact plasma CCK
concentrations compared to SFA or water infusions. In agreement with these latter
studies, no treatment effects on plasma CCK concentrations were detected herein,
suggesting that CCK is not the sole mediator of the decreased DMI in PUFAsupplemented cattle. Nevertheless, camelina meal contains several other anti-nutritional
compounds that may impair DMI and were not evaluated herein, such as sinapines,
tannins, and saponins (Mawson et al., 1993; Ahlin et al., 1994). It is also important to
note that, based on DMI and treatment composition, camelina meal was included at 6.3 %
of diet DM for CAM steers, which is below the inclusion limit established by the FDA
(10 %; Taasevigen, 2010).
In summary, inclusion of camelina meal into forage-based diets reduced total
DMI in beef steers. However, ruminal in situ forage degradability parameters and plasma
CCK concentrations were not affected. Therefore, additional research is needed to
understand the mechanisms by which supplementation with camelina meal, and PUFA in
general, reduces DMI in forage-fed cattle.
Experiment 2
No treatment x day interaction was detected (P ≥ 0.11) on serum FA content.
Steers receiving CAM had greater mean plasma concentrations of oleic (P < 0.01),
linoleic (P = 0.02), linolenic (P = 0.05), ω-3 (P = 0.05), ω-6 (P = 0.01), PUFA (P = 0.01),
and total (P = 0.02) FA compared to CO steers (Table 3.4). No treatment effects were
detected (P = 0.30) on plasma concentrations of erucic acid, which can be present at
48
significant amounts in camelina meal (2 to 5% of total FA; Putnam et al., 1993) and has
been shown to induce myocardial lipidosis in non-ruminants (Kramer et al., 1990).
Supporting our findings, previous research reported that PUFA supplementation
increased total FA and PUFA concentrations in plasma of forage-fed beef cattle (Lake et
al., 2007; Scholljegerdes et al., 2007; Cooke et al., 2011). In addition, Moriel et al. (2011)
reported that beef heifers supplemented with camelina meal had greater plasma
concentrations of oleic, linoleic, linolenic, PUFA, and total FA compared to nonsupplemented cohorts. Moriel et al. (2011) also reported that plasma concentration of
erucic acid in heifers receiving camelina meal were, although negligible, greater
compared to non-supplemented cohorts. However, the camelina meal utilized by Moriel
et al. (2011) contained 2.6 % of erucic acid (total FA basis), whereas the camelina meal
used herein only contained 0.77 % of erucic acid (total FA basis; Table 3.2) which may
explain the similar plasma concentrations of erucic acid among CAM and CO steers.
Our group recently demonstrated that steers receiving CRH infusion at 0.1 µg/kg
of BW to induce a neuroendocrine stress response experienced an acute-phase reaction
including increased RT and plasma concentrations of haptoglobin and ceruloplasmin
(Cooke and Bohnert, 2011). Therefore, this research model was utilized herein to
evaluate the potential nutraceuticals effects of camelina meal. No treatment effects were
observed for plasma cortisol concentrations (P = 0.28; Table 3.5). In both treatments,
plasma cortisol peaked (time effect, P < 0.01; data not shown) at 0.5 h relative to CRH
infusion, indicating that CAM and CO steers experienced a similar neuroendocrine stress
response (Cooke and Bohnert, 2011). No treatment effects were detected (P = 0.60) for
RT (Table 3.5), although RT peaked (time effect, P < 0.01; data not shown), similarly to
49
Cooke and Bohnert (2011), for both treatments at 8 h relative to CRH infusion. Steers
receiving CAM tended (P = 0.10; Table 3.5) to have reduced mean plasma haptoglobin
concentrations compared to CO cohorts (1.54 vs. 1.73 absorbance @ 450 nm x 100,
respectively; SEM = 0.07). No time effect (P ≥ 0.57) was detected for plasma
haptoglobin in CAM and CO steers, indicating that the CRH infusion did not stimulate an
increase in plasma concentrations of this acute-phase protein. Nevertheless, the aforesaid
tendency for a treatment effect suggests that baseline plasma haptoglobin concentrations
were reduced in CAM compared to CO steers. A treatment x time interaction (P < 0.01;
Figure 3.1) was detected for plasma ceruloplasmin. Similar to Cooke and Bohnert (2011),
ceruloplasmin concentrations increased following CRH infusion for both treatments (time
effects; P < 0.01), whereas CAM steers had reduced ceruloplasmin concentrations
compared to CO cohorts at 6, 18, 42, 120, 144, and 168 h.
Previous studies from our group demonstrated that PUFA supplementation
reduced the acute-phase response induced by stressful procedures such as transport and
feedlot entry (Araujo et al., 2010; Cooke et al., 2011). These outcomes were attributed to
immunomodulatory effects of PUFA, more specifically ω-3 and ω-6 FA. However,
linolenic acid and its ω-3 derivates promote synthesis of eicosanoids that do not elicit the
acute-phase protein response, whereas linoleic acid and its ω-6 derivates promote the
synthesis of PGE 2 , a potent stimulator of pro-inflammatory and acute-phase responses
(Yaqoob and Calder, 1993; Carroll and Forsberg, 2007; Schmitz and Ecker, 2008). In the
present experiment, CAM steers had increased plasma concentrations on linoleic,
linolenic, ω-3, and ω-6 FA compared to CO steers. Cattle requirements for linoleic,
linolenic, and their respective FA derivates are still unknown. Therefore, it cannot be
50
concluded if CAM steers had reduced baseline haptoglobin concentrations, as well as
lessened CRH-induced increase in plasma ceruloplasmin compared with CO steers
because of the additional supply of ω-3, ω-6, or both FA groups.
Thyroid hormones are essential for control of basal metabolic rate and normal
development of animals (Paxton, 1986). Camelina meal contains glucosinolates, which
are compounds that can impair function of the thyroid gland in cattle (Lardy and Kerley,
1994). Mawson et al. (1994) reported that glucosinolates reduce circulating
concentrations of T 3 and T 4 and may over stimulate TSH secretion, causing hypertrophy
of thyroid tissues. Conversely, in the present study, no treatment effects were detected for
serum concentrations of T 3 , T 4 , and TSH (P ≥ 0.55; Table 3.5). Lardy and Kerley (1994)
reported that beef steers supplemented with rapeseed meal, which contained elevated
amounts of glucosinolates, had decreased serum concentrations of T 4 compared to nonsupplemented cohorts. However, Moriel et al. (2011) reported that serum concentrations
of T 3 and T 4 were not impaired in heifers supplemented with camelina meal.
Concentration of glucosinolates in camelina are relatively low (22 μmol/g; Lange et al.,
1995) compared with rapeseed meal (90 to 140 μmol/g; Lardy and Kerley, 1994), which
explains the lack of detrimental effects of camelina meal on bovine thyroid function
reported herein and by Moriel et al. (2011).
In summary, camelina meal supplementation to beef steers increased plasma
concentrations of PUFA but not erucic acid, did not impair thyroid gland function
stimulated by a TRH challenge, and reduced plasma concentrations of ceruloplasmin
following a CRH challenge.
51
Experiment 3
During the PC phase, CAM steers had reduced (P < 0.01; data not shown) forage
and total DMI compared to CO cohorts (2.23 vs. 2.46 and 3.07 vs. 3.35 % of BW,
respectively; SEM = 0.04). Hence, ADG during PC tended (P = 0.10; Table 3.6) to be
reduced for CAM steers compared to CO cohorts. However, no treatment effects were
detected (P = 0.24; Table 3.6) on G:F during PC. As calculated in Exp. 1, CAM steers
consumed camelina meal at 8.5 % of diet DM during PC, which is below the limit
established by the FDA (Taasevigen, 2010). These findings support previous studies from
our research group indicating that PUFA supplementation reduced DMI in feeder cattle,
but did not impair feed efficiency parameters (Araujo et al., 2010; Cooke et al., 2011). In
the present experiment, hay and treatments were not mixed. As expected, mean treatment
intake during PC was reduced (P = 0.01; data not shown) in CAM compared to CO
steers, but similar to the offered amount (1.90 vs. 2.01 kg, respectively; SEM = 0.03).
Therefore, treatment effects detected for forage intake during PC should not be attributed
to reduced palatability of the CAM treatment (Lardy and Kerley, 1994). Similarly to Exp.
1, supplemental fat was included at 2.6 and 1.4 % of diet DM for CAM and CO during
PC, respectively, which is less than the limit suggested by Hess et al. (2008) to prevent
impaired forage intake. Therefore, based on the results reported in Exp. 1, reduced forage
and total DMI during PC should not be attributed to altered forage digestibility and CCK
concentrations in CAM steers.
During the PC phase, a treatment x day interaction was detected (P = 0.02; Figure
3.2) for plasma linolenic acid (samples collected on d -28, -15, and 0). No additional
treatment effects were detected (P ≥ 0.18; Table 3.7) for plasma FA during PC, including
52
erucic acid concentrations (P = 0.21). Based on results from Exp. 2 and FA profile of
treatments (Tables 3.1 and 3.2), it was expected that CAM steers would have greater
plasma concentrations of linoleic acid, ω-3, ω-6, PUFA, and total FA compared to CO
cohorts. According to treatment and hay intake of each pen during PC, feed nutritional
analysis, and the CPM-Dairy model (Version 3.08.01), average daily duodenal flow of
linoleic and linolenic acid per steer were greater (P < 0.01; data not shown) for CAM
compared to CO cohorts (14.2 vs. 10.3 g/d of linoleic acid, SEM = 0.2; and 4.21 vs. 1.23
g/d of linolenic acid, SEM = 0.06; respectively). Plasma FA profile typically reflects the
duodenal FA flow (Archibeque et al., 2005; Scholljegerdes et al., 2007; Hess et al.,
2008). However, other physiological mechanisms that may alter plasma FA profile and
explain the lack of treatment effects on linoleic acid, ω-3, ω-6, and PUFA, such as tissue
incorporation and enzymatic activity (Archibeque et al., 2005; Scholljegerdes et al.,
2007), were not evaluated in the present experiment. Nevertheless, supporting our current
findings, Moriel et al. (2011) observed an increase in plasma linolenic acid
concentrations of beef heifers fed camelina meal for a 60-d period.
No treatment effects were detected for plasma concentrations of cortisol (P =
0.74), ceruloplasmin (P = 0.66), and RT (P = 0.56) immediately prior to (d 0) and after
transportation (d 1; Table 3.6). However, CAM steers had reduced (P = 0.04) plasma
haptoglobin concentrations during the same period compared to CO cohorts (Table 3.6).
Similar cortisol concentrations between treatments suggest that CAM and CO steers
experienced a similar neuroendocrine stress response prior to and after transport
(Crookshank et al., 1979; Sapolsky et al., 2000). Circulating haptoglobin typically
increases 3 d after transport and feedlot entry in feeder steers (Arthington et al., 2008;
53
Araujo et al., 2010). Therefore, as in Exp. 2, treatment effects detected for plasma
haptoglobin suggest that CAM steers had reduced baseline haptoglobin concentrations
compared to CO steers. This outcome can be attributed to the greater concentrations of
plasma linolenic acid in CAM steers during PC, given that this FA favors antiinflammatory reactions (Yaqoob and Calder, 1993; Schmitz and Ecker, 2008).
No PC treatment × FR treatment interactions (P ≥ 0.20) were detected for any of
the performance and physiological variables evaluated during FR. A PC treatment effect
was detected (P < 0.01) on forage and total DMI during FR phase because steers that
received CAM during PC, independent of the treatment during FR, had reduced forage
and total DMI compared to steers that received CO (Table 3.8). The reasons for this
continued decrease in forage and total DMI in steers receiving CAM during PC is
unknown and deserves further investigation. However, steers receiving CAM during
preconditioning tended (PC treatment effect; P = 0.10) to have improved G:F compared
to CO cohorts during FR (Table 3.8). Supporting this outcome, previous research from
our group reported that PUFA supplementation during preconditioning enhanced growing
lot performance of feeder steers (Cooke et al., 2011). No additional PC treatment effects
were detected (P ≥ 0.22) on FR performance, physiological responses (Table 3.8), and
plasma FA concentrations (data not shown). In addition, no FR treatment effects were
detected (P ≥ 0.21) on FR performance (Table 3.9), concurring with previous research
(Araujo et al., 2010) reporting similar ADG and DMI in newly-received cattle
supplemented or not with PUFA. Similarly to Exp. 1 and PC, CAM steers consumed
camelina meal at 7.1 % of diet DM during FR, which is below the limit established by the
FDA (Taasevigen, 2010).
54
A FR treatment effect was detected for plasma oleic acid (P < 0.01), linoleic acid
(P < 0.05), linolenic acid (P < 0.01), and PUFA (P < 0.05) because CAM steers had
greater mean concentrations of these FA compared to CO steers during FR (Table 3.10).
Similar tendencies for FR treatment effects were detected for plasma concentrations of ω3 (P = 0.06) and ω-6 (P = 0.07; Table 3.10). No treatment effects were detected for
plasma concentrations of erucic acid during FR (P = 0.26; Table 3.10), supporting the
results detected during the PC phase and Exp. 2. According to treatment and hay intake
of each pen during FR, feed nutritional analysis, and the CPM-Dairy model (Version
3.08.01), average daily duodenal flow of linoleic and linolenic acid per steer during FR
were greater (P < 0.01; data not shown) for CAM steers compared to CO cohorts (18.09
vs. 12.44 g/d of linoleic acid, SEM = 0.22; and 5.45 vs. 1.56 g/d of linolenic acid, SEM =
0.06; respectively). Hence, FR treatment effects detected on plasma FA during FR reflect
the greater PUFA content of the CAM treatment, supporting the outcomes from Exp. 2
and results reported by our (Cooke et al., 2011) and other research groups (Lake et al.,
2007; Scholljegerdes et al., 2007).
Day effects (P < 0.01; Table 3.11) were detected for plasma haptoglobin and
ceruloplasmin in samples collected from d 0 to d 29 of the study. Similar to our previous
efforts (Araujo et al., 2010; Cooke et al., 2010), haptoglobin concentrations peaked on d
4 whereas ceruloplasmin peaked on d 1 relative to transport (Table 3.11), indicating that
transport and feedlot entry stimulated an acute-phase protein reaction in steers from both
treatments. No FR treatment effects were detected (P = 0.98) for plasma cortisol
concentrations (Table 3.9) during FR, suggesting that steers receiving CAM or CO
experienced a similar neuroendocrine stress response after transport and feedlot entry
55
(Crookshank et al., 1979; Sapolsky et al., 2000). However, FR treatment effects were
detected for RT (P = 0.02), plasma haptoglobin (P = 0.02) and ceruloplasmin (P = 0.05)
during the FR phase. Steers receiving CAM had reduced mean RT and plasma
concentrations of haptoglobin and ceruloplasmin (Table 3.9), concurring with previous
research from our group reporting that PUFA supplementation lessened the acute-phase
response induced by transport and feedlot entry (Cooke et al., 2011). Araujo et al. (2010)
also reported that PUFA supplementation during PC and FR prevented the haptoglobin
response to transport and feedlot entry, but did not improve FR performance parameters
as in the present experiment. Still, circulating concentrations of acute-phase proteins
during feedlot receiving have been negatively associated with performance (Qiu et al.,
2007; Cooke et al., 2009) and positively associated with incidence of respiratory diseases
(Berry et al., 2004) in overtly healthy cattle. Therefore, development of management
strategies that prevent or alleviate the acute-phase response is essential for optimal
performance, health, and efficiency parameters in beef operations (Duff and Galyean,
2007). Similar to Exp. 2, FR treatment effects detected on RT and plasma haptoglobin
and ceruloplasmin can be attributed to immunomodulatory effects of PUFA. However,
CAM steers had greater plasma concentrations of linoleic acid, linolenic acid, and their
respective derivates during FR (Table 3.10), which are known to favor or prevent,
respectively, inflammatory and acute-phase reactions (Yaqoob and Calder, 1993; Schmitz
and Ecker, 2008). Therefore, it cannot be concluded that steers receiving CAM during FR
had reduced RT and plasma concentrations of haptoglobin and ceruloplasmin compared
to CO cohorts because of the additional supply of ω-3, ω-6, or both FA groups.
56
In summary, camelina meal supplementation during PC increased plasma
linolenic acid and reduced baseline plasma haptoglobin concentrations, feed intake, and
ADG during PC, but benefited feed efficiency during FR. Camelina meal
supplementation during FR increased plasma concentrations of linoleic acid, linolenic
acid, and their respective derivates, decreased the acute-phase protein reaction induced by
transport and feedlot entry, but did not improve FR performance.
Overall conclusions
Camelina meal supplementation to feeder cattle within the limits established by the
FDA did not impair thyroid gland function nor increase plasma concentration of erucic
acid, which are two of the main health concerns associated with this feed ingredient.
However, steers receiving camelina meal had impaired forage and total DMI, but these
outcomes were not associated with altered ruminal forage disappearance or circulating
CCK concentrations. Camelina meal supplementation lessened the acute-phase protein
reaction associated with neuroendocrine stress induced by a CRH challenge and transport
and feedlot entry. Also, it benefited feed efficiency parameters if offered during
preconditioning. Therefore, camelina meal is a feasible feed ingredient to alleviate stressinduced inflammatory reactions and potentially promote cattle welfare and productivity
in beef operations.
57
Table 3.1. Ingredient composition, nutrient profile, and intake of treatments offered
during Exp. 1, 2, and 31.
Exp. 1 and 3
Exp. 2
Item
CO
CAM
CO
CAM
Corn
83.0
68.0
83.0
68.0
Soybean Meal
14.0
--
14.0
--
Camelina Meal
--
29.0
--
29.0
Mineral and vitamin mix2
3.0
3.0
3.0
3.0
NE g ,4 Mcal/kg
1.46
1.54
1.47
1.54
NE m ,4 Mcal/kg
2.01
2.11
2.02
2.12
CP, %
15.5
16.7
15.6
16.8
NDF, %
13.9
19.5
13.9
19.5
Ether extract, %
5.20
9.70
5.20
9.70
Linoleic acid, %
2.60
3.38
2.60
3.39
Linolenic acid, %
0.07
1.15
0.07
1.14
PUFA, %
2.67
4.53
2.67
4.53
SFA + MUFA
2.27
3.32
2.28
3.33
Ca, %
0.47
0.66
0.42
0.61
P, %
0.62
0.82
0.59
0.79
2.20
2.04
1.65
1.52
Ingredients, % as-fed
Nutrient Profile3, DM basis
Daily intake5
DM, kg
58
NE g 4, Mcal
3.21
3.14
2.43
2.34
NE m 4, Mcal
4.42
4.30
3.33
3.22
CP, kg
0.34
0.34
0.26
0.26
NDF, kg
0.31
0.39
0.23
0.30
Ether extract, kg
0.11
0.19
0.09
0.15
Linoleic acid, g
57.2
68.9
42.9
51.5
Linolenic acid, g
1.54
23.5
1.14
17.4
PUFA, g
58.7
92.4
44.1
68.9
SFA + MUFA, g
49.9
67.7
37.6
60.3
Ca, g
10.3
13.4
6.9
9.3
P, g
13.6
16.7
9.74
12.0
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Cattleman’s Choice (Performix Nutrition Systems; Nampa, ID); contained 14% Ca,
10% P, 16% NaCl, 1.5% Mg, 3200 ppm Cu, 65 ppm I, 900 ppm Mn, 140 ppm Se, 6000
ppm Zn, 136 IU/g of vitamin A, 13 IU/g of vitamin D 3 , and 0.05 IU/g of vitamin E.
3
Values obtained from a commercial laboratory wet chemistry analysis (Dairy One
Forage Laboratory, Ithaca, NY). Fatty acid content was determined based on Table 3.2,
and according to the procedures described by Folch et al. (1957) and Hayat et al.
(2009).
4
Calculated with the following equations (NRC 1996): NE g = 1.42 ME – 0.174 ME2 +
0.0122 ME3 – 0.165; NE m = 1.37 ME – 0.138 ME2 + 0.0105 ME3 – 1.12. Given that
ME = 0.82 × DE, and 1 kg of TDN = 4.4 Mcal of DE.
5
Estimated from the concentrate consumption of individual experimental unit.
59
Table 3.2. Fatty acid (FA) profile of feedstuffs offered to cattle during Exp. 1, 2, and 31.
Fatty Acid, %
Corn
Soybean Meal
Palmitic acid (16:0)
11.4
16.0
8.3
21.8
Stearic acid (18:0)
2.1
5.0
2.9
5.8
Oleic acid (18:1)
32.8
14.1
21.7
11.3
Linoleic acid (18:2)
52.5
56.4
28.8
13.8
Linolenic acid (18:3)
1.3
8.5
24.2
26.0
Erucic acid (22:1 n-9)
0.00
0.00
1
Camelina Meal
0.77
As % of total FA. All feedstuffs were analyzed for FA content according to the
procedures described by Folch et al. (1957) and Hayat et al. (2009).
Hay
0.00
60
Table 3.3. Plasma concentration of CCK, and ruminal in situ disappearance parameters
of mixed alfalfa-grass hay incubated in forage-fed steers offered supplements containing
(CAM; n = 5) or not (CO; n = 4) camelina meal in Exp. 21.
Item
CO
CAM
SEM
P-Value
Ruminal degradation rate, %/h
DM
8.58
7.91
0.5
0.35
NDF
7.39
7.49
0.6
0.91
Effective degradability,2 %
DM
64.9
64.3
1.00
0.57
NDF
70.9
70.1
1.00
0.55
26.78
22.72
2.88
0.35
Plasma CCK,3 pg/mL
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Calculated by fixing ruminal passage rate at 0.046/h (Poore et al., 1990) and using the
model proposed by Ørskov and McDonald (1979).
3
Blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 9, and 12 h relative to treatment
feeding (0 h).
61
Table 3.4. Serum fatty acids (FA) concentrations (mg/ml of plasma) of steers offered
supplements containing (CAM; n = 7) or not (CO; n = 7) camelina meal during Exp. 21,2.
Treatments
Item
CO
CAM
SEM
P-Value
Stearic (18:0)
0.196
0.210
0.006
0.14
Oleic (18:1)
0.184
0.229
0.006
< 0.01
Linoleic (18:2 n-6)
0.196
0.230
0.009
0.02
Linolenic (18:3 n-3)
0.079
0.097
0.006
0.05
Arachidonic (20:4 n-6)
0.008
0.009
0.001
0.78
Eicosapentaenoic (20:5 n-3)
0.007
0.008
0.001
0.30
Erucic acid (22:1 n-9)
0.015
0.017
0.001
0.30
Docosahexaenoic (22:6 n-3)
0.007
0.008
0.006
0.28
Total SFA
0.455
0.471
0.013
0.41
Total MUFA
0.368
0.397
0.020
0.31
Total ω-3
0.094
0.114
0.006
0.05
Total ω-6
0.207
0.243
0.009
0.01
ω-3: ω-6 ratio
0.633
0.492
0.130
0.45
PUFA
0.301
0.359
0.013
0.01
Total FA
1.135
1.274
0.041
0.02
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Blood samples were collected once on d 25 and 35 relative to beginning of treatments.
62
Table 3.5. Physiological responses of steers offered supplements containing (CAM; n =
7) or not (CO; n = 7) camelina meal during Exp. 21.
Treatments
Item
CO
CAM
SEM
P-Value
CRH challenge2
Plasma cortisol, ng/mL
20.4
25.8
3.40
0.28
Rectal temperature, ºC
39.10
39.10
0.05
0.61
1.73
1.54
0.07
0.10
3.23
4.11
1.17
0.61
98.10
115.8
20.60
0.55
2.13
2.14
0.11
0.95
Plasma haptoglobin, absorbance @ 450 nm × 100
TRH challenge3
Plasma triiodothyronine, ng/mL
Plasma thyroxine, ng/mL
Plasma thyrotropin-stimulating hormone, ng/mL
1
CO = grain-based supplement without the addition of camelina meal; CAM = grain-
based supplement with the addition of camelina meal.
2
Blood samples were collected hourly from -2 to 0 h and 4 to 8 h, and every 30 min from
0 to 4 h samples relative to corticotropin-releasing hormone (CRH) challenge (0.1 µg/kg
of BW). Rectal temperatures were recorded by every 30 min from −2 to 8 h relative to
CRH challenge.
3
Blood samples were collected hourly from -2 to 0 h and 4 to 8 h, and every 30 min from
0 to 4 h samples relative to thyrotropin-releasing hormone (TRH) challenge (0.33 µg/kg
of BW).
63
Table 3.6. Performance and physiological responses during preconditioning (d -28 to 0)
of steers offered supplements containing (CAM; n = 10) or not (CO = 10) camelina meal
in Exp. 31.
Treatments
Item
CO
CAM
SEM
P-Value
Shrunk BW (d -31), kg
220
221
2.00
0.61
Shrunk BW (d 1), kg
231
229
2.00
0.56
Average Daily Gain,2 kg/d
0.37
0.26
0.04
0.10
Feed efficiency,3 kg/kg
0.056
0.044
0.006
0.24
Rectal temperature, oC
39.20
39.20
0.03
0.56
Haptoglobin, absorbance @ 450 nm × 100
1.80
1.65
0.05
0.04
Ceruloplasmin, mg/dL
21.90
22.30
0.70
0.66
Cortisol, ng/mL
39.40
41.80
5.20
0.74
Performance traits
Physiological responses4
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Calculated using shrunk BW on d -31 and d 1.
3
Calculated for dividing the total DM consumed from d -28 to d 0 into the total shrunk
BW gain achieved over this time period.
4
All steers were transported for 24 h d 0. Physiological measurements were obtained on d
0 (before loading into the truck) and d 1 (immediately after unloading) relative to
transport.
64
Table 3.7. Plasma FA concentrations (mg/ml of plasma) during preconditioning (d -28
to 0) of steers offered supplements containing (CAM; n = 10) or not (CO; n = 10)
camelina meal in Exp. 31,2.
Treatments
Item
CO
CAM
SEM
P-Value
Stearic (18:0)
0.259
0.247
0.012
0.47
Oleic (18:1)
0.268
0.287
0.016
0.40
Linoleic (18:2 n-6)
0.199
0.215
0.008
0.18
Arachdonic (20:4 n-6)
0.009
0.009
0.001
0.92
Eicosapentaenoic (20:5 n-3)
0.029
0.022
0.005
0.40
Erucic acid (22:1 n-9)
0.018
0.019
0.001
0.30
Docosahexaenoic (22:6 n-3)
0.013
0.010
0.001
0.26
Total SFA
0.628
0.596
0.029
0.44
Total MUFA
0.460
0.487
0.020
0.36
Total ω-3
0.088
0.097
0.008
0.45
Total ω-6
0.225
0.232
0.007
0.54
ω-3: ω-6 ratio
3.146
2.853
0.237
0.39
PUFA
0.342
0.356
0.014
0.47
Total FA
1.430
1.434
0.060
0.96
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Blood samples were collected on d -28, d -15, and d 0 of preconditioning.
65
Table 3.8. Performance and physiological responses during feedlot receiving (d 1 to d
29) of steers offered preconditioning supplements (from d -28 to d 0) containing (CAM; n
= 10) or not (CO; n = 10) camelina meal in Exp. 31.
Treatments
Item
CO
CAM
SEM
P-Value
Shrunk BW (d 29), kg
281
279
3.00
0.69
Forage DMI, % of BW
2.61
2.45
0.03
< 0.01
Total DMI, % of BW
3.35
3.19
0.03
< 0.01
Average Daily Gain,2 kg/d
1.71
1.77
0.05
0.43
Feed Efficiency,3 kg/kg
0.215
0.231
0.006
0.10
Rectal temperature, ºC
39.14
39.05
0.05
0.23
Plasma cortisol, ng/mL
30.90
27.60
3.30
0.48
Plasma haptoglobin, 450 nm × 100
1.85
1.86
0.10
0.96
Plasma ceruloplasmin, mg/dL
20.60
20.80
0.50
0.83
Performance traits
Physiological responses4
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Calculated using shrunk BW on d 1 and d 29.
3
Calculated for dividing the total DM consumed from d 1 to d 29 into the total shrunk
BW gain achieved over this time period.
4
All steers were transported for 24 h on d 0. Rectal temperature was assessed on d 4 and
7 relative to transport. Blood samples were collected on d 4, 7, 10, 14, 21, and 29 relative
to transport.
66
Table 3.9. Performance and physiological responses during feedlot receiving (d 1 to d
29) of steers offered supplements containing (CAM; n = 10) or not (CO; n = 10) camelina
meal in Exp. 31.
Treatments
Item
CO
CAM
SEM
P-Value
Shrunk BW (d 29), kg
281
279
3.00
0.59
Forage DMI, % of BW
2.52
2.55
0.03
0.67
Total DMI, % of BW
3.29
3.26
0.32
0.52
Average Daily Gain,2 kg/d
1.79
1.70
0.05
0.21
Feed Efficiency,3 kg/kg
0.229
0.217
0.006
0.22
Rectal temperature, ºC
39.19
39.01
0.05
0.02
Plasma cortisol, ng/mL
29.20
29.30
3.31
0.98
Plasma haptoglobin, absorbance @ 450 nm × 100
2.02
1.69
0.09
0.02
Plasma ceruloplasmin, mg/dL
21.50
19.90
0.50
0.05
Performance traits
Physiological responses4
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
Calculated using shrunk BW on d 1 and d 29.
3
Calculated for dividing the total DM consumed from d 1 to d 29 into the total shrunk
BW gain achieved over this time period.
4
All steers were transported for 24 h on d 0. Rectal temperature was assessed on d 4 and
7 relative to transport. Blood samples were collected on d 4, 7, 10, 14, 21, and 29 relative
to transport.
67
Table 3.10. Plasma FA concentrations (mg/ml of plasma) during feedlot receiving (d 1 to
d 29) of steers offered supplements containing (CAM; n = 10) or not (CO; n = 10)
camelina meal in Exp. 31,2.
Treatments
Item
CO
CAM
SEM
P-Value
Stearic (18:0)
0.265
0.254
0.016
0.61
Oleic (18:1)
0.242
0.289
0.014
0.03
Linoleic (18:2 n-6)
0.242
0.260
0.006
0.05
Linolenic (18:3 n-3)
0.086
0.118
0.006
0.01
Arachdonic (20:4 n-6)
0.008
0.009
0.001
0.52
Eicosapentaenoic (20:5 n-3)
0.016
0.007
0.004
0.08
Erucic acid (22:1 n-9)
0.017
0.018
0.001
0.26
Docosahexaenoic (22:6 n-3)
0.008
0.008
0.0005
0.67
Total SFA
0.606
0.564
0.037
0.44
Total MUFA
0.441
0.460
0.020
0.52
Total ω-3
0.110
0.134
0.008
0.06
Total ω-6
0.256
0.275
0.007
0.07
ω-3: ω-6 ratio
2.642
2.257
0.175
0.14
PUFA
0.388
0.429
0.014
0.05
Total FA
1.435
1.455
0.066
0.83
1
CO = grain-based supplement without the addition of camelina meal; CAM = grainbased supplement with the addition of camelina meal.
2
All steers were transported for 24 h on d 0. Blood samples were collected on d 4, d 14,
and d 29 relative to transport.
68
Table 3.11. Day effects on plasma concentrations of acute-phase proteins of transported steers (n = 60) in Exp. 31,2.
Day of the study
Item
0
1
4
7
10
14
21
29
SEM
P-Value
Haptoglobin, 450 nm x 100
1.79a,c
1.65a,c
2.22b
2.18b
1.92a
1.51c
1.88a
1.42c
0.13
< 0.01
Ceruloplasmin, mg/dL
21.3ac
22.8b
22.2a,b
22.7b
22.7b
20.3c
18.7d
17.7e
0.56
< 0.01
1
Steers loaded into a commercial livestock trailer on d 0, transported for 24 h, and unloaded on d 1. Blood samples were
collected immediately prior to (d 0) and 1, 4, 7, 10, 14, 21, and 29 d relative to beginning of transport.
2
Within rows, values bearing different superscripts differ (P < 0.05).
69
Figure 3.1. Plasma ceruloplasmin concentrations of beef steers offered supplements containing (CAM; n = 7) or not (CO; n
= 7) camelina meal and receiving an i.v. corticotropin-releasing hormone (CRH) infusion (0.1 µg/kg of BW) at h 0 in Exp 2.
A treatment x time interaction (P < 0.01) was detected. Treatment comparison within hour: † P < 0.05, * P < 0.01.
†
*
†
†
*
*
*
70
Figure 3.2. Plasma linolenic acid concentrations (± SEM) during preconditioning (d -28
to 0) of steers offered supplements containing (CAM; n = 10) or not (CO; n = 10)
camelina meal in Exp 3. A treatment x day interaction was detected (P = 0.02). Treatment
comparison within day: † P < 0.05, * P < 0.01.
*
†
71
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