University of Veterinary Medicine Hannover Effects of monensin and

Transcription

University of Veterinary Medicine Hannover
Effects of monensin and essential oils on ruminal
fermentation, performance, energy metabolism and immune
parameters of dairy cows during the transition period
Thesis
Submitted in partial fulfilment of the requirements for the degree
-Doctor of Veterinary MedicineDoctor medicinae veterinariae
( Dr. med. vet. )
by
Caroline Drong
Haltern am See
Hannover 2016
Academic supervision:
1. Prof. Dr. agr. habil. Dr. med. vet. Sven Dänicke
Institute of Animal Nutrition
Federal Research Institute for Animal Health
Friedrich-Loeffler-Institut, Braunschweig
2. Prof. Dr. med. vet. Jürgen Rehage
Clinic for Cattle
University of Veterinary Medicine, Hannover
1. Referee:
Prof. Dr. med. vet. Jürgen Rehage
2. Referee:
Prof. Dr. med. vet. Korinna Huber
Day of the oral examination:
23.05.2016
Meiner Familie gewidmet,
in Liebe und Dankbarkeit
Parts of this thesis have already been accepted for publication in the following journals:
1. C. Drong, U. Meyer, D. von Soosten, J. Frahm, J. Rehage, G. Breves, S. Dänicke.
Effect of monensin and essential oils on performance and energy metabolism of transition
dairy cows.
Journal of Animal Physiology and Animal Nutrition, 2016, Volume 100,
DOI:10.1111/jpn.12401
2. C. Drong, U. Meyer, D. von Soosten, J. Frahm, J. Rehage, H. Schirrmeier, M. Beer, S.
Dänicke.
Effects of monensin and essential oils on immunological, haematological and biochemical
parameters of cows during the transition period.
Journal of Animal Physiology and Animal Nutrition, 2016, In Press,
DOI:10.1111/jpn.12494
Furthermore, results of this thesis were presented in form of oral presentations at the following
conferences:
1. Effects of monensin and essential oils on the energy metabolism of periparturient dairy
cows.
C. Drong, U. Meyer, D. von Soosten, J. Rehage, S. Dänicke.
69. Jahrestagung der Gesellschaft für Ernährungsphysiologie, 10.-12.03.2015, Göttingen,
Germany, Proc. Soc. Nutr. Physiol. 24, p. 82.
2. Effects of a monensin releasing bolus and a blend of essential oils on white blood cell
profile and function of periparturient dairy cows.
C. Drong, U. Meyer, D. von Soosten, J. Frahm, J. Rehage, S. Dänicke.
70. Jahrestagung der Gesellschaft für Ernährungsphysiologie, 08.-10.03.2016, Hannover,
Germany, Proc. Soc. Nutr. Physiol. 25, p. 88.
CONTENTS
CONTENTS
INTRODUCTION .......................................................................................................................... 1
BACKGROUND............................................................................................................................. 3
1.
2.
Transition period ................................................................................................................. 3
1.1
Metabolism .................................................................................................................... 3
1.2
Immune system and health............................................................................................. 7
Monensin ............................................................................................................................ 11
2.1
Characteristics and mode of action in rumen ............................................................... 11
2.2
Effects on performance and health of dairy cows ........................................................ 12
3. Essential Oils ...................................................................................................................... 13
3.1
Characteristics and mode of action in rumen ................................................................ 13
3.2
Effects on performance and health of dairy cows ......................................................... 16
SCOPE OF THE THESIS ........................................................................................................... 18
PAPER I ........................................................................................................................................ 21
Effect of monensin and essential oils on performance and energy metabolism
of transition dairy cows.
Journal of Animal Physiology and Animal Nutrition, DOI: 10.1111/jpn.12401
PAPER II ...................................................................................................................................... 53
Effects of monensin and essential oils on immunological, haematological and
biochemical parameters of cows during the transition period.
Journal of Animal Physiology and Animal Nutrition, DOI: 10.1111/jpn.12494
GENERAL DISCUSSION ........................................................................................................... 87
SUMMARY................................................................................................................................. 104
CONTENTS
ZUSAMMENFASSUNG ........................................................................................................... 107
REFERENCES ........................................................................................................................... 110
DANKSAGUNG ......................................................................................................................... 131
ABBREVIATIONS
ABBREVIATIONS
(cited in Introduction, Background and General Discussion)
AA
Amino acids
AST
Aspartate-aminotransferase
ATP
Adenosine triphosphate
AP
ante partum
BCS
Body condition score
BHB
Beta-hydroxybutyrate
BVDV
Bovine Viral Diarrhea virus
CoA
Coenzyme A
CPT
Carnitine palmitoyltransferase
CRC
Controlled-release capsule
d
Day
DIM
Days in milk
DMI
Dry matter intake
DNA
Deoxyribonucleic acid
EB
Energy balance
ECM
Energy-corrected milk
Ig
Immunoglobulin
GGT
Gamma-glutamyltransferase
GLDH
Glutamine-dehydrogenase
HMG
3-hydroxy-3-methylglutaryl
HAP
Hyper-ammonia producing
LFI
Liver functionality index
LPS
Lipopolysaccharides
mRNA
Messenger ribonucleic acid
NEFA
Non-esterified fatty acids
NEL
Net energy for lactation
ABBREVIATIONS
NOX
NADPH-oxidase
PBMC
Peripheral blood mononuclear cells
PC
Pyruvate carboxylase
PEPCK
Phosphoenolpyruvate carboxykinase
PMN
Polymorphonuclear leukocytes
PP
post partum
ROS
Reactive oxygen species
SCFA
Short-chain fatty acids
TAG
Triacylglycerides
TCA
Tricarboxylic acid
TMR
Total mixed ration
VLDL
Very low density lipoprotein
INTRODUCTION
INTRODUCTION
The weeks around parturition of the dairy cow, commonly known as the transition period, are
characterized by physical, endocrine and metabolic changes in preparation for calving and the
onset of lactation (MALLARD et al. 1998). As genetic progress and management improvements
proceed to raise milk performance of the dairy herds, metabolism of the cow faces dramatic
challenges. The increasing nutrient demand, especially the placental and mammary uptake of
maternal glucose for growth of the fetus and the onset of lactation, in times of reduced feed
intake at calving result in a negative energy balance (EB) (BELL 1995). Metabolic adaptations
are aimed at counterbalancing the inadequate energy supply from feed intake by a massive
mobilization of fatty acids from the adipose tissues. Although a negative EB in early lactation can
be seen as quite normal for ruminants as a result of the homeorhetic regulation (INGVARTSEN
2006), any complications in the modern high-yielding dairy cow relevant for the adaptation
process like stress, disease, incorrect management or insufficient feeding can have wide-ranging
consequence for cow health and productivity during the whole lactation cycle. In fact, the highest
number of production diseases can be found in early lactation (INGVARTSEN 2006) while
elevated concentrations of the blood metabolites non-esterified fatty acids (NEFA) and the ketone
beta-hydroxybutyrate (BHB) seem to be aetiologically involved in an impaired immune function
around calving (CONTRERAS and SORDILLO 2011).
Therefore, great interest in dairy nutrition research has focused on intervention measures
modulating rumen fermentation to improve energy metabolism, performance and health during
this period. One approach is the modulation of the ruminal short-chain fatty acid (SCFA) profile
towards an increased propionate production. As the main precursor of hepatic gluconeogenesis
(SEAL and REYNOLDS 1993), this should increase availability of glucose for the cow around
calving.
The ionophore antimicrobial drug monensin has been successfully tested for this property
(BERGEN and BATES 1984) and was widely used until the ban on antibiotics as feed additives
in
the
European
Union
(Directive
1831/2003/CCE,
European
Commission,
2003).
Simultaneously, the research in natural alternatives to monensin was greatly enhanced meanwhile
1
INTRODUCTION
fermentation-modulating effects of essential oils caught great interest. Although results are not
consistent, essential oils are also assigned the attributes to increase propionate production in
rumen fermentation (CALSAMIGLIA et al. 2007). Reports about the subsequent effects on
performance parameters are inconsistent and often use different compounds and/or doses. There
is especially little information available if essential oils are able to improve the energy status of
cows analogous to monensin.
Recently, monensin was relaunched in the EU as a controlled-release capsule (CRC) indicated for
over-conditioned transition cows. It was shown to diminish incidence rates of ketosis, displaced
abomasum and mastitis whereby underlying mechanisms beside an improved energy status are
rarely examined, especially its direct effects on immune cell populations and function.
Considering health, the therapeutic potential of essential oils is well-known since ancient times.
Different compounds of essential oils have successfully been tested for anticancer, antibacterial,
antiviral, antioxidative property and in the treatment and prevention of cardiovascular diseases
including atherosclerosis and thrombosis in humans (EDRIS 2007) and an enhanced
immunocompetence and health of gut and a better performance of broilers and pigs (MICHIELS
et al. 2010, TIIHONEN et al. 2010), but studies on the effects on the immune system of cows are
very rare.
Therefore, a recently established animal model that enables generating animal groups being in a
ketogenic metabolic status by a specific combination of the factors high body condition score
(BCS) at the beginning of the transition period, overfeeding in the dry period and a decelerated
energy supply post partum (PP) (SCHULZ et al. 2014) was used for assessment of monensin and
essential oils and their effects on ruminal fermentation, performance and health of transition dairy
cows.
2
BACKGROUND
BACKGROUND
1. Transition period
1.1 Metabolism
The transition period of the cow includes late gestation and early lactation and can commonly be
defined as the time from 3 weeks ante partum (AP) until 3 weeks PP (GRUMMER 1995).
Meanwhile, the high-yielding dairy cow is confronted with a massive change in the metabolic,
endocrine and immune status. The reduction of feed intake around calving is accompanied by an
increase of the nutrient demand for the growth of the fetus and the initiated lactation (Grummer
1995) and leads to a state of negative EB. The requirements for glucose and metabolizable energy
increase 2- to 3-fold in the weeks around parturition (DRACKLEY et al. 2001). Endocrine and
metabolic mechanisms coordinating the partitioning of nutrients and energy during pregnancy
and early lactation have been well characterized (BAUMAN and CURRIE 1980,
INGVARTSEN and ANDERSEN 2000). They involve two types of regulation, homeostasis and
homeorhesis. Homeostasis regulates the maintenance of physiological equilibrium despite
changing environmental conditions. To mention one important example, blood glucose
concentration is maintained relatively constant, mainly by insulin and glucagon, as its supply is
of critical importance for many tissues and physiological processes. Homeorhesis means the
coordination of physiological processes in support of a dominant physiological state or chronic
situation as it is described for pregnancy and lactation (BAUMAN and CURRIE 1980).
Regarding the example of glucose concentration in blood, mammary utilization of glucose
primarily for lactose synthesis is markedly increased with the onset of lactation. To support an
adequate glucose supply, hepatic rates of gluconeogenesis are increased and glucose uptake,
utilization and oxidation by adipose tissue and muscle are reduced. In this context, somatotropin
presents a key homeorhetic hormone involved in mechanisms attenuating tissue response to
insulin (BELL 1995), known as insulin resistance (HOLTENIUS and HOLTENIUS 1996).
Furthermore, an increased somatotropin to insulin ratio in early lactation leads to a stimulation of
NEFA release from adipose tissue, which are a major source of energy to the cow during this
time (INGVARTSEN 2006). The sensitivity to lipolytic signals of norepinephrine and
3
BACKGROUND
epinephrine is greatly enhanced while lipogenesis is essentially shut down (THEILGAARD et al.
2002, INGVARTSEN 2006). The conversion of NEFA to ketone bodies in the liver is an
additional strategy in times of negative EB as ketone bodies can be oxidized by the heart, kidney,
skeletal muscle, mammary gland and gastrointestinal tract of ruminants and can serve as
substrates for mammary fatty acid synthesis (HEITMANN et al. 1987). However, a glucose
deficit together with excessive fatty acid mobilization und ketogenesis can lead to serious
metabolic disorders and an impaired health and productivity of transition dairy cows. In fact, the
highest incidence of production illnesses in dairy cows can be found in early lactation
(INGVARTSEN 2006).
The liver is situated at the crossroad of metabolism and plays a key role in the coordination of
nutrient fluxes during the transition from late gestation to lactation (DRACKLEY et al. 2001)
representing the concept of homeorhesis (BAUMAN and CURRIE 1980).
Two important areas of liver metabolism with distinct effects on cow performance and health are
metabolism of NEFA and gluconeogenesis.
After release, NEFA can be used as a source of milk fat synthesis in mammary gland, reesterified to triacylglycerides (TAG) or partially oxidized to ketone bodies in the liver or
completely oxidized by skeletal muscle or liver as an energy source (DRACKLEY 1999,
ROCHE et al. 2009). Additional to mitochondrial beta-oxidation in the liver, there is an
alternative pathway of beta-oxidation found within the peroxisomes that may be induced during
times of increased flow of fatty acids to the liver (GRUM et al. 1994, DRACKLEY et al. 2001).
However, in times of massive body reserve mobilization when uptake of NEFA into liver
exceeds the oxidation and secretion ability, the fatty liver syndrome of periparturient cows may
occur. It is defined as a multifactorial disease accompanied by a diminished metabolic function,
decreased health status and reproductive performance of the cow (BOBE et al. 2004). In fact,
rates of TAG synthesis of ruminants are similar to rates of non-ruminants but a lower capacity for
synthesis and secretion of TAG as very low density lipoproteins (VLDL) was reported and made
co-responsible for the accumulation in liver (KLEPPE et al. 1988, DRACKLEY et al. 2001). It
has been object of research to locate factors that regulate the disposition of NEFA between
4
BACKGROUND
oxidation and esterification in the liver (DRACKLEY 1999). In this context, a key role is
attributed to the enzyme carnitine palmitoyltransferase (CPT)-1 which controls the entry of
NEFA into the mitochondria for beta-oxidation to carbon dioxide or ketone bodies (DRACKLEY
1999). Its total activity in mitochondria increases from dry period to early lactation (DANN et al.
2000) and is inhibited by malonyl-coenzyme A (CoA) and methylmalonyl-CoA (BRINDLE et al.
1985). Concentration of malonyl-CoA, a fatty acid synthesis intermediate, is regulated by activity
of acetyl-CoA carboxylase which is active during well-feed conditions characterized by high
insulin to glucagon ratios and inactive during insulin-deficient states (ZAMMIT 1996) and may
serve as an energy balance-sensitive regulatory mechanism for NEFA uptake into mitochondria
(DRACKLEY et al. 2005). Likewise, CPT-1 is less sensitive to inhibition by malonyl-CoA
during situations of low insulin or insulin resistance (ZAMMIT 1996). Methylmalonyl-CoA is
produced during metabolism of propionate and may link supply of propionate from the ruminal
fermentation with the need for NEFA oxidation (ZAMMIT 1990). In fact, in vitro studies showed
that high concentrations of propionate inhibit fatty acid oxidation in bovine liver slices (JESSE et
al. 1986).
Besides the NEFA supply to liver and the activity of CPT-1 to promote entry into mitochondria,
ketogenesis is regulated by intramitochondrial activity of 3-hydroxy-3-methylglutaryl (HMG)CoA synthase which forms the regulatory step in conversion of acetyl-CoA to ketone bodies
(HEGARDT 1999). Propionate has been attributed an antiketogenic effect as an abundant supply
of propionate inhibits HMG-CoA synthase via an increased pool size of the inhibitor succinylCoA (DRACKLEY et al. 2001). Moreover, propionate stimulates insulin secretion that is a potent
regulator of lipogenesis and an antagonist of the lipolytic action of growth hormone (VERNON
and POND 1997, RHOADS et al. 2004).
As mentioned before, glucose demand of the cow at calving and early lactation exceeds glucose
supply from digestible energy intake by up to 500 g/day (d) (DRACKLEY et al. 2001), leading to
an enhanced gluconeogenesis mainly in liver. It contributes up to about 70% of the total glucose
flux in high producing cows (HUNTINGTON 1997). Propionate is the major glucogenic
precursor taken up by the liver with an estimated proportion of up to 32 to 73% of
gluconeogenesis, followed by amino acids (AA) with 10 to 30% and lactate with 15% (SEAL and
5
BACKGROUND
REYNOLDS 1993) while glycerol may contribute as much as 15% to 20% to glucose demand
around parturition (GRUMMER 1995). Capacity of liver tissue isolated at d 1 and d 21 PP to
convert propionate to glucose was 19% and 29% greater, respectively, than at d -21 AP
(OVERTON et al. 1998). Although propionate is the main precursor of hepatic gluconeogenesis
(SEAL and REYNOLDS 1993), its utilization is linked to nutrient supply from rumen and may
therefore be limited in times of low dry matter intake (DMI) around parturition (DRACKLEY et
al. 2001). Moreover, an in vitro study showed that fat accumulation decreases gluconeogenesis
from propionate in bovine hepatocytes (CADÓRNIGA-VALIÑO et al. 1997). Fatty liver was
proposed to be related to an inhibited hepatic ureagenesis with subsequent increased blood
ammonia concentrations (ZHU et al. 2000) that also decrease gluconeogenesis from propionate
(OVERTON et al. 1999) and so may present a possible indirect link between fatty liver and a
diminished gluconeogenesis.
Alanine und glutamine are the two AA with greatest contribution to glucose synthesis
(BERGMAN and HEITMANN 1978). The AA from gastrointestinal tract, skeletal muscle and
other tissue proteins play a significant role as gluconeogenesis increases during early lactation
(BELL et al. 2000). In fact, OVERTON et al. (1998) found an even 98% and 50% greater
conversion of alanine to glucose in liver tissue isolated at d +1 and +21 PP, respectively, than at d
-21 AP. The supply of AA from gastrointestinal tract and skeletal muscle is a determinant factor
of their contribution to gluconeogenesis (DANFÆR et al. 1995).
Lactate utilization for gluconeogenesis represents rather a recycling of carbon as it is formed
either during catabolism of glucose in peripheral tissues or by partial catabolism of propionate by
visceral epithelial tissue (DRACKLEY et al. 2001).
Glycerol is released from adipose tissue as a consequence of lipolysis and has therefore also some
recycling character although this can be seen as a recycling over a whole lactation cycle and not
on minute-to-minute basis as for lactate (DRACKLEY et al. 2001). The extent of body fat
mobilization in early lactation and dietary circumstances therefore likely determine the role of
glycerol in peripartal gluconeogenesis.
Pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) are two potential
rate-limiting enzymes for hepatic gluconeogenesis from precursors that enter the glucogenic path
6
BACKGROUND
prior to the triose phosphates. While carbon of propionate is metabolized to oxaloacetate via
succinyl-CoA through the tricarboxylic acid (TCA) cycle, the carbon of lactate and some
glucogenic AA like alanine are first metabolized to pyruvate and then to oxaloacetate by PC. A
second fate of pyruvate in liver is the conversion to acetyl-CoA and further metabolism through
the TCA cycle or partially oxidation to ketone bodies similar to NEFA. The enzyme PEPCK
catalyzes the conversion of oxaloacetate to phosphoenolpyruvate with subsequent glucose
formation. Its expression in nonruminants is stimulated in fasted states by glucagon and
glucocorticoids and reduced in fed states characterized by high insulin levels (PILKIS and
GRANNER 1992) and was shown to be induced by NEFA in rat hepatocytes (Massillon et al.
2003). GREENFIELD et al. (2000) examined the messenger ribonucleic acid (mRNA)
expression of PC and PEPCK in the liver of transition dairy cows around parturition and found a
7.5-fold increase in abundance of PC mRNA after calving until d +28 while they only found a
moderate increase in PEPCK mRNA by d +28 and +56. They associated the increase in PC
mRNA abundance, reflecting an increase in enzyme activity, with an elevated utilization of
lactate and/or AA for gluconeogenesis in early lactation. Hence, the increase in PC mRNA
abundance on day of calving was hypothesized to be an adaptive mechanism to maintain glucose
output and simultaneously minimize ketogenesis from non-lipid precursors (GREENFIELD et al.
2000).
1.2 Immune system and health
Early lactation features the highest incidence of production illnesses in dairy cows
(INGVARTSEN 2006). INGVARTSEN et al. (2003) analyzed date of former epidemiological
studies and stated a common lactational incidence rate for dystocia (1 - 2.1%), periparturient
paresis (0.2 - 8.9%), ketosis (0.2 - 10%), left displaced abomasum (0.6 – 6.3%), retained placenta
(3.1 – 13%), ovarian cysts (3.1 to 13%), metritis (2.2 – 43.8%), mastitis (2.8% - 39%) and
lameness (1.8 – 60%) whereby great variability is likely due to varying definitions of the
examined disease. The incidence of subclinical ketosis with BHB concentrations of 1.2 – 2.9
mmol/L amounted to 43.2% with a peak prevalence at 5 days in milk (DIM) (28.9%) and was
7
BACKGROUND
related to higher risk of displaced abomasum, removing from the herd, a decreased milk
production and conceiving to first service (MCART et al. 2012). The risk of ketosis increases
with parity (GRÖHN et al. 1984) and a BCS of 3.5 or more at calving (GILLUND et al. 2001). A
Dutch study indicates that a moderate fatty liver infiltration (more than 50 mg TAG in 1 g wet
liver tissue) can be found in about 50% of cows in early lactation (JORRITSMA et al. 2000).
Important risk factors are high BCS and overfeeding in late lactation and dry period (FRONK et
al. 1980) and low DMI around calving (BERTICS et al. 1992) with subsequent high rates of lipid
mobilization. Hormonal changes and a higher incidence of disease are other contributing factors
to an increased mobilization of NEFA at calving (GOFF AND HORST 1997). Fatty liver was
associated with an increased risk of displaced abomasum, ketosis, mastitis and metritis (BOBE et
al. 2004) and a deteriorated reproductive performance (WENSING et al. 1997).
Earlier research has stated a great number of changes in immune cell populations and functions
that may be underlying mechanisms of an increased susceptibility to infectious diseases in early
lactation (ZERBE et al. 2000). An impaired phagocytic function of blood lymphocytes and
neutrophils has been reported for the time around calving (NEWBOULD 1976, ISHIKAWA
1987, DETILLEUX et al. 1995) whereby high circulating concentrations of ketone bodies and
NEFA seem to be aetiologically involved.
LACETERA et al. (2005) linked a massive lipomobilization at calving to an alteration of
lymphocyte functions and suggested that especially high condition cows are at high risk of
infection in the periparturient period. Likewise, they tested the influence of various
concentrations of NEFA on lymphocyte functions of heifers in vitro and stated a diminished
deoxyribonucleic acid (DNA) synthesis, Immunoglobulin (Ig) M and Interferon-γ secretion
(LACETERA et al. 2004) as well as a negative impact on peripheral blood mononuclear cell
(PBMC) proliferation (LACETERA et al. 2010). Analogous to that, a study of SCALIA et al.
(2006) provided evidence for the regulation of viability and reactive oxygen species (ROS)
generation of bovine polymorphonuclear leukocytes (PMN) by high concentrations of NEFA in
vitro. SURIYASATHAPORN et al. (1999) investigated the chemotaxis capacity of bovine
leukocytes and stated that it is lower in leukocytes from cows with high values of BHB and that it
8
BACKGROUND
is impaired in an environment with high concentrations of ketone bodies in vitro. Similarly,
HOEBEN et al. (1999) described an inhibiting effect of ketone bodies on the proliferation of
bovine bone marrow cells and on oxidative burst activity of PMN.
An impaired function of lymphocytes was additionally shown to be related to a decreased T-cell
population (SHAFER-WEAVER et al. 1996) or a shift in the ratio between lymphocytes
subpopulations (helper vs. cytotoxic cells) at calving (SAAD et al. 1989). HARP et al. (1991)
reported an increased proportion of CD4+ cells after calving while CD8+ proportion did not
change in 8 multiparous Holstein cows. Contrary, KIMURA et al. (1999) investigated
lymphocyte populations of 8 periparturient Jersey cows and found a decline by 25% in CD4 +
lymphocytes that reached a nadir at parturition but no statistical changes in proportion of CD8+
lymphocytes or the CD4+:CD8+ ratio. Finally, VAN KAMPEN and MALLARD (1997) detected
a decrease in CD4+ and CD8+ subset proportions, especially between 3 weeks AP and the week
of calving. Although results were quite different between studies, all found changes relative to
calving that may influence the immune response to and recovery from infection and disease
(TAYLOR et al. 1995, BRODERSEN and KELLING 1999). Interestingly, KIMURA et al.
(2002) showed that all T-lymphocyte subtypes decreased at parturition in intact cows in contrast
to no changes in mastectomized animals. This further emphasizes the great influence of the onset
of lactation with metabolic and endocrine changes on immune cell populations and certainly
more components of the immune system.
ROS are formed normally as by-products of cellular metabolism but they are also part of the host
defense mechanisms against infectious diseases (MILLER et al. 1993). Oxidative burst describes
the massive ROS production in context of the phagocytosis process mediated by the
multicomponent enzyme NADPH-oxidase (NOX) (DAHLGREN and KARLSSON 1999).
Besides, ROS were reported to be involved in the expression of cell signaling molecules
(DEVASAGAYAM et al. 2004) and in the optimization of the inflammatory response
(KVIETYS and GRANGER 2012). However, an imbalance between ROS production and
availability of antioxidative defenses is known as oxidative stress (SIES 1991) and can lead to
peroxidative damage of lipids, proteins, polysaccharides, DNA and other macromolecules and
therefore alter cell function (MILLER et al. 1993).
9
BACKGROUND
Dairy cows are confronted with massive oxidative stress in transition period as they experience
dramatic physiological changes with an increased oxygen metabolism that may lead to a
depletion of important antioxidative defenses (BERNABUCCI et al. 2002, SORDILLO and
AITKEN 2009). This may be another contributing factor to periparturient health disorders and
influence metabolic status in dairy cattle (MILLER et al. 1993, BERNABUCCI et al. 2005).
According to that, there have been shown modulatory effects on bovine inflammatory responses
by several micronutrients with antioxidative capabilities. Supplementation of vitamin E and/or
selenium reduced the incidence of mastitis and retained placenta in dairy cows (SPEARS and
WEISS 2008, SORDILLO and AITKEN 2009). A high BCS AP and great BCS loss PP together
with high concentrations of NEFA and BHB in early lactation are associated with a higher
sensitivity to oxidative stress (BERNABUCCI et al. 2005). Besides, NEFA are known to activate
the NOX-dependent ROS production of neutrophils (SCHÖNFELD and WOJTCZAK 2008).
Finally, transition period is characterized by inflammatory conditions that are likely a result of
proinflammatory cytokine release as a consequence of metabolic and environmental stress,
infection or endotoxin release from the rumen because of feeding practices (BERTONI et al.
2008). Main effects concern nutrient partitioning, anorexia, reproductive activity, lipolysis and
liver synthesis where an acute phase response is induced (FLECK 1989, DRACKLEY et al.
2005). It is characterized by induction of acute phase proteins synthesis (e.g. haptoglobin and
ceruloplasmin) and the impairment of hepatic synthesis of negative acute phase proteins such as
albumin and retinol binding protein (FLECK 1989). In fact, an increased mRNA abundance for
several proteins in liver involved in the inflammatory response at d 1 PP was observed (LOOR et
al. 2005). Uncontrolled inflammation is a dominant factor in early lactation disorders like metritis
and mastitis and is highly influenced by an altered lipid metabolism and oxidative stress
(SORDILLO et al. 2009).
10
BACKGROUND
2. Monensin
2.1 Characteristics and mode of action in rumen
Monensin belongs to the ionophore antimicrobial drugs and was first discovered as an active
compound produced by a strain of Streptomyces cinnamonensin (AGTARAP et al. 1967).
Besides their application as coccidiostats for poultry, ionophores were used in ruminant nutrition
for improvement of feed efficiency and growth promotion (RUSSELL and STROBEL 1989)
until the ban on antibiotics as feed additives in the European Union in 2003 (Directive
1831/2003/CCE, European Commission). Recently, monensin was re-launched in the European
Union as a CRC indicated for transition dairy cows.
a)
b)
Figure 1: Schematic representation (a) and complete crystal sturcture (b) of monensic acid.
From: LOWICKI and HUCZYSKI (2013).
Ionophores are compounds of moderate molecular weight that form lipid-soluble complexes with
polar cations, especially K+, Na+, Ca2+, Mg2+ and biogenic amines (PRESSMAN 1976) and act as
vehicles for transporting ions across biological membranes (PRESSMAN and FAHIM 1982)
what provides them toxic properties against many bacteria, protozoa and fungi. Ion exchange
among the cell membrane is dependent on selective cation affinities of the ionophore and cation
gradients among the membrane (RUSSELL and STROBEL 1989). Monensin is an antiporter
with a high selectivity for Na+ but it also has the ability to translocate K+. RUSSELL (1987)
examined the mode of action of monensin in cultures of the Gram-positive rumen bacteria
11
BACKGROUND
Streptococcus bovis and reported mainly a K⁺ efflux and an intracellular accumulation of protons
(H⁺) after addition of monensin. To maintain ion balance and intracellular pH, he proposed that
the cells expel the excess of protons via utilization of adenosine triphosphate (ATP) until the
depletion of the ATP pools lead to a growth inhibition. Gram-negative bacteria which are
associated with succinate and propionate production possess a natural protective barrier against
monensin as the outer membrane is impermeable for hydrophobic substances and molecules with
sizes of ionophores (RUSSELL and STROBEL 1989). Gram-positive bacteria that produce
primary acetate, butyrate, hydrogen, lactate, ammonia and formate are inhibited by ionophores
(BERGEN and BATES 1984).
Therefore, a major effect proposed for monensin is a shift of the ruminal fermentation pattern
towards an increased propionate and decreased acetate production. Likewise, long-term
continuous culture fermentation studies showed an increased propionate and a decreased acetate
and butyrate proportion after addition of monensin (BUSQUET et al. 2005) similar to in vivo
results (VAN MAANEN et al. 1978, SAUER et al. 1998). Additionally, an increased production
of propionate as a reductive step in the context of anaerobic fermentation in the rumen may
redirect hydrogen away from methane production and therefore contribute to a reduced methane
emission of the cow and a concomitant reduction of loss of feed energy (VAN NEVEL and
DEMEYER 1977,
VAN NEVEL and DEMEYER 1996). In fact, there has been shown a
diminished methane production in cattle after supplementation of monensin (THORNTON and
OWENS 1981, ODONGO et al. 2007). Monensin was shown to inhibit Gram-positive “hyperammonia producing” (HAP) bacteria known to produce high amounts of ammonia (RUSSELL et
al. 1988, PASTER et al. 1993). Analogous to that, MCGUFFEY et al. (2001) reported that
protein and AA degradation in the rumen were reduced in vitro with monensin leading to the
assumption, that more protein of dietary origin reaches the small intestine.
2.2 Effects on performance and health of dairy cows
Supplementation of monensin in transition dairy cattle has been examined to a large extent in the
last decades. Fortunately, a recent meta-analysis examined data of studies providing monensin
12
BACKGROUND
orally as a controlled-release capsule (CRC) or top dress to summarize the effects of treatment
across studies and to investigate factors explaining potential heterogeneity of response
(DUFFIELD et al. 2008 a, b, c). Main influence factors were delivery method, stage of lactation,
dose and diet besides herd, BCS and genetic merit.
The metabolic effects of monensin include a reduction of blood concentrations of BHB by 13%,
of NEFA by 7%, an increase of glucose by 3% and of urea by 6%. No effects were detected for
cholesterol, calcium, milk urea or insulin. Greatest effects of monensin on BHB concentration
were found in early lactation (DUFFIELD et al. 2008 a).
Impacts of monensin on production parameters were a decrease in DMI by 0.3 kg/d, an increase
in 305-d milk yield by 0.7 kg/d and a 2.5% improved milk production efficiency. While milk fat
yield was not changed, the percentage was decreased by 0.13%. Milk protein percentage was
decreased by 0.03% while yield was increased by 0.016 kg/d with monensin. No effects were
detected for milk lactose. These results indicate an improvement of energy metabolism and milk
production efficiency by monensin.
Evaluation of effects on health and reproduction data showed a decreased risk of ketosis and
displaced abomasum by 25% and mastitis by 9% while no effects were found for milk fever,
lameness, dystocia, retained placenta, metritis, first-service conception risk or days to pregnancy.
3. Essential Oils
3.1 Characteristics and mode of action in rumen
Essential oils are volatile, complex secondary metabolites obtained from plant material whereby
steam distillation is the most commonly method used for commercial production of these
aromatic oily liquids. Of the over 3000 different known essential oils, 300 are of commercial
importance (BURT 2004). In nature, they are responsible for odor and color of plants. They play
an important part in the communication between plants and their environment and in the
protection of plants as antibacterials, antivirals, antifungals and insecticides (DEANS and
RITCHIE 1987, SMITH-PALMER et al. 1998, BAKKALI et al. 2008). The main compounds of
essential oils are included in two chemical groups that are synthesized through different
13
BACKGROUND
metabolic pathways from precursors of the plant’s primary metabolism: terpenoids and
phenylpropanoids.
a)
b)
Figure 2: Chemical structures of the essential oil compounds eugenol (a) und thymol (b) that are
part of the product CRINA® Ruminants, DSM, Basel, Switzerland. From: BAKKALI et al.
(2008).
Essential oils can accumulate in the lipid bilayer of bacteria because of their lipophilic character
due to the cyclic hydrocarbons structure. The subsequent changes in membrane structure and
fluidity cause a leakage of ions across the cell membrane and a decreased transmembrane ionic
gradient. In most cases, bacteria can equalize the decreasing transmembrane ionic gradient by
using ionic pumps which leads to a depletion of energy and hence growth inhibition (SIKKEMA
et al. 1994). ULTEE (1999) investigated the Gram-positive bacterium Bacillus cereus and
reported a depletion of intracellular ATP pool after treatment with carvacrol, the major essential
oils of thyme and oregano that can be related either to a reduced rate of ATP synthesis or an
increased ATP hydrolysis. Additionally, he described secondary effects of essential oils like
inhibition of enzymes and reducing metabolic activity. Further modes of action proposed for
essential oils are the hydroxyl group of phenols acting as a transmembrane carrier of cations that
is similar to ionophores (ULTEE et al. 2002) or the inhibition of the synthesis of proteins, RNA
and DNA of the cell by allicin, a component found in garlic (FELDBERG et al. 1988).
Gram-positive bacteria are more sensitive to antimicrobial effects of essential oils than Gramnegative bacteria (SMITH-PALMER et al. 1998, CHAO et al. 2000). NIKAIDO and HIROSHI
(1985) considered the strong hydrophilicity of the outer membrane due to the presence of
lipopolysaccharides (LPS) as a natural permeability barrier against essential oils. But the outer
membrane of Gram-negative bacteria is not a barrier for all hydrophobic substances as small
molecules with a high hydrogen-bonding capacity can inhibit the growth of these microorganisms
14
BACKGROUND
as described for thymol and carvacrol (HELANDER et al. 1998,
GRIFFIN et al. 1999).
Unfortunately, this activity against Gram-positive and –negative bacteria reduces the selectivity
of these compounds against specific populations, making the modulation of rumen microbial
fermentation more difficult. Moreover, there is a great variation in results of actual in vivo and in
vitro rumen fermentation and dairy performance studies as different combinations of essential
oils or their purified components and different doses were administered. Interactions between
components may lead to antagonistic, additive or synergistic effects (BASSOLÉ and JULIANI
2012) and feed composition and animal physiology may play an additional role.
Based on aforementioned and as we used a defined, patented mixture of natural and natureidentical essential oil compounds in our study, further discussion will concentrate on this
commercial product of blended essential oils. It includes thymol, guaiacol, eugenol, vanillin and
limonene as its main components on an organic carrier (CRINA® Ruminants, DSM,
Switzerland). These 5 essential oils were tested separately in vitro by CASTILLEJOS et al.
(2006). Most of these compounds demonstrated their antimicrobial activity by decreasing total
SCFA concentration at high doses. Eugenol at 5 mg/L rumen fluid in 24-h batch fermentation
reduced the proportion of acetate and the acetate to propionate ratio while at 500 mg/d it reduced
the proportion of propionate. In a continuous culture fermenter study with different doses of
eugenol und thymol (6 days of adaptation and 3 days of sampling) only a dose of 5 mg/L of
thymol tended to reduce acetate proportion and increased proportion of butyrate without
decreasing total SCFA concentration whereas thymol and eugenol at 500 mg/L the decrease in
acetate and increase in propionate proportion was accompanied by a decreased total SCFA
production. Results underline that effects on rumen fermentation are highly dependent upon the
applied doses and that there might be a potential adaptation of microorganisms to essential oils
supplementation suggesting that short-term in vitro studies should be interpreted with caution
(CASTILLEJOS et al. 2006).
Reported changes in SCFA production in the rumen after supplementation of CRINA® are
inconsistent (PATRA 2011). CASTILLEJOS (2005) reported an increased total SCFA
concentration without affecting individual SCFA proportions in vitro at 1.5 mg/l of CRINA® and
an increased acetate to propionate ratio in rumen fluid of sheep at 110 mg/d of CRINA®
15
BACKGROUND
(CASTILLEJOS et al. 2007). No changes in total and individual proportions of SCFA production
were found in sheep receiving 110 mg/d of CRINA® (NEWBOLD et al. 2004). A reduced
methane production could not be verified in vivo at 1 g/d of CRINA® (BEAUCHEMIN and
MCGINN 2006) or 1 and 2 g/d of CRINA® (TOMKINS et al. 2015) like it was reported for
example for thymol in vitro (EVANS and MARTIN 2000). Examinations of the effect of
CRINA® on microbial populations in ruminal fluid showed an inhibition of HAP bacteria
accompanied by a decreased AA deamination similar to monensin (MCINTOSH et al. 2003,
WALLACE 2004). But that effect varied as not all HAP species were equally sensitive
(MCINTOSH et al. 2003) and the reduction in the number of HAP was higher when a low
protein diet was fed (WALLACE et al. 2002). Commonly, essential oils show a less distinct
effect on deamination than monensin due to the assumption that EO effect fewer species
(WALLACE 2004).
3.2 Effects on performance and health of dairy cows
Only a few studies have been conducted in vivo to evaluate the influence of CRINA® on
ruminant metabolism and performance. A study of TASSOUL and SHAVER (2009) was the only
one situated in transition period as they fed 1.2g/cow/d of CRINA® to 40 Holstein cows from 3
weeks AP until 15 weeks in lactation. Essential oils supplementation decreased DMI in the
lactation period by 1.8 kg/d. Metabolic parameters like glucose, NEFA and BHB stayed
unaltered, as did milk yield. Milk protein content was 0.15% less for essential oils. BENCHAAR
et al. (2006) supplemented CRINA® to 4 ruminal cannulated lactating Holstein cows (98 ± 7
DIM) in a Latin-square-design. A dose of 2 g/d increased ruminal pH (6.50 vs. 6.39) but had no
effect on milk production or ruminal fermentation parameters. Similarly, a dose of 750 mg/d in
lactating Holstein cows (98 ± 7 DIM) only resulted in an increased ruminal pH (6.40 vs 6.30) and
higher milk lactose content (4.78% vs. 4.58%) while the rest of ruminal and milk parameters
were unaltered (BENCHAAR et al. 2007). A study with 30 lactating Holstein cows and heifers
(118 ± 70 DIM) provided 1.2 g/d of CRINA® for 9 weeks and detected an increased DMI by 1.9
kg/d and an increased milk production of 2.7 kg 3.5% fat-corrected milk/d. In this study, all cows
16
BACKGROUND
underwent a 2 weeks adaptation period when they all received 0.6 g/d of CRINA® before being
allocated to control and essential oils treatment, probably making the comparability to other
studies more difficult.
The therapeutic properties of plants and spices are known since ancient times (EDRIS 2007).
Different compounds of essential oils have successfully been tested for anticancer, antibacterial,
antiviral, antioxidative property and in the treatment and prevention of cardiovascular diseases
including atherosclerosis and thrombosis in humans (EDRIS 2007) and an enhanced
immunocompetence and health of gut and a better performance of broilers and pigs (MICHIELS
et al. 2010, TIIHONEN et al. 2010), but studies on the effects on the immune system of cows are
very rare. ANASSORI et al. (2015) investigated the influence of raw garlic and garlic oil on
blood profile of 4 ruminal cannulated rams in a Latin-square-design with 28-d periods and found
no effects on blood BHB, NEFA, glucose, total triglycerides, cholesterol, total protein, albumin
and urea nitrogen but an increase in insulin concentration. A study with 20 Baluchi lambs (3
month old) receiving 400 mg/d of a mixture of EO containing thymol, carvacrol, eugenol,
limonene and cinnamaldehyde detected no change in plasma concentration of glucose, urea, total
protein and cholesterol while triglyceride concentration was lower (MALEKKHAHI et al. 2015).
17
SCOPE OF THE THESIS
SCOPE OF THE THESIS
A smooth periparturient transition from late gestation to early lactation is the basis for a healthy
and economic lactation period of the dairy cow. Ionophore antimicrobial drugs have been
successfully tested for beneficial effects on energy metabolism, performance and health of dairy
cows via a modulation of ruminal fermentation. However, the relaunch of monensin in the
European Union as a pharmaceutical for transition cows after the ban as a feed additive in 2006
aroused public attention in the light of possible residues in milk and meat, bacterial development
of antibiotic resistances and as Kexxtone® is attributed a doping-relevant character to mask
husbandry, feeding and management deficits in modern dairy cow farming. The quest for natural
alternatives to monensin is subject of recent research whereby positive experiences have been
made with dietary use of essential oils in different animal species and also in some parts of cattle
nutrition.
A commonly accepted theory is that monensin alters ruminal fermentation patter towards an
increased propionate production. As propionate is the major precursor of hepatic
gluconeogenesis, this should contribute to an improved energy metabolism and better
performance and health of treated cows. Essential oils may also alter rumen fermentation
similarly although results are inconsistent. Accordingly, the hypothesis of the here presented
study was, that essential oils have comparable antiketogenic effects as the ionophore monensin.
Thus, the effects of monensin and essential oils on ruminal fermentation and protozoa
populations, milk performance and their energy household were studied in dairy cows in the
transition period (PAPER I).
In addition, it was hypothesized that antiketogenic effects of monensin and essential oils will
improve humoral and cellular immune cell function of dairy cows in the transition period.
Although effects on incidences of production illnesses have been evaluated after monensin
supplementation, less information is available for underlying mechanism and possible direct or
indirect effects of monensin or essential oils on immune cell function. Hence, we evaluated a
broad spectrum of immune parameters including blood metabolites (PAPER I, II), white and
18
SCOPE OF THE THESIS
red blood cell profile as well as function parameters of PBMC and PMN and antibody production
after Bovine Viral Diarrhea virus (BVDV) vaccination (PAPER II).
Figure 3: Scheme of the collected data of the present trial as it is presented in Paper I and II.
In this context, an animal model was applied that generated groups with different degrees of
metabolic stress at calving due to feeding strategies and body condition management in the weeks
around calving. A total of 60 multiparous German Holstein cows with a mean parity of 2.3 ± 1.4
(Standard Deviation) were allocated 6 weeks AP to either high condition (n = 45) or low
condition group (LC, n = 15) according to their BCS. High condition cows were overfed in the
dry period with a 60% concentrate proportion in the daily ration in comparison to 20% in the LC
group. After calving, the concentrate proportion was raised from initially 30% to 50% in all
19
SCOPE OF THE THESIS
cows. This increase was decelerated (3 vs. 2 weeks) in high condition cows to further stimulate
PP lipolysis. The high condition cows were subdivided into 1 control group (HC, n = 15), one
group receiving a monensin CRC (HC/MO, n = 15) and 1 group receiving a commercial blend of
essential oils (HC/EO, n = 15). All cows remained on treatment until 56 days in milk (DIM).
20
PAPER I
PAPER I
Effect of monensin and essential oils on performance and energy metabolism
of transition dairy cows
C. Drong1, U. Meyer1, D. von Soosten1, J. Frahm1, J. Rehage2, G. Breves3 and S. Dänicke1
1
Institute of Animal Nutrition, Friedrich-Loeffler-Institut, Federal Research Institute for Animal
Health, Braunschweig, Germany
2
3
Clinic for Cattle, University of Veterinary Medicine, Foundation, Hannover, Germany
Institute of Physiology, University of Veterinary Medicine, Foundation, Hannover, Germany
Journal of Animal Physiology and Animal Nutrition (JAPAN)
Volume 100
DOI: 10.1111/jpn.12401
Printed with kind permission of John Wiley and Sons
21
PAPER I
Summary
The present work examined preventive effects of a dietary and a medical intervention measure on
post partum (p.p.) ketogenesis in dairy cows over-conditioned in late pregnancy. 60 German
Holstein cows were allocated 6 weeks ante partum (a.p.) to 3 high body condition score (BCS)
groups (BCS 3.95 ± 0.08) and 1 low BCS group (LC, BCS 2.77 ± 0.14). Concentrate proportion
in diet a.p. was higher (60% vs. 20%) and increase in proportion p.p. from 30% up to 50%
decelerated (3 vs. 2 weeks) in high BCS groups. High BCS cows received a monensin controlledrelease capsule (CRC) (HC/MO), a blend of essential oils (HC/EO) or formed a control group
(HC). Performance parameters and energy status were evaluated in 3 periods (day (d) -42 until
calving, 1 until 14 days in milk (DIM), 15 until 56 DIM). Feed efficiency was 65% and 53%
higher in HC/MO than in LC (p < 0.001) and HC group (p = 0.002) in the second period. Milk fat
content was higher in HC/EO (5.60 vs. 4.82%, p = 0.012) and milk urea higher in HC/MO (135
mg/kg) than in LC cows (107 mg/kg, p < 0.001). Increased p.p. levels of non-esterified fatty
acids in serum were found in HC (p = 0.003), HC/MO (p = 0.068) and HC/EO (p = 0.002) in
comparison to LC cows. Prevalence of subclinical and clinical ketosis was 54% and 46%,
respectively, in HC group. Monensin decreased the prevalence to 50% and 7%, respectively.
Ruminal fermentation pattern showed higher proportions of propionate (23.43 mol% and 17.75
mol%, respectively, p < 0.008) and lower acetate:propionate ratio (2.66 vs. 3.76, p < 0.001) in
HC/MO than HC group. Results suggest that a monensin CRC improved energy status and feed
efficiency of transition dairy cows while essential oils failed to elicit any effect.
Introduction
In the transition period, the high-yielding dairy cow is confronted with a massive change in the
metabolic status. The reduction of feed intake is accompanied by an increase of the nutrient
demand for the growth of the fetus and the initiated lactation (GRUMMER 1995).
As glucose demand of the cow exceeds glucose supply from digestible energy, gluconeogenesis
in liver is stimulated. Propionate is the major glucogenetic precursor taken up by the liver, with
an estimated proportion of up to 32-73% of gluconeogenesis (SEAL and REYNOLDS 1993).
22
PAPER I
Metabolic adaptations are aimed at counterbalancing the negative energy balance by a massive
mobilization of fatty acids from the adipose tissues.
The limited capacity of the liver to metabolize non-esterified fatty acids (NEFA) and the lack of
oxaloacetate are responsible for the production of ketone bodies from acetyl-CoA instead of
using it for β-oxidation in the liver. Although the conversion of NEFA to ketone bodies in the
liver may be an additional strategy in times of negative energy balance as ketone bodies can be
oxidized by the heart, kidney, skeletal muscle, mammary gland and gastrointestinal tract of
ruminants (HEITMANN et al. 1987), a glucose deficit together with excessive fatty acid
mobilization und ketogenesis can lead to serious metabolic disorders and an impaired health and
productivity of transition dairy cows (BERGMAN 1971).
There has been massive interest in studying effects and mechanisms of essential oils on ruminal
fermentation since the ban on antibiotics as feed additives in the European Union (Directive
1831/2003/CCE, European Commission, 2003). Recently, monensin was launched in the EU as a
controlled-release capsule (CRC) indicated for transition over-conditioned dairy cows.
Numerous reviews currently summarized the impact of monensin and essential oils on ruminal
fermentation, metabolism and performance of dairy cows (MCGUFFEY et al. 2001,
IPHARRAGUERRE and CLARK 2003, CALSAMIGLIA et al. 2007, PATRA 2011). Monensin
belongs to the ionophore antibiotics and was first discovered as an active compound produced by
a strain of Streptomyces cinnamonensin (AGTARAP et al. 1967). Ionophores form lipid- soluble
complexes with polar cations (PRESSMAN 1976) and act as vehicles for transporting ions across
biological membranes (PRESSMAN and FAHIM 1982) what provides them toxic properties
against many bacteria, protozoa and fungi.
As Gram-negative bacteria are resistant to monensin because of the presence of an outer
membrane (RUSSELL and STROBEL 1989), the ruminal fermentation pattern changes towards
an increased production of propionate with a concomitant reduction in methane (BERGEN and
BATES 1984, SAUER et al. 1998). Additionally, ruminal degradation of peptides and amino
acids is reduced (MCGUFFEY et al. 2001,
IPHARRAGUERRE and CLARK 2003). The
impacts of monensin on transition cows include an improved energy status, feed efficiency and
animal health (DUFFIELD et al. 2008 a, b, c).
23
PAPER I
Essential oils are volatile, complex secondary metabolites obtained from plants by steam or
hydro-distillation and naturally-occurring play an important part in the communication between
plants and their environment and in the protection of plants as antibacterials, antivirals,
antifungals and insecticides (DEANS and RITCHIE 1987,
SMITH-PALMER et al. 1998,
BAKKALI et al. 2008). Modes of action of essential oils are an accumulation in the lipid bilayer
of bacteria and subsequent changes in membrane structure and fluidity (SIKKEMA et al. 1994),
an inhibition of enzymes (ULTEE et al. 1999), acting as transmembrane carriers of cations
similar to ionophores (ULTEE et al. 2002) and an inhibition of the synthesis of proteins, RNA
and DNA of the cells (FELDBERG et al. 1988). Although Gram-negative bacteria are less
sensitive to essential oils (SMITH-PALMER et al. 1998, CHAO et al. 2000) due to a strong
hydrophilicity of the outer membrane (NIKAIDO and VAARA 1985), a growth inhibition was
reported for Gram-positive and -negative bacteria (HELANDER et al. 1998, GRIFFIN et al.
1999) reducing the selectivity of these compounds against specific bacterial populations and
consequently making the modulation of rumen microbial fermentation more difficult.
Although results are not consistent, essential oils are also assigned the attributes to increase
propionate and decrease methane production and to modify peptidolysis and deamination in
rumen fermentation (CALSAMIGLIA et al. 2007, PATRA 2011). Reports about the subsequent
effects on performance parameters are inconsistent and often use different compounds and/or
doses. There is especially little information available if essential oils are able to improve the
energy status of cows.
Therefore, a recently established animal model that enables generating animal groups being in a
ketogenic metabolic status by a specific combination of the factors high BCS at the beginning of
the transition period, overfeeding in the dry period and a decelerated energy supply p.p.
(SCHULZ et al. 2014) was used for testing of possible effects of monensin and essential oils on
the energy status and performance of transition dairy cows.
24
PAPER I
Material and Methods
Experimental Design
The experiment was carried out at the experimental station of the Institute of Animal Nutrition,
Friedrich-Loeffler-Institute (FLI), Braunschweig, Germany.
60 multiparous German Holstein cows were allocated 6 weeks a.p. to 2 experimental groups by
the main criterion BCS (5-point scale) (EDMONSON et al. 1989) and further consideration of
milk yield, milk composition and body weight of the previous lactation period. Low BCS group
(LC) was formed by 15 cows with a mean BCS of 2.77 ± 0.14 (± SD) and a mean parity of 1.7 ±
0.9 (± SD), representing a positive control group. The remaining 45 cows were assigned to high
BCS group with a mean BCS of 3.95 ± 0.08 (± SD). The cows of high BCS group were assigned
to a negative control group (HC, n = 15, parity 2.5 ± 1.4) and 2 treatment groups receiving either
monensin (HC/MO, n = 15, parity 2.6 ± 1.3) or essential oils (HC/EO, n = 15, parity 2.4 ± 1.6).
All cows remained on treatment until 56 days in milk (DIM). The experiment was divided into 3
periods. Period 1 (d -42 until calving), period 2 (1 until 14 DIM) and period 3 (15 until 56 DIM).
Ingredients and chemical composition of the experimental diet is shown in Tab. 1. In the first
period, LC cows were fed according to the recommendations of the German Society of Nutrition
Physiology (GFE, 2001) with an energetic adequate ration of 80% roughage (50% maize silage,
50% grass silage) and 20% concentrate based on DM content. The high BCS groups received an
energetic oversupply through an increased concentrate feed proportion of 60% of the daily ration.
After calving, all cows were fed with a standardized total mixed ration (TMR) adjusted for
lactation requirement with an initial concentrate feed proportion of 30%. This ratio was raised
stepwise to 50% of the daily ration. This increase was decelerated (3 vs. 2 weeks) in the high
BCS groups to additionally stimulate p.p. lipolysis (SCHULZ et al. 2014). TMR was provided ad
libitum via self-feeding stations (type RIC, Insentec B.V., Marknesse, The Netherlands).
25
PAPER I
Table 1 Ingredients and chemical composition of concentrate and roughage of the experimental diet. High BCS (HC)
cows were fed a concentrate proportion of 60%; low BCS (LC) cows was fed a concentrate proportion of 20% in the
prepartum diet. Post calving, the concentrate proportion in the diet was increased from 30% to 50% within 2 weeks
in LC group and within 3 weeks in HC groups. HC/MO was administered a monensin controlled-release capsule 3
weeks antepartum (a.p.), HC/EO received 1 g/d of a blend of essential oils from week 3 a.p. until 56 days in milk.
CON†
Ingredient, %
Soybean meal
Rapeseed meal
Wheat
Corn
Dryed sugarbeet pulp
Soybean oil
Vitamin/mineral premix**
Vitamin/mineral premix††
Calcium carbonate
Essential oils (EO)
15.8
11.0
46.0
21.2
5.0
1.0
-
Concentrate
EO‡
Pre§
Post
15.8
11.0
46.0
21.1
5.0
1.0
0.1
15.8
11.0
44.0
20.8
5.0
1.0
1.2
1.2
-
10.0
20.0
46.2
20.8
1.0
2.0
-
¶
Roughage*
Maize silage
Grass silage
Analysed chemical profile
Dry matter (DM), g/kg
857
856
868
868
355
380
Nutrient, g/kg of DM
Crude ash
36
34
46
53
39
113
Crude protein
195
197
196
195
72
115
Ether extract
41
38
44
38
31
25
Crude fiber
57
51
57
52
197
317
NDF
184
182
181
174
422
581
Energy‡‡, MJ/kg of DM
ME
14.4
14.4
13.2
13.8
11.4
9.2
NEL
9.3
9.2
8.3
8.9
7.0
5.4
Values are Means; * Roughage consisted of 50% maize silage, 50% grass silage on dry matter basis; † Control
concentrate (CON) was provided at 1 kg/d/cow in LC, HC, HC/MO; ‡ Essential oils concentrate (EO) was provided
at 1 kg/d/cow in HC/EO from d -21 until d 56 relative to calving, so that every cow received 1 g/d of the product
(CRINA® ruminants, DSM, Basel, Switzerland); § Concentrate prepartum (Pre) was provided in a 20:80 concentrate
to roughage ratio in LC and in a 60:40 concentrate to roughage ratio in the HC groups in the TMR; ¶ Concentrate
postpartum (Post) was provided in a 30:70 concentrate to roughage and changed stepwise to 50:50 within 2 weeks in
LC and 3 weeks in HC groups; ** For dry dairy cows. Ingredients per kg mineral feed: 60 g Ca; 100. 5 g Na; 80 g P;
50 g Mg; 7 g Zn; 4.8 g Mn; 1.3 g Cu; 100 mg I; 40 mg Se; 30 mg Co; 800,000 IU vitamin A; 100,000 IU vitamin D 3;
1500 mg vitamin E; †† For lactating dairy cows. Ingredients per kg mineral feed: 140 g Ca; 120 g Na; 70 g P; 40 g
Mg; 6 g Zn; 5.4 g Mn; 1 g Cu; 100 mg I; 40 mg Se; 25 mg Co; 1,000,000 IU vitamin A; 100,000 IU vitamin D 3;
1,500 mg vitamin E; ‡‡ Calculation based on nutrient digestibilities measured with wethers GfE (1991) and on
equations for calculation of energy content in feedstuffs published by GfE (2001, 2008).
26
PAPER I
In addition, concentrate was provided by a computerized concentrate feeding station (Insentec,
B.V., Marknesse, The Netherlands). The HC/EO treatment included a blend of essential oils
(BEO, CRINA® ruminants, DSM, Basel, Switzerland) in the pelleted concentrate, with the target
to provide 1g/cow BEO per day from d -21 (-22 ± 7 (mean ± SD)) a.p., while other groups
received a control concentrate. The product contains a patented mixture of natural and
synthesized essential oils compounds, including thymol, eugenol, vanillin, guaiacol and limonene
(MCINTOSH et al. 2003). HC/MO was administered a monensin CRC (Kexxtone, Elanco®, Bad
Homburg, Germany) d -21 (-19 ± 5 (mean ± SD)) before expected calving, supposed to release
steadily 335mg monensin /d for a period of 95 d.
The classification of a cow as healthy, subclinical ketotic or clinical ketotic was based on the
thresholds assumed by SCHULZ et al. (2014) with β-hydroxybutyrate (BHB) values in blood
serum > 1.2 mmol/L indicative for subclinical ketosis and BHB > 2.5 mmol/L indicative for
clinical ketosis.
Measurements and Sample Collection
During the study, samples of grass and maize silage were taken twice a week and samples of
concentrate were collected once a week over a collective period of 4 weeks. The individual dry
matter intake (DMI) was recorded for the whole experimental period (computerised feeding
station: Type RIC, Insentec, B.V., Marknesse, The Netherlands). The BCS was evaluated every
experimental week by a 5-point-scale (EDMONSON et al. 1989). Animals were weighted every
week before calving. After calving, body weight (BW) was recorded twice a day post-milking.
Milking took place twice per day at 0530 and 1530 h. Meanwhile, milk yield was measured using
automatic milk counters (Lemmer Fullwood GmbH, Lohmar, Germany). Samples of milk were
collected twice a week and stored at 4°C until analysis.
Blood samples were taken on d -42, -14, -7, -3, 1, 7, 14, 21, 28, 35, 42 and 56 relative to calving
from the coccygeal vein by using serum tubes. After centrifugation (Heraeus Varifuge®, 3.0R,
2000g, 15°C, 15 min), serum was stored at -80°C until analysis.
Liver samples of approximately 30 mg of tissue each were biopsied directly after blood sampling
on d -42, -14, 7, 21 and 56 relative to calving using an automated spring-loaded biopsy device
27
PAPER I
(Bard Magnum®, Bard, UK) with a 16-gauge needle under local anesthesia (procaine
hydrochloride, Isocaine 2%, Selectavet, Weyarn-Holzolling, Germany). In total approximately
100 mg of tissue were stored at -80°C.
Samples of rumen fluid were taken by using an oral rumen tube and a hand vacuum pump from
the ventral sac of the rumen on d -42, -14, 7, 14, 21, 35, 42 and 56 relative to calving. After
centrifugation (Heraeus Varifuge®, 5000 rpm, 5 min), 1 ml of sulfuric acid (25%) was added to
10 ml of the supernatant and the samples were centrifuged again (Heraeus Varifuge®, 5000 rpm,
20 min). After the addition of 1 drop of mercuric chloride, samples were stored at -20°C until
analysis. Additionally, 15 ml of rumen fluid were mixed with 15 ml of a methylgreen-formalin
solution (OGIMOTO and IMAI 1981) and stored at 4°C for counting of the protozoal density.
Analyses
TMR and concentrate were analyzed for DM, crude ash, crude protein, ether extract and neutral
detergent fiber (NDF) according to the suggestions of the Association of German Agricultural
Analysis and Research Centres (VDLUFA, 2007).
Milk samples were analyzed for fat, protein, lactose and urea concentration using an infrared
milk analyser (Milkoscan FT 6000, Foss Electric, Hillerød, Denmark).
Blood samples were analyzed for serum concentrations of BHB, NEFA and glucose, using an
automatic analyzing system, based on photometric measurement (Eurolyser, Type VET CCA,
Salzburg, Austria).
The liver tissue was analyzed for the content of total lipid (TL) using a gravimetrical method. TL
was extracted from homogenized tissue samples with hexane: isopropanol and expressed in mg/g
fresh liver weight (mixing ratio 3:2, continual agitation for 24 h, 20°C) (STARKE et al. 2010).
Immediately after collection of rumen fluid pH was measured using a glass electrode (pH 525,
WTW, Weilheim, Germany). Ruminal ammonia nitrogen (NH3) was analyzed according to DIN
38406-E5-2 (1998). Short-chain fatty acids (SCFA) were determined according to Koch et al.
(2006) using a gas chromatograph (Hewlett Packard®, Böblingen, Gaschromatograph 5890 II).
Protozoa were counted using a Fuchs-Rosenthal chamber under an optical microscope and
differentiated into entodiniomorpha and holotricha.
28
PAPER I
Calculations
Net energy requirement for maintenance (NEM) and lactation (NEL) and milk energy
concentration were calculated based on the equations published by the German Society of
Nutrition Physiology (GFE, 2001) as follows:
NEM (MJ NEL/d) = 0.293 × BW0.75,
Milk energy concentration (MJ NEL/kg) = 0.38 × milk fat (%) + 0.21 × milk protein (%) + 0.95,
Energy requirement for lactation NEL (MJ NEL/d) = [milk energy concentration (MJ NEL/d) +
0.086] × milk yield (kg/d).
Fat-corrected milk was calculated based on the equation of GAINES (1928):
4 % FCM (kg/d) = [(milk fat (%) x 0.15) + 0.4] x daily milk yield (kg/d).
Energy-corrected milk was calculated based on the equation of SJAUNJA et al. (1990):
ECM (kg/d) = milk yield (kg/d) × [(38.3 × milk fat (g/kg) + 24.2 × milk protein (g/kg) + 16.54 ×
milk lactose (g/kg) + 20.7) / 3140].
The net energy balance was calculated as follows:
Net energy balance (MJ NEL/d) = energy intake (MJ NEL/d) – [NEM (MJ NEL/d) + NEL (MJ
NEL/d)]
During the last 6 weeks before calving, additionally 13 MJ NEL/d with an increase to 18 MJ
NEL/d for the last 3 weeks, were regarded as requirement for gestation in the calculation of net
energy balance.
Daily DMI multiplied by energy content of the TMR resulted in daily energy intake. Energycorrected milk divided by DMI resulted in feed efficiency (kg/kg).
29
PAPER I
Statistical analyses
All statistical analyses were performed using the Software SAS (Version 9.2). Parameters of
performance as DMI, milk yield and milk components were reduced to weekly means.
Performance parameters, clinical chemistry and rumen fermentation characteristics were analysed
using the MIXED procedure for repeated measures (LITTELL et al. 1998) in a compound
symmetry covariance structure. The model contained experimental treatment and period as fixed
effects and the interaction between these factors. The cow within treatment was considered as a
random effect. Effects were declared as a trend when p-values were ≤ 0.10 and significant when
P-values were ≤ 0.05 after Tukey test. Results are presented as LSMeans ± Standard Error of
Means (SEM) unless otherwise stated.
Results
Out of 60 cows, 55 animals completed the entire trial and 1 cow finished the experiment at day
28 because of an abomasal replacement. Additionally, 4 cows were excluded because of illnesses
that were not associated with the experimental treatments. Causes were abomasal displacement
and multisystemic health problems around calving (3 animals).
Over the trial, the prevalence of subclinical and clinical ketosis was 57% and 7% in LC, 54% and
46% in HC, 50% and 7% in HC/MO and 33% and 53% in HC/EO, respectively.
BCS was higher in HC, HC/MO and HC/EO than in LC group before calving and in the second
period (p < 0.001). In period 3, BCS was higher in HC/EO than in LC group (p = 0.01, Tab. 2).
Course of BW tended to be dependent on treatment (p = 0.055) as cows of HC/EO were heavier
than cows of LC treatment (p = 0.065). Additionally, there was a treatment x period interaction
detected (p < 0.001), as BW of HC/MO (p = 0.029) and HC/EO (p = 0.024) cows was higher than
in LC cows in period 1 (Tab. 2).
30
PAPER I
Table 2 Body condition score (BCS), body weight (BW), dry matter intake (DMI), net energy intake and net energy
balance of the experimental groups during the experimental period from day (d) -42 relative to calving until 56 days
in milk (DIM). High BCS (HC) cows were fed a concentrate proportion of 60%; low BCS (LC) cows were fed a
concentrate proportion of 20% in the prepartum diet. Post calving, the concentrate proportion in the diet was
increased from 30% to 50% within 2 weeks in LC group and within 3 weeks in HC groups. HC/MO was
administered a monensin controlled-release capsule 3 weeks antepartum (a.p.), HC/EO received 1 g/d of a blend of
essential oils from week 3 a.p. until 56 DIM.
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period Trt*Period
BCS
d -42 until calving
2.86b
4.02aA
4.15aA
4.13aA
0.12
d 1 until 14 DIM
2.75b
3.61aB
3.63aB
3.78aB
0.13
<0.001 <0.001
<0.001
d 15 until 56 DIM
2.61b
3.14abC
3.07abC
3.25aC
0.12
BW, kg
d -42 until calving
725bA
787abA
800aA
801aA
15
B
B
B
d 1 until 14 DIM
628
669
680
680B
15
0.055
<0.001
<0.001
d 15 until 56 DIM
601B
619C
611C
622C
15
DMI, kg/d
d -42 until calving
13.60bB
15.84abA
16.86aA
16.30abA
0.74
B
B
B
d 1 until 14 DIM
14.10
11.89
10.55
11.66B
0.87
0.726
<0.001
<0.001
A
A
A
A
d 15 until 56 DIM
17.69
16.05
15.27
15.03
0.73
Net energy intake, MJ NEL/d
d -42 until calving
79.57bB
106.06aA 115.55aA 112.74aA
5.41
B
B
B
B
d 1 until 14 DIM
92.58
76.08
69.39
78.10
6.50
0.994
<0.001
<0.001
d 15 until 56 DIM
117.57A
106.74A
101.90A
101.24A
5.37
Net energy balance, MJ NEL/d
d -42 until calving
34.20bA
69.17aA
68.72aA
70.70aA
5.54
d 1 until 14 DIM
-41.14aB -52.22abB -82.48bC -71.06abC
7.88
0.071
<0.001
<0.001
aB
abB
abB
bB
d 15 until 56 DIM
-16.98
-22.58
-36.74
-37.40
4.73
Values are LSMeans; abc Least square means within row with different superscripts differ (p < 0.05); ABC Least square
means within column with different superscripts differ (p < 0.05); * Standard Error of Means (SEM), averaged;
†
Treatment (Trt): High condition (HC, n = 13) cows were fed a concentrate proportion of 60%; low condition (LC, n
= 14) cows a concentrate proportion of 20% in the prepartum diet. Postcalving, the concentrate proportion in the diet
was increased from 30% to 50% within 2 weeks in LC group and 3 weeks in HC groups. HC/MO (n = 14) was
administered a monensin CRC d -21, HC/EO (n = 15) received 1 g/d of a blend of essential oils from d -21 until 56
DIM.
A treatment x period interaction was detected for DMI (p < 0.001), driven primarily by a 3.26 kg
higher DMI per day of HC/MO cows than of LC cows in the first period (p = 0.088, Tab. 2).
The observed treatment x period interaction concerning net energy intake (p < 0.001) was due to
a higher value in HC, HC/MO and HC/EO than in LC group before calving (p < 0.05, Tab. 2).
31
PAPER I
The calculated net energy balance (EB) tended to differ according to treatment (p = 0.071) and a
treatment x period interaction was detected (p < 0.001). In period 1, EB of HC, HC/MO and
HC/EO cows was higher than of LC cows (p < 0.05). HC/MO cows showed a more negative EB
in comparison to LC cows in period 2 (p = 0.013). In period 3, EB of HC/EO cows were more
negative than in LC group (p = 0.096, Tab. 2).
Milk parameters are presented in Tab.3. Milk yield, FCM, ECM, content and yield of milk
protein and milk lactose did not differ between the treatments. Milk fat content was
approximately 0.78% higher in HC/EO than in LC group (p = 0.012) and a trend for a treatment x
period interaction was observed (p = 0.075). Milk fat yield was not different between the
treatments. The fat:protein ratio was higher in HC/EO (p = 0.001) and tended to be higher in
HC/MO (p = 0.067) than in LC group over the experimental trial. Milk urea showed differences
with regard to treatment (p < 0.001), as it was higher in HC/MO than in HC group (p < 0.001)
and tended to be higher in HC/EO than in HC group (p = 0.051). Milk urea levels were
significantly higher in HC/MO cows compared to their HC counterparts in both periods (p <
0.05). The milk energy concentration tended to be different between the treatments (p = 0.065),
as it was higher in HC/EO than in LC cows (p = 0.038), and a trend for a treatment x period
interaction was found (p = 0.082). Overall, milk energy output did not differ between the
treatments. Feed efficiency (ECM/DMI) was influenced by treatment (p < 0.001). It was higher
in HC/MO and HC/EO than in LC (p < 0.05) and in HC/MO than in HC treatment (p < 0.001).
Additionally, there was a trend for a higher feed efficiency in HC/EO than in HC group (p =
0.081). A treatment x period interaction was detected (p = 0.015), as feed efficiency was 65% and
53% higher in HC/MO than in LC and HC group, respectively, in the second period (p < 0.05).
32
PAPER I
Table 3 Milk performance parameters of the experimental groups during the experimental period from day (d) 1
until 56 days in milk (DIM). High BCS (HC) cows were fed a concentrate proportion of 60%; low BCS (LC) cows
were fed a concentrate proportion of 20% in the prepartum diet. Post calving, the concentrate proportion in the diet
was increased from 30% to 50% within 2 weeks in LC group and within 3 weeks in HC groups. HC/MO was
administered a monensin controlled-release capsule (CRC) 3 weeks antepartum (a.p.), HC/EO received 1 g/d of a
blend of essential oils from week 3 a.p. until 56 DIM.
Treatment
P-Value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period Trt*Period
Milk yield, kg/d
d 1 until 14 DIM
29.28B
27.21
31.18
29.91
1.79
0.430
<0.001
0.538
A
d 15 until 56 DIM
32.50
29.75
33.66
31.09
1.66
4% fat-corrected milk, kg/d
d 1 until 14 DIM
36.55
34.91
40.75
40.61
2.47
0.454
<0.001
0.317
d 15 until 56 DIM
34.33
33.18
35.68
35.58
2.13
Energy-corrected milk (ECM), kg/d
d 1 until 14 DIM
34.97
33.73
38.27
38.18B
2.28
0.608
<0.001
0.356
d 15 until 56 DIM
32.91
31.45
33.74
33.29A
1.99
ECM/dry matter intake, kg/kg
d 1 until 14 DIM
2.35b
2.54b
3.88a
3.13ab
0.21
<0.001 <0.001
0.015
d 15 until 56 DIM
1.86
1.96
2.26
2.30
0.15
Milk fat content, %
d 1 until 14 DIM
5.29A
5.63A
6.13A
6.27A
0.26
0.022
<0.001
0.075
B
B
B
d 15 until 56 DIM
4.35
4.69
4.34
4.94B
0.17
Milk fat yield, kg/d
d 1 until 14 DIM
1.60
1.60
1.87B
1.88B
0.13
0.369
<0.001
0.319
A
d 15 until 56 DIM
1.42
1.41
1.49
1.53A
0.10
Milk protein content, %
d 1 until 14 DIM
3.42B
3.40B
3.30A
3.29A
0.08
0.178
<0.001
0.739
A
A
B
d 15 until 56 DIM
2.92
2.85
2.72
2.82B
0.05
Milk protein yield, kg/d
d 1 until 14 DIM
1.02
0.96A
1.04A
0.99A
0.06
0.690
<0.001
0.680
B
B
d 15 until 56 DIM
0.95
0.85
0.91
0.88B
0.05
Milk lactose content, %
d 1 until 14 DIM
4.60
4.63
4.52B
4.49B
0.05
0.170
<0.001
0.279
A
d 15 until 56 DIM
4.68
4.75
4.70
4.66A
0.04
Milk lactose yield, kg/d
d 1 until 14 DIM
1.40
1.30
1.41B
1.37
0.09
0.666
<0.001
0.740
d 15 until 56 DIM
1.53
1.42
1.58A
1.45
0.08
Milk urea, mg/kg
d 1 until 14 DIM
114.8ab
96.8b
156.9aB
128.3ab
10.6
<0.001 <0.001
0.243
d 15 until 56 DIM
98.3ab
73.9b
112.1aA
98.9ab
7.3
33
PAPER I
Table 3 (Continued)
Treatment
P-Value
HC
MO
Item
LC
Control
EO
SEM*
Trt†
Period Trt*Period
Milk fat : protein ratio
d 1 until 14 DIM
1.56
1.65
1.86
1.91
0.08
0.002
0.006
0.214
d 15 until 56 DIM
1.49
1.65
1.60
1.76
0.08
Milk energy concentration, MJ/kg
d 1 until 14 DIM
3.68A
3.80A
3.97A
4.03A
0.10
0.065
<0.001
0.082
B
B
B
B
d 15 until 56 DIM
3.22
3.33
3.17
3.42
0.07
Milk energy output, MJ/d
d 1 until 14 DIM
113.63
109.48
124.56
124.31A
7.36
0.570
<0.001
0.352
B
d 15 until 56 DIM
107.61
102.44
110.55
108.89
6.43
†
DIM.
Serum concentrations and course of time of metabolic parameters and liver fat content are
presented in Tab.4 and Fig.1. BHB in blood serum varied relative to treatment (p = 0.003), as it
was higher in HC/EO than in LC and HC/MO group (p < 0.05). In period 3, HC/EO cows
showed an 80% and 122% higher value than LC and HC/MO, respectively (p < 0.05). This also
resulted in a treatment x period interaction (p = 0.008). NEFA differed relative to treatment (p =
0.001) and a period x treatment interaction was detected (p = 0.013). It was higher in HC and
HC/EO (p < 0.05) and tended to be higher in HC/MO than in LC group (p = 0.068). In period 2,
NEFA of HC, HC/MO and HC/EO were 50.7%, 40.3% and 49.3% higher than in LC cows,
respectively, while in period 3, a difference was only observed between HC, HC/EO and LC (p <
0.05). Glucose and liver fat content did not differ between the treatments. Except for glucose, all
described parameters were significantly influenced by period.
34
PAPER I
Table 4 Concentrations of β-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), glucose and liver fat
content of the experimental groups during the experimental period from d -42 until 56 days in milk (DIM). High
BCS (HC) cows were fed a concentrate proportion of 60%; low BCS (LC) cows were fed a concentrate proportion of
20% in the prepartum diet. Post calving, the concentrate proportion in the diet was increased from 30% to 50%
within 2 weeks in LC group and within 3 weeks in HC groups. HC/MO was administered a monensin controlledrelease capsule (CRC) 3 weeks antepartum (a.p.), HC/EO received 1 g/d of a blend of essential oils from week 3 a.p.
until 56 DIM.
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period
Trt*Period
BHB, mmol/L
d -42 until calving
0.71
0.70B
0.71
0.77B
0.16
d 1 until 14 DIM
1.04
1.67A
1.12
1.66A
0.16
0.003
<0.001
0.008
d 15 until 56 DIM
0.94b
1.32abAB
0.76b
1.69aAB
0.14
NEFA, mmol/L
d -42 until calving
0.33B
0.34C
0.33C
0.36C
0.05
bA
aA
aA
d 1 until 14 DIM
0.67
1.01
0.94
1.00aA
0.06
0.001
<0.001
0.013
d 15 until 56 DIM
0.44bB
0.68aB
0.57abB
0.67aB
0.05
Glucose, mg/dL
d -42 until calving
55.3
58.8
56.9
56.1
2.0
d 1 until 14 DIM
55.1
54.7
59.3
54.2
2.1
0.170
0.217
0.601
d 15 until 56 DIM
54.1
56.7
56.4
51.5
1.7
Liver fat content, mg/g
d -42 until calving
57
65
50B
55B
22
A
A
d 1 until 14 DIM
143
165
146
202
24
0.108
<0.001
0.261
d 15 until 56 DIM
100
142
103AB
183A
20
†
DIM.
Rumen fermentation characteristics and protozoa population are presented in Tab.5. Total SCFA
in rumen fluid, NH3, proportion of acetate and valerate and protozoa population and density were
not influenced by treatment. A treatment x period interaction was observed for rumen pH (p =
0.041), as pH tended to be higher in HC/MO than in LC group (p = 0.098) in the second period.
Propionate proportion was 32% higher in HC/MO than in HC cows (p = 0.008). Proportion of
butyrate was higher in HC than in HC/MO (p = 0.034) and HC/EO group (p = 0.031). A higher
valerate proportion in LC treatment than in HC/MO in the third period (p = 0.024) resulted in a
35
PAPER I
treatment x period interaction (p = 0.013). Isovalerate proportion tended to be higher in HC/MO
than in HC group (p = 0.062) and a trend for a treatment x period interaction was detected (p =
0.077). The acetate:propionate ratio was higher in HC (p < 0.001) and tended to be higher in LC
(p = 0.083) than in HC/MO cows. The period had an influence on pH, total SCFA, the proportion
of acetate and propionate and the acetate:propionate ratio (p < 0.05) and tended to influence NH3
(p = 0.066). The pH tended to be higher in the first (p = 0.054) and was higher the third (p =
0.041) than in the second period. The total SCFA tended to be higher in the second (p = 0.064)
and were higher in the third (p = 0.026) than in the first period. While acetate proportion was
higher in the first period than in the second (p = 0.003) and third one (p = 0.007), a higher
propionate proportion was observed in period 2 (p = 0.012) and 3 (p = 0.036) than in the first one.
The acetate:propionate ratio was higher in the first period than in periods 2 (p = 0.002) and 3 (p =
0.003).
Table 5 Rumen fermentation parameters and protozoa population of the experimental groups during the
experimental period from d -42 until 56 days in milk (DIM). High BCS (HC) cows were fed a concentrate proportion
of 60%; low BCS (LC) cows were fed a concentrate proportion of 20% in the prepartum diet. Post calving, the
concentrate proportion in the diet was increased from 30% to 50% within 2 weeks in LC group and within 3 weeks in
HC groups. HC/MO was administered a monensin controlled-release capsule (CRC) 3 weeks antepartum (a.p.),
HC/EO received 1 g/d of a blend of essential oils from week 3 a.p. until 56 DIM.
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period
Trt*Period
pH
d -42 until calving
7.35
7.13
7.16
7.33
0.20
d 1 until 14 DIM
6.63
7.15
7.27
6.97
0.19
0.599
0.018
0.041
d 15 until 56 DIM
7.07
7.23
7.24
7.06
0.18
NH3, mg/100 g
d -42 until calving
3.60
4.60
2.82
6.57
1.45
d 1 until 14 DIM
2.71
2.10
2.17
3.31
1.37
0.130
0.066
0.562
d 15 until 56 DIM
3.15
2.24
3.62
4.07
1.24
Total short-chain fatty acids, mmol/L
d -42 until calving
45.49
57.98
67.42
52.95
9.10
d 1 until 14 DIM
70.56
59.26
62.64
71.42
8.74
0.896
0.034
0.115
d 15 until 56 DIM
71.02
62.22
66.65
68.37
8.14
Acetate, mol %
d -42 until calving
68.99
68.77
61.76
68.05
2.70
d 1 until 14 DIM
60.11
65.14
60.91
61.89
2.55
0.106
0.004
0.177
d 15 until 56 DIM
60.65
64.01
62.08
63.72
2.32
36
PAPER I
Table 5 (Continued)
Treatment
HC
Control
MO
P-value
Item
LC
EO
SEM*
Trt†
Period
Trt*Period
Propionate, mol %
d -42 until calving
16.34
15.26
22.73
18.12
2.55
d 1 until 14 DIM
22.68
18.61
24.41
23.22
2.40
0.012
0.016
0.539
d 15 until 56 DIM
22.12
19.39
23.15
21.02
2.17
Butyrate, mol %
d -42 until calving
13.51
15.17
12.97
11.98
1.06
d 1 until 14 DIM
15.07
14.72
12.64
12.73
1.00
0.005
0.807
0.610
d 15 until 56 DIM
14.60
14.31
12.60
13.30
0.91
Valerate, mol %
d -42 until calving
0.05
0.28
0.71
0.96
0.33
d 1 until 14 DIM
1.02
0.54
0.31
1.00
0.31
0.207
0.127
0.013
a
ab
b
ab
d 15 until 56 DIM
1.16
0.81
0.52
0.80
0.28
Isovalerate, mol %
d -42 until calving
1.33
0.76
1.89
1.40
0.30
d 1 until 14 DIM
1.33
1.06
1.80
1.41
0.29
0.060
0.400
0.077
d 15 until 56 DIM
1.49
1.58
1.70
1.23
0.27
Acetate : Propionate
d -42 until calving
4.11
4.44
2.72
3.60
0.40
d 1 until 14 DIM
2.82
3.52
2.54
2.83
0.38
0.001
0.002
0.163
d 15 until 56 DIM
2.88
3.33
2.71
3.17
0.34
Protozoa population
Total Protozoa, 103/ml
d -42 until calving
53.46
128.27
54.22
66.28
33.47
d 1 until 14 DIM
112.71
48.00
63.21
83.41
18.01
0.895
0.977
0.183
d 15 until 56 DIM
81.22
52.48
94.03
70.36
9.76
Entodiniomorpha, 103/ml
d -42 until calving
53.16
125.18
52.44
63.12
32.86
d 1 until 14 DIM
111.56
46.44
62.18
82.40
75.64
0.870
0.965
0.184
d 15 until 56 DIM
79.55
50.89
91.62
69.14
9.57
Holotricha, 103/ml
d -42 until calving
0.48
3.12
1.65
3.30
1.49
d 1 until 14 DIM
1.34
1.52
0.90
1.02
0.79
0.746
0.432
0.581
d 15 until 56 DIM
1.73
1.57
2.38
1.23
0.43
†
DIM.
37
PAPER I
Discussion
In the current study, the effect of monensin and essential oils on performance parameters and the
energy status of periparturient cows was evaluated by using the ketosis model by SCHULZ et al.
(2014). The combination of high BCS a.p., overfeeding in the dry period and a decelerated
energy supply at the beginning of lactation was applied to create animal groups being subjected
to a ketogenic metabolic status.
Regarding initial BCS 6 weeks before calving, it was possible to form 2 BCS groups with a
difference in body condition with an average of more than 1.0 point on scale in our experiment (p
< 0.001). In the course of the trial until parturition, it was even possible to further enhance body
condition in all HC groups while BCS in LC stayed nearly on the same level. This BCS increase
in HC groups was probably a consequence of the higher net energy intake (p < 0.001) and the
resulting energy balance a.p. (p < 0.001). The experimental design induced a massive
mobilization of body fat depots as NEFA concentration was more than 50% higher in HC than in
LC group in the p.p. periods. Additionally, the liver fat content, although not significant between
groups, was higher in the liver of cows of HC than of LC in early lactation. According to the
classification of ketosis by SCHULZ et al. (2014), the prevalence of either subclinical or clinical
ketosis was 36% higher in HC than in LC group. In summary, the implemented trial seems to
offer adequate conditions for testing monensin and essential oils and their effects on performance
parameters and energy metabolism. The restricted energy supply in the high BCS groups in the
first 3 weeks after parturition led to a more negative energy balance and a loss in body condition
in those groups.
DMI decreased in all groups, as expected, around calving and was not influenced by treatment.
Similar results for a monensin CRC have been reported in previous literature (GREEN et al.
1999, FAIRFIELD et al. 2007, MULLINS et al. 2012). A meta-analysis of 59 studies showed a
decrease in DMI of 2% (DUFFIELD et al. 2008 b) after addition of monensin, similar to the
results of a review by IPHARRAGUERRE and CLARK (2003). The difference to the present
results may be explained by the low number of cows in a single study to statistically detect such a
small effect and to different experimental designs and diets fed, as most of the studies reporting
an effect on DMI used TMR with forages DM proportions of 50% (IPHARRAGUERRE and
38
PAPER I
CLARK 2003). Regarding essential oils, TASSOUL and SHAVER (2009) reported a decrease in
DMI of 1.8 kg/d p.p., when 1.2 g/d BEO was fed to 40 multiparous Holstein cows 3 weeks a.p..
BENCHAAR et al. (2007) suggested that the effect of essential oils on DMI may be highly
dependent on the supplemented dose.
2.5
Period 3
Period 2
Period 1
BHB, mmol/L
2
1.5
1
0.5
0
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
1.4
NEFA, mmol/L
1.2
1
0.8
0.6
0.4
0.2
0
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
-42 -35 -28 -21 -14 -7
0
7
14 21 28
Experimental day relative to calving
35
42
49
56
Liver fat content, mg/g
250
200
150
100
50
0
Figure 1 Change of β-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA) and total liver fat content (Means)
during the experiment. Cows received a low concentrate (∆) diet with a concentrate: roughage ratio of 20:80 (n=14)
or a high concentrate (●) diet with a concentrate:roughage ratio of 60:40 ante partum. Postpartal
concentrate:roughage ratio was changed stepwise from 30:70 to 50:50 within 2 weeks in the low concentrate group
(∆) and 3 weeks in the high concentrate groups (●). Treatment included no supplementation (─) (n=13), the
administration of a monensin controlled-release capsule (∙∙∙) (n=14) at day (d) -21 and the addition of 1 g/d/cow of a
blend of essential oils (---) (n=15) from d -21 until d 56 relative to calving.
39
PAPER I
The evoked mobilization of body fat depots caused higher levels of NEFA in all HC groups than
in LC group in the first 2 weeks of lactation. Thereafter, HC/MO showed values similar to LC
cows giving evidence for a reduction of body fat mobilization in monensin supplemented cows.
After the release from fat depots, NEFA can be used as an energy source of milk fat synthesis in
mammary gland, re-esterified to triglycerides or partially oxidized to ketone bodies in the liver or
completely oxidized by skeletal muscle or liver as an energy source (DRACKLEY 1999,
ROCHE et al. 2009). The finding that monensin-supplemented cows mobilized adipose tissues
similar to the HC counterparts but had concentrations of BHB (1.12 mmol/L and 0.76 mmol/L in
the postpartal periods, respectively) and liver fat (146 mg/g and 103 mg/g in the postpartal
periods, respectively) similar to LC cows (BHB 1.04 mmol/L and 0.74 mmol/L, liver fat content
143 mg/g and 100mg/g in the postpartal periods, respectively) is indicative for a more favorable
utilization of circulating NEFA.
In this context, the importance of the liver in transition period is emphasized as it sits at the
crossroad of metabolism and plays a key role in coordination of nutrient fluxes (DRACKLEY et
al. 2001) and inflammatory conditions in the liver play a role for periparturient performance and
health (SORDILLO et al. 2009) in dairy cows. Thus, an improved liver metabolism may be a
contributing factor to the present results.
A reducing effect of monensin on BHB concentration in blood serum has been consistently
reported in literature. GREEN et al. (1999) reported a decrease in BHB by 35% after the
application of a monensin CRC 3 weeks a.p., which is in accordance with the meta-analysis by
DUFFIELD et al. (2008 a) who reported an average decrease of 13%.
In contrast, BHB of essential oils supplemented cows was 80% higher than in LC cows (p =
0.013) and even more than twice as high as in HC/MO cows (p < 0.001) in the last period
showing that ketogenesis in these cows was rather further increased by essential oils or utilization
of BHB was diminished in the third period. Comparable studies evaluating blood parameters
representative for the energy status of essential oils supplemented cows are, to our knowledge,
very rare. ANASSORI et al. (2015) investigated different doses of raw garlic and garlic oil in 4
ruminally fistulated rams in a Latin square design (28-day periods) and reported no influence of
treatment on BHB, NEFA and glucose.
40
PAPER I
Furthermore, essential oils increased milk fat content (p = 0.012) in comparison to LC group
which is in contrast to recent literature reporting no influence of BEO on milk composition
(BENCHAAR et al. 2006, TASSOUL and SHAVER 2009). As summarized by BAUMAN and
GRIINARI (2003), the contribution of fatty acids from the body fat stores to milk fat synthesis
increases proportional to the energy deficit and the mammary uptake of NEFA is directly related
to their circulating concentration. HC/EO cows started lactation with a high milk fat percentage,
as they suffered from highly negative energy balance. Overall, the difference between our results
and mentioned studies may be related to the current experimental design, as a negative energy
balance was provoked and could not be compensated by a supplementation of essential oils.
Furthermore, HC/EO had a higher milk fat: protein ratio (p = 0.001) than LC cows. This might be
related to a high lipolysis and an increased incidence of ketosis (HEUER et al. 1999).
Long-term continuous culture fermentation studies (BUSQUET et al. 2005, CASTILLEJOS et
al. 2006) have reported a shift of the ruminal microflora towards Gram-negative bacteria in the
presence of monensin, as Gram-positive bacteria are more sensitive because of the absence of an
outer membrane (RUSSELL and STROBEL 1989). As the fermentation pattern changes towards
increased propionate and decreased acetate and butyrate proportions in short-chain fatty acid
profile and propionate is the major precursor of gluconeogenesis (SEAL and REYNOLDS 1993),
a commonly accepted theory is that a higher glucose production in the liver leads to a diminished
body fat mobilization and therefore inhibits ketogenesis and the accumulation of fatty acids in the
liver. In fact, we could detect a higher proportion of ruminal propionate (p = 0.008), a lower
proportion of butyrate (p = 0.033) and a diminished acetate:propionate ratio (p < 0.001) in
monensin supplemented cows than in HC cows.
Although no differences between groups were observed in the present trial, glucose was reported
to be higher in cows receiving a monensin CRC a.p. (DUFFIELD et al. 1998 a, GREEN et al.
1999), while other studies detected no change, too (MULLINS et al. 2012). As DUFFIELD et al.
(2008 a) reported only a moderate increase by 3% in a meta-analysis and glucose is a wellregulated blood parameter in cows, differences in result are likely dependent on sample
management and cow-individual parameters.
41
PAPER I
Considering essential oils, the proposed mechanism of a shift of the ruminal fermentation pattern
towards an increased propionate production could not be supported by our results as all
fermentation parameters, except for a lower butyrate proportion (p = 0.031), were not different to
HC group. Summaries of diverse actual research results imply that especially the change of
ruminal fermentation is highly dependent on the component and the dose fed (CALSAMIGLIA
et al. 2007, PATRA 2011). Therefore, the applied mixture of essential oils may not be beneficial
under the present conditions for the energy status of transition dairy cows, while other
combinations or solitary application of promising substances may achieve better results. There is
more research necessary to test promising essential oils from in vitro investigations in in vivo
experiments.
Essential oils and monensin did not affect milk yield. The lacking effects of essential oils on milk
yield agree with the results of TASSOUL and SHAVER (2009), who observed no influence of
1.2 g/d BEO on milk yield of 40 multiparous Holstein cows. Similarly, a dose of 2 g/d BEO did
not alter milk yield in 4 ruminally cannulated Holstein cows (BENCHAAR et al. 2006).
Effects of monensin on milk production are not consistent in literature. Recent studies about the
application of a monensin CRC 3 weeks a.p. found no effect on milk production (GREEN et al.
1999, FAIRFIELD et al. 2007, MULLINS et al. 2012). DUFFIELD et al. (1999) reported an
increased milk production in a double-blind, randomized clinical trial including 1010 cows and
heifers, especially in fat cows (BCS ≥ 4.0). There was no influence of monensin on milk
production visible under the present conditions. This may be related to the restricted number of
cows in our study or diet-dependent.
In fact, the feed efficiency (ECM/DMI) was 65% (p < 0.001) and 50% (p = 0.002) higher than in
LC and HC group, respectively, in the first 2 weeks of lactation in the HC/MO group. In the
context of the anaerobic fermentation in the rumen, monensin may redirect hydrogen from
methane to propionate production and may therefore contribute to a reduced loss of feed energy
(VAN NEVEL and DEMEYER 1996). A reduced methane production has already been shown in
cows after monensin supplementation (ODONGO et al. 2007).
Monensin increased milk urea in high conditioned cows under the present conditions. This is not
in agreement with the results of DUFFIELD et al. (2008 a), who detected no influence of
42
PAPER I
monensin on milk urea in a meta-analysis. MULLINS et al. (2012) found also an increase in milk
urea N, in a trial with 41 multiparous Holsteins fed 400 mg/d monensin top-dressed to the diet.
An increased milk urea is related to a higher N concentration in blood (BRODERICK and
CLAYTON 1997). DUFFIELD et al. (1998 b) found a higher blood urea in the first 2 weeks of
lactation in 1010 heifers and cows administered a monensin CRC 3 weeks a.p.. A trend towards
higher blood urea in the p.p. period was also found in a trial with 52 primi- and multiparous
Holstein cows after a.p. application of a CRC (GREEN et al. 1999). A higher blood urea N is
likely a consequence of the protein sparing effect of monensin. As Gram-positive bacteria are
more sensitive to monensin, the fermentation of “hyper-ammonia producing” (HAP) species
(RUSSELL et al. 1988, PASTER et al. 1993) that produce high amounts of ammonia is reduced.
MCGUFFEY et al. (2001) summarized that protein degradation, ammonia accumulation and
microbial nitrogen in the rumen were reduced in vitro with monensin. This leads to the
assumption, that more protein of dietary origin reaches the small intestine after monensin
supplementation. The higher absorption rate of amino acids may contribute to higher blood
concentrations of glucose, as they may be used for the hepatic synthesis of glucose. Amino acids
are considered to exert a significant role in gluconeogenesis in the early p.p. period (BELL et al.
2000). Furthermore, the conversion of alanine, as one of the amino acids with greatest
contribution to gluconeogenesis, to glucose was found to be 98% higher at 1 d and 50% higher at
21 d p.p. than at 21 d a.p. (OVERTON et al. 1998). The subsequent deamination before
gluconeogenesis could therefore contribute to the increased concentrations of blood urea, as
presumed by DUFFIELD et al. (1998 b). However, NH3 in rumen fluid was not found to be
influenced by treatment under the present conditions.
Essential oils tended to increased milk urea (p = 0.051) analogous to monensin, but to a lesser
extent. BENCHAAR et al. (2006) found no increase in milk urea in 4 ruminally cannulated
Holstein cows fed 2 g/d BEO, as did TASSOUL and SHAVER (2009). 110 mg/d BEO was found
to reduce the deamination of amino acids measured in vitro in rumen fluid removed from sheep
by 25% (NEWBOLD et al. 2004). In accordance with this, MCINTOSH et al. (2003) observed an
inhibiting effect of essential oils on the rate of deamination of amino acids in a trial with 4
ruminally cannulated Holstein cows receiving 1 g/d BEO. Essential oils were added to pure
43
PAPER I
cultures of rumen microorganism, leading to a growth inhibition of some of the HAP species
(RUSSELL et al. 1988, PASTER et al. 1993, MCINTOSH et al. 2003). As some of the HAP
bacteria sensitive to monensin were not affected by essential oils, MCINTOSH et al. (2003)
hypothesized that this may explain why essential oils are less effective in inhibiting ammonia
production than monensin.
Conclusion
The animal model generated animal groups experiencing different manifestations of fat
mobilization and ketogenesis. Dairy cows with a high condition around calving may profit from a
monensin CRC administered 3 weeks a.p., as the energy status was improved. Additionally, the
feed efficiency (ECM/DMI) was increased. The supplemented dose of a blend of essential oils
showed no effect on energy status and performance of transition dairy cows under the current
conditions.
References
AGTARAP, A., J. W. CHAMBERLIN, M. PINKERTON, and L. K. STEINRAUF (1967):
Structure of monensic acid, a new biologically active compound.
ACS Cent Sci. 89, 5737-5739
ANASSORI, E., B. DALIR-NAGHADEH, R. PIRMOHAMMADI, and M. HADIAN (2015):
Changes in blood profile in sheep receiving raw garlic, garlic oil or monensin.
J. Anim. Physiol. Anim. Nutr. (Berl) 99, 114-122
BAKKALI, F., S. AVERBECK, D. AVERBECK, and M. IDAOMAR (2008):
Biological effects of essential oils – A review.
Food Chem. Toxicol. 46, 446-475
BAUMAN, D. E., and J. M. GRIINARI (2003):
Nutritional regulation of milk fat synthesis.
Annu. Rev. Nutr. 23, 203-227
44
PAPER I
BELL, A. W., W. S. BURHANS, and T. R. OVERTON (2000):
Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy
cows.
Proc. Nutr. Soc. 59, 119-126
BENCHAAR, C., H. PETIT, R. BERTHIAUME, T. WHYTE, and P. CHOUINARD (2006):
Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation,
milk production, and milk composition in dairy cows.
J. Dairy Sci. 89, 4352-4364
BENCHAAR, C., H. PETIT, R. BERTHIAUME, D. OUELLET, J. CHIQUETTE, and P.
CHOUINARD (2007):
Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk
production, and milk composition in dairy cows fed alfalfa silage or corn silage.
J. Dairy Sci. 90, 886-897
BERGEN, W. G., and D. B. BATES (1984):
Ionophores: Their effect on production efficiency and mode of action.
J. Anim. Sci. 58, 1465-1483
BERGMAN, E. N. (1971):
Hyperketonemia-ketogenesis and ketone body metabolism.
J. Dairy Sci. 54, 936-948
BRODERICK, G. A., and M. K. CLAYTON (1997):
A statistical evaluation of animal and nutritional factors influencing concentrations of milk urea
nitrogen.
J. Dairy Sci. 80, 2964-2971
BUSQUET, M., S. CALSAMIGLIA, A. FERRET, P. CARDOZO, and C. KAMEL (2005):
Effect of garlic oil and four of its compounds on rumen microbial fermentation.
J. Dairy Sci. 88, 4393-4404
CALSAMIGLIA, S., M. BUSQUET, P. CARDOZO, L. CASTILLEJOS, and A. FERRET
(2007):
Invited review: Essential oils as modifiers of rumen microbial fermentation.
J. Dairy Sci. 90, 2580-2595
45
PAPER I
CASTILLEJOS, L., S. CALSAMIGLIA, and A. FERRET (2006):
Effect of essential oil active compounds on rumen microbial fermentation and nutrient flow in in
vitro systems.
J. Dairy Sci. 89, 2649-2658
CHAO, S. C., D. G. YOUNG, and C. J. OBERG (2000):
Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses.
J. Essent. Oil Res. 12, 639-649
DEANS, S., and G. RITCHIE (1987):
Antibacterial properties of plant essential oils.
Int. J. Food Microbiol. 5, 165-180
DRACKLEY, J. K. (1999):
Biology of dairy cows during the transition period: The final frontier?
J. Dairy Sci. 82, 2259-2273
DRACKLEY, J. K., T. R. OVERTON, and G. N. Douglas (2001):
Adaptations of glucose and long-chain fatty acid metabolism in liver of dairy cows during the
periparturient period.
J. Dairy Sci. 84, Supplement, 100-112
DUFFIELD, T., D. SANDALS, K. LESLIE, K. LISSEMORE, B. MCBRIDE, J. LUMSDEN, P.
DICK, and R. BAGG (1998 a):
Efficiency of monensin for the prevention of subclinical ketosis in lactating dairy cows.
J. Dairy Sci. 81, 2866-2873
DICK, and R. BAGG (1998 b):
Effect of prepartum administration of monensin in a controlled-release capsule on postpartum
energy indicators in lactating dairy cows.
J. Dairy Sci. 81, 2354-2361
DUFFIELD, T., K. LESLIE, D. SANDALS, K. LISSEMORE, B. MCBRIDE, J. LUMSDEN,
P. DICK, and R. BAGG (1999):
Effect of prepartum administration of monensin in a controlled-release capsule on milk
production and milk components in early lactation.
J. Dairy Sci. 82, 272-279
46
PAPER I
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 a):
A meta-analysis of the impact of monensin in lactating dairy cattle. Part 1: Metabolic effects.
J. Dairy Sci. 91, 1334-1346
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 b):
A meta-analysis of the impact of monensin in lactating dairy cattle. Part 2: Production effects.
J. Dairy Sci. 91, 1347-1360
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 c):
A meta-analysis of the impact of monensin in lactating dairy cattle. Part 3: Health and
reproduction.
J. Dairy Sci. 91, 2328-2341
EDMONSON, A. J., I. J. LEAN, L. D. WEAVER, T. FARVER, and G. WEBSTER (1989):
A body condition scoring chart for Holstein dairy cows.
J. Dairy Sci. 72, 68-78
FAIRFIELD, A., J. PLAIZIER, T. DUFFIELD, M. LINDINGER, R. BAGG, P. DICK, and B.
MCBRIDE (2007):
Effects of prepartum administration of a monensin controlled release capsule on rumen pH, feed
intake, and milk production of transition dairy cows.
J. Dairy Sci. 90, 937-945
FELDBERG, R., S. CHANG, A. KOTIK, M. NADLER, Z. NEUWIRTH, D. SUNDSTROM,
and N. THOMPSON (1988):
In vitro mechanism of inhibition of bacterial cell growth by allicin.
Antimicrob. Agents Chemother. 32, 1763-1768
GAINES, W. L. (1928):
The energy basis of measuring milk yield in dairy cows.
Illinois AES Bull. 308, 401-438
GESELLSCHAFT FÜR ERNÄHRUNGSPHYSIOLOGIE (GFE, 1991):
Leitlinien für die Bestimmung der Verdaulichkeit von Rohnährstoffen an Wiederkäuern:
Herausgegeben vom Ausschuss für Bedarfsnormen der Gesellschaft für Ernährungsphysiologie,
Frankfurt.
47
PAPER I
Empfehlung zur Energie- und Nährstoffversorgung von Milchkühen und Aufzuchtrindern.
DLG-Verlag, Frankfurt am Main
Communications of the committee for requirement standards of the Society of Nutrition
Physiology: New equations for predicting metabolisable energy of grass and maize products for
ruminants.
Proc. Nutr. Soc. 17, 191-198
GREEN, B. L., B. W. MCBRIDE, D. SANDALS, K. E. LESLIE, R. BAGG, and P. DICK
(1999):
The impact of a monensin controlled-release capsule on subclinical ketosis in the transition dairy
cow.
J. Dairy Sci. 82, 333-342
GRIFFIN, S. G., S. G. WYLLIE, J. L. MARKHAM, and D. N. LEACH (1999):
The role of structure and molecular properties of terpenoids in determining their antimicrobial
activity.
Flavour Fragr. J. 14, 322-332
GRUMMER, R. R. (1995):
Impact of changes in organic nutrient metabolism on feeding the transition dairy cow.
J. Anim. Sci. 73, 2820-2833
HEITMANN, R. N., D. J. DAWES, and S. C. SENSENIG (1987):
Hepatic ketogenesis and peripheral ketone body utilization in the ruminant.
J. Nutr. 117, 1174-1180
HELANDER, I. M., H.-L. ALAKOMI, K. LATVA-KALA, T. MATTILA-SANDHOLM, I.
POL, E. J. SMID, L. G. GORRIS, and A. VON WRIGHT (1998):
Characterization of the action of selected essential oil components on Gram-negative bacteria.
J. Agric. Food Chem. 46, 3590-3595
HEUER, C., Y. H. SCHUKKEN, and P. DOBBELAAR (1999):
Postpartum body condition score and results from the first test day milk as predictors of disease,
fertility, yield, and culling in commercial dairy herds.
J. Dairy Sci. 82, 295-304
48
PAPER I
IPHARRAGUERRE, I. R., and J. H. Clark (2003):
Usefulness of ionophores for lactating dairy cows: A review.
Anim. Feed Sci. Technol. 106, 39-57
KOCH, M., E. STROBEL, C. C. TEBBE, J. HERITAGE, G. BREVES, and K. HUBER (2006):
Transgenic maize in the presence of ampicillin modifies the metabolic profile and microbial
population structure of bovine rumen fluid in vitro.
Br. J. Nutr. 96, 820-829
LITTELL, R., P. HENRY, and C. AMMERMAN (1998):
Statistical analysis of repeated measures data using SAS procedures.
J. Anim. Sci. 76, 1216-1231
MCGUFFEY, R., L. RICHARDSON, and J. WILKINSON (2001):
Ionophores for dairy cattle: Current status and future outlook.
MCINTOSH, F., P. WILLIAMS, R. LOSA, R. WALLACE, D. BEEVER, and C. NEWBOLD
(2003):
Effects of essential oils on ruminal microorganisms and their protein metabolism.
Appl. Environ. Microbiol. 69, 5011-5014
MULLINS, C. R., L. K. MAMEDOVA, M. J. BROUK, C. E. MOORE, H. B. GREEN, K. L.
PERFIELD, J. F. SMITH, J.P. HARNER, and B.J. BRADFORD (2012):
Effects of monensin on metabolic parameters, feeding behavior, and productivity of transition
dairy cows.
J. Dairy Sci. 95, 1323-1336
NEWBOLD, C., F. MCINTOSH, P. WILLIAMS, R. LOSA, and R. WALLACE (2004):
Effects of a specific blend of essential oil compounds on rumen fermentation.
NIKAIDO, H., and M. VAARA (1985):
Molecular basis of bacterial outer membrane permeability.
Microbiol. Rev. 49, 1
49
PAPER I
ODONGO, N. E., R. BAGG, G. VESSIE, P. DICK, M. M. OR-RASHID, S. E. HOOK, J. T.
GRAY, E. KEBREAB, J. FRANCE, and B. W. MCBRIDE (2007):
Long-term effects of feeding monensin on methane production in lactating dairy cows.
J. Dairy Sci. 90, 1781-1788
OGIMOTO, K., and S. IMAI (1981):
Atlas of rumen microbiology.
Japan Scientific Societies Press, Tokyo
OVERTON, T., J. DRACKLEY, G. DOUGLAS, L. EMMERT, and L. CLARK (1998):
Hepatic gluconeogenesis and whole-body protein metabolism of periparturient dairy cows as
affected by source of energy and intake of the prepartum diet.
J. Dairy Sci. 81, 295
PASTER, B. J., J. B. RUSSELL, C. YANG, J. CHOW, C. R. WOESE, and R. TANNER (1993):
Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius,
Clostridium sticklandii, and Clostridium aminophilum sp..
Int. J. Syst. Bacteriol. 43, 107-110
PATRA, A. K. (2011):
Effects of essential oils on rumen fermentation, microbial ecology and ruminant production.
Asian J. Anim. Vet. Adv. 6, 416–428
PRESSMAN, B. C. (1976):
Biological applications of ionophores.
Annu. Rev. Biochem. 45, 501-530
PRESSMAN, B. C., and M. FAHIM (1982):
Pharmacology and toxicology of the monovalent carboxylic ionophores.
Annu. Rev. Pharmacol. Toxicol. 22, 465-490
ROCHE, J. R., N. C. FRIGGENS, J. K. KAY, M. W. FISHER, K. J. STAFFORD, and D. P.
BERRY (2009):
Invited review: Body condition score and its association with dairy cow productivity, health, and
welfare.
J. Dairy Sci. 92, 5769-5801
50
PAPER I
RUSSELL, J., H. STROBEL, and G. CHEN (1988):
Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia
production.
RUSSELL, J. B., and H. STROBEL (1989):
Effect of ionophores on ruminal fermentation.
SAUER, F., V. FELLNER, R. KINSMAN, J. KRAMER, H. JACKSON, A. LEE, and S. CHEN
(1998):
Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added
to the diet.
J. Anim. Sci. 76, 906-914
SCHULZ, K., J. FRAHM, U. MEYER, S. KERSTEN, D. REICHE, J. REHAGE, and S.
DÄNICKE (2014):
Effects of prepartal body condition score and peripartal energy supply of dairy cows on postpartal
lipolysis, energy balance and ketogenesis: An animal model to investigate subclinical ketosis.
J. Dairy Res. 81, 257–266
SEAL, C., and C. REYNOLDS (1993):
Nutritional implications of gastrointestinal and liver metabolism in ruminants.
Nutr. Res. Rev. 6, 185-208
SIKKEMA, J., J. DE BONT, and B. POOLMAN (1994):
Interactions of cyclic hydrocarbons with biological membranes.
J. Biol. Chem. 269, 8022-8028
SJAUNJA, L., L. BAEVRE, L. JUNKKARINEN, J. PEDERSEN, and J. SETÄLÄ (1990):
A nordic proposal for an energy corrected milk (ECM) formula.
EAAP Public 50, 156-157
SMITH-PALMER, A., J. STEWART, and L. FYFE (1998):
Antimicrobial properties of plant essential oils and essences against five important food-borne
pathogens.
Lett. Appl. Microbiol. 26, 118-122
51
PAPER I
SORDILLO, L. M., G. CONTRERAS, and S. L. AITKEN (2009):
Metabolic factors affecting the inflammatory response of periparturient dairy cows.
Anim. Health Res. Rev. 10, 53-63
STARKE, A., A. HAUDUM, R. BUSCHE, M. BEYERBACH, S. DÄNICKE, and J. REHAGE
(2010):
Technical note: Analysis of total lipid and triacylglycerol content in small liver biopsy samples in
cattle.
J. Anim. Sci. 88, 2741-2750
TASSOUL, M., and R. SHAVER (2009):
Effect of a mixture of supplemental dietary plant essential oils on performance of periparturient
and early lactation dairy cows.
J. Dairy Sci. 92, 1734-1740
ULTEE, A., E. KETS, and E. SMID (1999):
Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus.
ULTEE, A., M. BENNIK, and R. MOEZELAAR (2002):
The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen
Bacillus cereus.
VAN NEVEL, C., and D. DEMEYER (1996):
Control of rumen methanogenesis.
Environ. Monit. Assess. 42, 73-97
ASSOCIATION OF GERMAN AGRICULTURAL ANALYSIS AND RESEARCH CENTERS
(VDLUFA, 1993):
Handbuch der landwirtschaftlichen Versuchs- und Untersuchungsmethodik (VDLUFA
Methodenbuch). Die chronologische Untersuchung von Futtermitteln.
3. Aufl. VDLUFA Verlag, Darmstadt
52
PAPER II
PAPER II
Effects of monensin and essential oils on immunological, haematological and
biochemical parameters of cows during the transition period
C. Drong1, U. Meyer1, D. von Soosten1, J. Frahm1, J. Rehage2, H. Schirrmeier3, M. Beer3
and S. Dänicke1
1
Institute of Animal Nutrition, Friedrich-Loeffler-Institut, Federal Research Institute for Animal
Health, Braunschweig, Germany
2
3
Clinic for Cattle, University of Veterinary Medicine, Foundation, Hannover, Germany
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for
Animal Health, Greifswald-Insel Riems, Germany
Journal of Animal Physiology and Animal Nutrition (JAPAN)
In press
DOI: 10.1111/jpn.12494
Printed with kind permission of John Wiley and Sons
53
PAPER II
Summary
Using a model to generate experimental groups with different manifestations of post partum
(p.p.) fat mobilization and ketogenesis, effects of a dietary and a medical intervention on
biochemical and hematological parameters, antibody titer, leukocytes subsets and function of
transition cows were examined. In total 60 German Holstein cows were allocated 6 weeks ante
partum (a.p.) to 3 high body condition score (BCS) groups (BCS 3.95) and 1 low BCS group
(LC, BCS 2.77). High BCS cows received a monensin controlled-release capsule (HC/MO), a
blend of essential oils (HC/EO) or formed a control group (HC). Parameters were evaluated in 3
periods (day (d) -42 until calving, 1 until 14 days in milk (DIM), 15 until 56 DIM).
Over the course of trial various parameters were influenced by period with greatest variability
next to calving. White blood cell count was higher in HC (8.42 x 103/µl) and HC/EO (8.38 x
103/µl) than in HC/MO group (6.81 x 103/µl) considering the whole trial. Supplementation of
monensin decreased aspartate aminotransferase in comparison to HC group similar to LC
treatment. Bilirubin concentration was nearly doubled in all high BCS cows in period 2. In period
3, essential oils increased γ-glutamyltransferase (80.4 Units/L) in comparison to all other groups
and glutamine dehydrogenase (61 Units/L) in comparison to LC (19 Units/L) and HC/MO group
(18 Units/L).
Results suggest that parameters were generally characterized by a high variability around calving.
Based on biochemical characteristics it appeared that HC cows seemed to have compromised
hepatocyte integrity when compared to LC cows. From the immune parameters investigated the
BVDV antibody response was more pronounced in HC/MO compared to HC/EO.
Introduction
The transition period of the high-yielding dairy cow extends from 3 weeks before to 3 weeks
after parturition (GRUMMER 1995). It is accompanied by a lowered responsiveness of the
immune system which seems to be related to physical, endocrine and metabolic changes of late
pregnancy, calving and lactation (MALLARD et al. 1998) and leads to the highest incidence of
production diseases in early lactation (INGVARTSEN et al. 2003). Especially high condition
transition cows are at high risk of immunosuppression and periparturient health disorders
54
PAPER II
(LACETERA et al. 2005). This seems to be partly accounted for by the negative energy balance
and related metabolic derailment like fatty liver syndrome and ketosis (TREVISI et al. 2011).
Commonly the high energy demand for the growth of the fetus and onset of lactation together
with a decreased dry matter intake lead to an unavoidable state of negative energy balance in
transition dairy cows (GRUMMER 1995). Consequential elevated levels of the serum
metabolites non-esterified fatty acids (NEFA) and ketone bodies seem to be aetiologically
involved in an impaired function of neutrophils and leukocytes around calving (CONTRERAS
and SORDILLO 2011). High concentrations of NEFA were reported to diminish proliferation,
antibody and cytokine secretion of bovine lymphocytes of heifers (LACETERA et al. 2004) as
well as viability and generation of reactive oxygen species (ROS) in bovine polymorphonuclear
leukocytes (PMN) (SCALIA et al. 2006). Similarly ketone bodies elicit an inhibitory effect on
chemotaxis of bovine leukocytes (SURIYASATHAPORN et al. 1999) and on respiratory burst
activity of bovine neutrophils (HOEBEN et al. 1997). Oxidative stress is another contributing
factor to inflammatory and immune dysfunction in dairy cattle (SORDILLO and AITKEN 2009).
It describes the imbalance between the rate of ROS production and the antioxidant mechanisms
in living organisms and can lead to peroxidative damage of lipids, proteins, polysaccharides,
DNA and other macromolecules and therefore alter cell function (MILLER et al. 1993). ROS
production of PMN is part of the normal host defenses against infectious diseases and is mediated
by the multicomponent enzyme NADPH-oxidase (NOX) (DAHLGREN and KARLSSON 1999).
However dairy cows are confronted with massive oxidative stress in the transition period as the
enhanced oxygen metabolism increases the rate of ROS production and may lead to a depletion
of important antioxidative defenses (SORDILLO and AITKEN 2009).
In order to investigate the immune system of high condition cows in a ketogenic metabolic status
we formed different animals groups with a diverse extent of postpartal body fat mobilization by
combination of the factors BCS before calving and concentrate proportion in the diet (DRONG et
al. 2015). We showed that a recently in the EU launched monensin controlled-release capsule
indicated for transition cows improved energy status and decreased ketogenesis in high
conditioned animals likely via an increased provision of ruminal propionate to hepatic
gluconeogenesis (SEAL and REYNOLDS 1993; DRONG et al. 2015). Simultaneously we could
55
PAPER II
not confirm analogous effects of the natural intervention essential oils on ruminal fermentation or
energy household of the cows (DRONG et al. 2015).
Still different compounds of essential oils have successfully been tested for anticancer,
antibacterial, antiviral, antioxidative property and in the treatment and prevention of
cardiovascular diseases including atherosclerosis and thrombosis in humans (EDRIS 2007) and
an enhanced immunocompetence and health of gut and a better performance of broilers and pigs
(MICHIELS et al. 2010; TIIHONEN et al. 2010), but studies on the effects on the immune
system of cows are very rare. Besides a reduction of the incidence of production-associated
diseases like mastitis, ketosis and displaced abomasum (DUFFIELD et al. 2008), monensin
improved chemotaxis of neutrophils obtained from monensin-treated cows in comparison to
control animals (STEPHENSON et al. 1996). An improved energy status, as found in the present
trial (DRONG et al. 2015), may therefore be a contributing factor to an improved immune
response around calving.
Therefore, the present work contributes to the topic of an immunosuppression around calving and
evaluates possible effects of the supplements on transition cow health.
Material and Methods
Experimental design
The experiment was carried out at the experimental station of the Institute of Animal Nutrition,
Friedrich-Loeffler-Institute (FLI) Braunschweig, Germany, in accordance with the Animal
Welfare Act concerning the protection of experimental animals with the approval of the Lower
Saxony State Office for Consumer Protection and Food Safety (LAVES, file no. 33.14-42502-0411/0444, Oldenburg, Germany). The animals were housed in free-stall barns and milked twice
daily.
The outline of the whole experiment is described in full detail in DRONG et al. (2015). In brief,
applying an animal model proposed by SCHULZ et al. (2014), 60 multiparous German Holstein
cows (mean parity 2.3 ± 1.4 (SD)) were allocated 42 days (d) ante partum (a.p.) to either high
body condition score (BCS) group (n = 45) with a mean BCS (EDMONSON et al. 1989) of 3.95
± 0.08 (SD) or to low BCS group (LC, n = 15) with a mean BCS of 2.77 ± 0.14 (SD). The high
56
PAPER II
BCS group was subdivided into 1 control group (HC, n = 15) and 2 groups receiving either
monensin (HC/Mo, n = 15) or essential oils (HC/EO, n = 15). Additionally every group was
portioned, one half of the animals receiving a vaccination against the Bovine Viral Diarrhea virus
(BVDV) (inactivated BVDV vaccine Bovilis BVD-MD, MSD Animal Health, Schwabenheim an
der Selz, Germany) into cervical musculature on d 42 a.p. (boost d 14 a.p.), and a second half
being vaccinated d 1 post partum (p.p.) (boost d 28 p.p.) except for the 5 ruminal-cannulated
cows (2 cows of LC, 1 cow of HC/MO, 2 cows of HC/EO group). All cows remained on
treatment until 56 days in milk (DIM). The experiment was divided into 3 periods: Period 1 (d 42 until calving), period 2 (1 until 14 DIM) and period 3 (15 until 56 DIM).
Ingredients and chemical composition of the experimental diet is shown by DRONG et al.
(2015). TMR was provided ad libitum via self-feeding stations (type RIC, Insentec B.V.,
Marknesse, The Netherlands). In addition, concentrate was provided by a computerized
concentrate feeding station (Insentec, B.V., Marknesse, The Netherlands). The HC/EO treatment
included a blend of essential oils (CRINA ruminants, DSM, Basel, Switzerland) in the pelleted
concentrate, with the target to provide 1 g essential oils/cow/d from d -21 (-22 ± 7 (mean ± SD))
a.p., while other groups received a control concentrate. The product contains a patented mixture
of natural and synthesized essential oils compounds including thymol, eugenol, vanillin, guaiacol
and limonene (MCINTOSH et al. 2003). The HC/MO group was administered a monensin CRC
(Kexxtone, Elanco, Bad Homburg, Germany) d -21 (-19 ± 5 (mean ± SD)) before calving,
supposed to release steadily 335 mg monensin/d for a period of 95 days. The classification of the
vaccination reaction as positive or negative was based on a threshold of >20% as suggested by
the supplier of the ELISA kit.
Measurements and sample collection
Blood samples were taken on d -42 (-40 ± 7 (mean ± SD)), -14 (-14 ± 3), -7 (-7 ± 1), -3 (-2 ± 1),
1, 7, 14, 21, 28, 35, 42 and 56 relative to calving from the jugular vein by using serum tubes for
the analysis of biochemical parameters and EDTA tubes for hematological analysis and flow
cytometry. Additionally samples from d -42, -14, -7, 1, 7, 14, 21, 28, 35, 42 and 56 relative to
calving were taken in serum and lithium heparin tubes for determination of BVDV antibody titer
57
PAPER II
and from d –42, -7, 1, 7, 14, 21 and 56 relative to calving for cell culture methods using
heparinised vacuum tubes.
After centrifugation (Heraeus Varifuge, 3.0R, 2000g, 15°C, 15 min), serum for determination of
biochemical parameters and serum and plasma for the detection of BVDV antibodies was stored
at -80°C until analysis. Additionally, whole blood was used for hematological analyses.
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by gradient
centrifugation using Ficoll separation solution after 1:1 dilution with phosphate buffered saline
(PBS) as described in detail by RENNER et al. (2011). The cells were frozen and stored at -80°C
until analysis.
Analyses
Hematology was performed in EDTA full blood using the automatic analyser Celltac-α (MEK
6450, Nihon Kohden, Qinlab Diagnostik, Weichs, Germany). White blood cell profile included
total leukocyte count (WBC), lymphocytes, granulocytes (basophil, neutrophil) and monocytes
whereby red blood cell profile included red blood cell count (RBC), hematocrit, hemoglobin and
the erythrocyte indices mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH)
and mean corpuscular hemoglobin concentration (MCHC).
Blood serum samples were analyzed for serum concentrations of triglycerides, albumin, total
protein, cholesterol, urea, γ-glutamyltransferase (GGT), aspartate aminotransferase (AST) and
glutamate dehydrogenase (GLDH) using an automatic analyzing system based on photometric
measurement (Eurolyser, Type VET CCA, 5020 Salzburg, Austria).
Cell metabolic activity and Concavalin A-stimulated proliferation of PBMC were evaluated using
the Alamar Blue (AB) assay which is based on the reduction of the nonfluorescent resazurin to
the fluorescent molecule resofurin by metabolically active cells. Thawed PBMC were first
washed and after centrifugation at 250 g for 8 minutes at room temperature, the pellet was
resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Biochrom AG, Berlin,
Germany) enriched with 5% fetal bovine serum (FBS, Biochrom AG, Berlin, Germany), 1 M
HEPES buffer (Biochrom AG, Berlin, Germany), 2 mM L-glutamine (Biochrom AG, Berlin,
Germany), 5 mM β-mercaptoethanol (Biochrom AG, Berlin, Germany), 100 U/mL penicillin and
58
PAPER II
0.1 mg/mL streptomycin solution (Biochrom AG, Berlin, Germany). Cell viability was
determined by tryptan blue exclusion technique in a Neubauer counting chamber and cell number
was adjusted to 1 x 106 cells/ml. The proportion of living cells mainly exceeded 60%. A 96-well
plate was filled quintuplicate with 100 µl cell suspension, Concavalin A (ConA, 2.5 µl/ml,
Sigma-Aldrich, Steinheim, Germany) and medium or only medium up to a total volume of 200
μl/well. After incubation for 69.5 hours at 37° and 5% CO2, plates were centrifuged at 200 g for 5
minutes and 100 µl of the supernatant were removed of each well before refilling with 11 µl AB
(AbD Serotec, Oxford, United Kingdom) in a ratio of 1:10. After a 2.5 hours incubation period,
the fluorescence of resofurin was measured (Tecan infinite M200, Groedig, Austria) at 540 nm
(excitation) and 590 nm (emission).
The determination of BVDV-specific antibodies was performed via a commercially available
indirect enzyme-linked immunosorbent assay (SVANOVA, Svanovir BVBV AB). A well-plate
coated with noninfectious BVDV-antigen was incubated with the serum sample and a positive
binding was visualised via an anti-bovine-conjugate and a substrate following the instructions of
the kit supplier. The extent of the reaction was determined photometrically at 450 nm by
measuring the optical density (OD) and a percentage of reaction was calculated as described by
the kit supplier.
For T-cell phenotyping, whole blood samples were double stained with monoclonal antibodies
for CD4 (mouse anti bovine CD4: FITC, BioRad, Hercules, CA, USA) and CD8 (mouse anti
bovine CD8: PE, BioRad, Hercules, CA, USA) or the corresponding isotype controls (mouse
IgG2a negative control: RPE and mouse IgG2b:FITC negative control, BioRad, Hercules, CA,
USA). After incubation for 30 min at room temperature red blood cells were lysed with lysis
buffer (BD Biosciences, San Jose, CA, USA) for 10 min at room temperature. Samples were
centrifuged (200 g, 5 min, 4°C), resuspended in HBS and measured by flow cytometry using
FACSCantoII (BD Biosciences, San Jose, CA, USA). Lymphoid population was identified
according to their side- and forward-scattering properties. At least 10,000 lymphocytes were
stored in list mode data files and the spillover of both fluorochromes (FITC and PE) was
compensated by the BD FACSDivaTM Software (BD Biosciences, San Jose, CA, USA). An
59
PAPER II
estimated number of T-cells of each phenotype were calculated using percentages obtained by
flow cytometer.
To analyze the capacity of PMN to elicit oxidative burst and the release of ROS, an assay was
employed that is based on the oxidation of the nonfluorescent dihydrorhodamine 123 (DHR) to
the fluorescent metabolite rhodamine 123 (R123) by hydrogen peroxide that can be detected and
measured by flow cytometry. The R123+ population describes the portion of PMN that converted
DHR to R123 namely via producing ROS. The mean fluorescence intensity (MFI) quantifies the
mean conversion of DHR per cell. Phorbol-12-mystristate-13-acetate (PMA) is an activator of
NOX by enhancing protein kinase C and thus stimulates production of ROS. Therefore whole
blood samples were incubated with 40 µM DHR (Molecular Probes Inc., Eugene, OR, USA)
alone or stimulated with 20 nM PMA (Sigma Aldrich Sigma-Aldrich, Steinheim, Germany) at
37°C for 15 min. After lysis of erythrocytes and fixation with lyse buffer (BD Pharm LyseTM, BD
Biosciences, San Jose, CA, USA) for 10 min, cells were washed with HEPES (4-(2hydroxyethyl)-1-piperazinethanesulfonic acid) buffered saline (HBS) and measured in duplicates
by flow cytometry using the FACSCantoII (BD Bioscienses, San Jose, CA, USA). At least
10,000 granulocytes were examined whereas the population of PMN was defined by its size and
granularity using forward and side scatter measurements.
Calculations
Proliferation of PBMC in vitro was calculated by the ratio between fluorescence of ConAstimulated PBMC and non-stimulated PBMC.
𝑆𝐼 =
𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑐𝑎𝑣𝑎𝑙𝑖𝑛 𝐴 − 𝑠𝑡𝑖𝑚𝑢𝑙𝑎𝑡𝑒𝑑 𝑃𝐵𝑀𝐶
𝐹𝑙𝑢𝑜𝑟𝑒𝑛𝑠𝑐𝑒𝑛𝑐𝑒 𝑜𝑓 𝑛𝑜𝑛𝑠𝑡𝑖𝑚𝑢𝑙𝑎𝑡𝑒𝑑 𝑃𝐵𝑀𝐶
The percentage of CD4+ divided by the percentage of CD8+ amounts to the ratio CD4+/CD8+.
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝐶𝐷4+
𝑅𝑎𝑡𝑖𝑜 𝐶𝐷4 /𝐶𝐷8 =
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝐶𝐷8+
+
+
60
PAPER II
Statistical analyses
All statistical analyses were performed using the MIXED procedure for repeated measures
(LITTELL et al. 1998) in SAS Software (Version 9.2). After testing various covariance
structures, compound symmetry covariance structure was used showing the lowest Akaike
information criterion (AICC). For biochemistry, hematology, parameters from cell culture and
flow cytometry the fixed effects included experimental treatment (LC, HC, HC/MO, HC/EO) and
period (1, 2, 3) and the interaction between these factors. For evaluation of the vaccination titer
experimental groups were split up according to vaccination time (a.p. vs. p.p.) before analysis.
The model contained experimental treatment and vaccination status (before vaccination, after
boost) as fixed effects and the interaction between these factors. The cow within treatment was
considered as a random effect. Effects were declared significant when p-values were ≤ 0.05 after
Tukey correction. Correlation coefficients were calculated by using the Statistica Software
(Version 12).
Results
Out of 60 cows, 55 animals completed the entire trial and one cow finished the experiment at day
28 (HC/EO group) because of an abomasal replacement. Additionally, 4 cows were excluded
because of illnesses that were not associated with the experimental treatments (1 cow of LC and
HC/MO group, 2 cows of HC group). Causes were abomasal displacement and multisystemic
health problems around calving (3 animals).
Hematological parameters are presented in Table 1. WBC count was 24% and 23% higher in HC
(p = 0.027) and HC/EO (p = 0.024), respectively, than in HC/MO group when all 3 periods were
pooled together. Additionally, it was dependent on period (p < 0.001) as it increased from period
1 to period 2 (p = 0.002) and decreased thereafter to values lower than in period 1 (p = 0.005).
Also the percentage of lymphocytes was dependent on the period (p < 0.001) as it was higher in
period 3 than in the previous periods (p < 0.001). The total lymphocyte count increased from
period 1 to period 2 (p = 0.040). The percentage of granulocytes in blood decreased from period
1 (p < 0.001) and 2 (p = 0.033) to period 3 while the total number was higher in periods 1 and 2
(4.3 x 103/µl and 4.8 x 103/µl, respectively) in comparison to period 3 (3.4 x 103/µl, p < 0.001).
61
PAPER II
The RBC showed no difference between the groups but it increased from period 1 to period 2 (p
= 0.003) and decreased thereafter to lower values in period 3 than in period 1 (p < 0.001). The
hemoglobin concentration was higher in the first 2 periods than in period 3 (p < 0.001), but no
effect of treatment was detectable. The hematocrit increased from period 1 to period 2 (p < 0.001)
and decreased in period 3 to lower values than in the first period (p < 0.001). Moreover,
hematocrit was higher in HC group (34.1%) than in LC group (31.6%, p = 0.019). MCV, MCH
and MCHC showed no differences in regard to treatment but they varied depending on period.
MCV increased in period 2 (p = 0.002) and decreased in period 3 (p < 0.001). MCH was higher
in the first 2 periods than in period 3 (p < 0.05) and MCHC was higher in period 2 than in period
3 (p = 0.016). Percentage and number of monocytes did not differ between groups and periods.
Table 1 Hematological variables during the trial from day (d) -42 relative to calving until 56 days in milk (DIM).
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period Trt*Period
3
White blood cells, 10 /µl
d -42 until calving
7.41AB
8.62
6.91
8.11B
0.50
abA
ab
b
d 1 until 14 DIM
8.63
9.06
7.24
9.89aA
0.52
0.014
<0.001
0.242
d 15 until 56 DIM
6.94B
7.59
6.27
7.14B
0.45
Lymphocytes, %
d -42 until calving
37.88
35.31
35.41B
35.75B
1.86
AB
d 1 until 14 DIM
37.03
37.75
38.18
35.07AB 1.90
0.715
<0.001
0.164
A
A
d 15 until 56 DIM
38.83
42.34
43.08
39.39
1.64
Lymphocytes, 103/µl
d -42 until calving
2.74
2.95
2.38
2.78
0.21
d 1 until 14 DIM
2.85
3.28
2.55
3.24
0.21
0.080
0.048
0.528
d 15 until 56 DIM
2.63
3.18
2.64
2.80
0.19
Monocytes, %
d -42 until calving
3.28
3.57
3.51
3.42
0.76
d 1 until 14 DIM
4.57
3.24
3.91
5.30
0.81
0.408
0.366
0.866
d 15 until 56 DIM
3.40
3.63
3.63
4.31
0.61
Monocytes, 103/µl
d -42 until calving
0.23
0.29
0.22
0.28
0.10
d 1 until 14 DIM
0.40
0.27
0.25
0.68
0.10
0.071
0.051
0.299
d 15 until 56 DIM
0.23
0.28
0.22
0.28
0.08
Granulocytes, %
d -42 until calving
52.35
53.53
54.75A
55.83
2.71
d 1 until 14 DIM
50.22
51.46
50.92AB
53.80
2.78
0.738
<0.001
0.298
d 15 until 56 DIM
51.57
45.46
45.80B
49.56
2.39
62
PAPER II
Table 1 (Continued)
Treatment
HC
Control
MO
P-value
Item
LC
EO
SEM*
Trt†
Period Trt*Period
3
Granulocytes, 10 /µl
d -42 until calving
3.97
4.70AB
3.91
4.70AB
0.40
d 1 until 14 DIM
4.84
4.95A
4.05
5.49A
0.41
0.121
<0.001
0.564
d 15 until 56 DIM
3.69
3.46B
2.99
3.60B
0.35
Red blood cells, 106/µl
d -42 until calving
5.64A
5.77A
5.49AB
5.49A
0.14
A
A
A
d 1 until 14 DIM
5.75
5.84
5.78
5.69A
0.14
0.810
<0.001
0.128
d 15 until 56 DIM
5.21B
5.25B
5.36B
5.16B
0.13
Hemoglobin, g/dL
d -42 until calving
10.55A
10.64AB
10.15
10.68
0.34
A
d 1 until 14 DIM
10.69
11.11A
10.76
10.94A
0.34
0.722
<0.001
0.439
B
B
B
d 15 until 56 DIM
8.98
9.64
9.70
9.36
0.30
Hematocrit, %
d -42 until calving
32.31A
35.14A
33.15B
33.62A
0.68
A
A
A
A
d 1 until 14 DIM
33.25
35.78
35.29
35.26
0.69
0.027
<0.001
0.274
d 15 until 56 DIM
29.12B
31.29B
31.42B
30.94B
0.64
Mean corpuscular volume, fL
d -42 until calving
57.70A
61.20A
60.50A
61.37A
1.31
A
A
A
A
d 1 until 14 DIM
58.23
61.38
61.37
62.26
1.31
0.163
<0.001
0.614
d 15 until 56 DIM
56.36B
59.63B
58.87B
60.21B
1.30
Mean corpuscular hemoglobin, pg
d -42 until calving
18.29
18.98
18.39
19.78
0.63
d 1 until 14 DIM
18.89
19.05
18.75
19.30
0.64
0.593
0.002
0.603
d 15 until 56 DIM
17.38
18.35
18.22
18.21
0.58
Mean corpuscular hemoglobin concentration, g/dL
d -42 until calving
31.13
30.31
30.56
31.05
0.25
d 1 until 14 DIM
30.73
30.50
30.15
30.75
0.24
0.207
0.021
0.142
d 15 until 56 DIM
30.46
30.51
30.33
30.21
0.20
† Treatment (Trt): High condition (HC, n = 13) cows were fed a concentrate proportion of 60%; low condition (LC,
n = 14) cows a concentrate proportion of 20% in the prepartum diet. Postcalving, the concentrate proportion in the
diet was increased from 30% to 50% within 2 weeks in LC group and 3 weeks in HC groups. HC/MO (n = 14) was
DIM.
Biochemical parameters are presented in Table 2. The highest concentration of albumin was
found in the third period except for HC/MO group, whereby a significant difference was detected
between the prepartal time and period 3 in HC/EO group (34.6 g/L vs. 37.6 g/L, p = 0.003).
63
PAPER II
Cholesterol concentration was lowest in the first 2 weeks of lactation and increased significantly
thereafter in the third period (p < 0.001). Total protein concentration was dependent on period (p
< 0.001) and a treatment x period interaction was found (p = 0.004) as it was highest in the third
period and lowest in the prepartal period in all groups. Concentration of triglycerides decreased
after calving (p < 0.001) and cows of LC showed higher concentrations than cows of HC/EO in
the prepartal period (24.5 mg/dL vs. 20.6 mg/dL, p = 0.037). Bilirubin concentration was
increased in HC (0.28 mg/dL, p = 0.028) and HC/EO (0.30 mg/dL, p = 0.003) cows in
comparison to LC (0.18 mg/dL) and it was higher in all HC groups than in LC in the first 2
weeks of lactation (p < 0.05). Blood urea concentration was higher in HC/MO cows in
comparison to the other groups (p < 0.05), especially in the first 2 periods. The enzymes AST,
GGT and GLDH all increased after calving whereas AST was highest immediately after calving
(p < 0.001) while the others reached their maximum in period 3 (p < 0.05). Cows of HC (92.8
Units/L) had higher AST concentrations than LC (66.4 Units/L) and HC/MO cows (70.1 Units/L)
over the whole trial (p < 0.05). Distinct differences were found between HC/EO group and all
other groups concerning GGT (p < 0.05) and between HC/EO (61.4 Units/L) and LC (18.8
Units/L, p = 0.027) and HC/MO treatment (18.5 Units/L, p = 0.024) for GLDH in the third
period.
Table 2 Biochemical metabolites and serum activities of γ-glutamyltransferase (GGT), aspartate aminotransferase
(AST) and glutamate dehydrogenase (GLDH) during the trial from day (d) -42 relative to calving until 56 days in
milk (DIM).
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period Trt*Period
Albumin, g/L
d -42 until calving
34.9
36.0
34.7
34.6B
0.8
d 1 until 14 DIM
34.3
35.6
36.1
36.0AB
0.8
0.690
<0.001
0.056
d 15 until 56 DIM
36.8
36.8
35.6
37.6A
0.7
Cholesterol, mg/dL
d -42 until calving
92.3B
81.7B
73.5B
76.5B
6.5
B
B
B
d 1 until 14 DIM
80.4
71.6
66.3
73.7B
6.7
0.174
<0.001
0.095
d 15 until 56 DIM
160.5A
142.5A
145.5A
162.7A
6.0
64
PAPER II
Table 2 (Continued)
Treatment
HC
Control
MO
P-value
Item
LC
EO
SEM*
Trt†
Period Trt*Period
Total protein, g/L
d -42 until calving
66.5B
66.4B
65.3B
62.2C
1.4
d 1 until 14 DIM
67.5AB
66.8B
69.1AB
67.4B
1.4
0.935
<0.001
0.004
d 15 until 56 DIM
71.4A
72.8A
70.7A
73.5A
1.3
Triglycerides, mg/dL
d -42 until calving
24.5aA
22.9abA
21.4abA
20.6bA
0.8
B
B
B
d 1 until 14 DIM
10.7
11.9
11.2
11.3B
0.8
0.180
<0.001
0.076
d 15 until 56 DIM
10.9B
11.7B
10.4B
10.9B
0.7
Bilirubin, mg/dL
d -42 until calving
0.14
0.15B
0.16B
0.21B
0.04
b
aA
aA
d 1 until 14 DIM
0.24
0.48
0.45
0.47aA
0.04
0.004
<0.001
0.026
B
B
B
d 15 until 56 DIM
0.15
0.22
0.18
0.24
0.03
Urea, mg/dL
d -42 until calving
18.6b
23.7abA
27.8a
23.4abA
1.3
ab
bAB
a
abA
d 1 until 14 DIM
23.3
21.5
28.3
24.3
1.4
<0.001
<0.001
0.014
d 15 until 56 DIM
18.9
15.8B
20.3
19.2B
1.1
AST, Units/L
d -42 until calving
56.3
62.0B
52.2B
62.0B
8.7
b
aA
abA
abA
d 1 until 14 DIM
71.8
128.8
92.0
107.0
8.8
0.007
<0.001
0.072
d 15 until 56 DIM
71.1
87.8B
66.2AB
86.2AB
8.0
GGT, Units/L
d -42 until calving
19.6
21.5
21.1
19.4B
8.5
d 1 until 14 DIM
21.3
24.4
21.6
25.8B
8.6
0.034
<0.001
0.007
d 15 until 56 DIM
26.3b
37.4b
31.2b
80.4aA
8.2
GLDH, Units/L
d -42 until calving
14.4
8.9
8.3
6.8
9.0
d 1 until 14 DIM
11.1
18.4
14.3
13.6
9.0
0.283
0.001
0.026
b
ab
b
a
d 15 until 56 DIM
18.8
23.6
18.5
61.4
8.6
†
DIM.
Evaluation of the AB-assay showed no influence of treatment on viability of non-stimulated or
proliferation of ConA-stimulated PBMC as well as on the SI, but all parameters were influenced
by period (Table 3). Basal viability of non-stimulated cells was higher in the second than in the
65
PAPER II
third period (p = 0.027) while the proliferation after ConA-stimulation increased from period 1 to
period 2 (p = 0.021) and 3 (p < 0.001, Table 3). Similarly SI was lower in period 1 (p < 0.001)
and 2 (p < 0.001) than in period 3 (Table 3). During course of trial the ConA-stimulated
proliferation peaked on d +1 in HC/EO group while it was d +14, d +21 and d +56 in LC, HC and
HC/MO treatment, respectively (Figure 1). The SI reached a nadir close to parturition, namely on
d -7 in HC/EO, d +1 in HC and d +7 in LC and HC/MO group and increased thereafter with a
maximum in LC group on d +21 (Figure 1).
Table 3 Function parameters of peripheral blood mononuclear cells (PBMC) during the trial from day (d) -42
relative to calving until 56 days in milk (DIM).
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period
Trt*Period
Stimulation index
d -42 until calving
4.41B
4.33
4.65
4.80
0.33
B
d 1 until 14 DIM
4.16
4.42
4.88
4.85
0.28
0.711
0.000
0.325
d 15 until 56 DIM
5.69A
5.37
5.52
5.29
0.28
Fluorescence : nonstimulated PBMC
d -42 until calving
4965AB
4897
4308
5460
455
A
d 1 until 14 DIM
6188
5522
5105
5192
357
0.533
0.008
0.170
d 15 until 56 DIM
4425B
4413
4938
5052
426
Fluorescence : mitogen-stimulated PBMC
d -42 until calving
19488
20604
19377B
22235
1401
d 1 until 14 DIM
23009
21513
23037AB
23742
1081
0.311
0.001
0.522
d 15 until 56 DIM
23662
22534
26204A
24062
1307
†
DIM.
66
PAPER II
8000
unstimulated PBMC
7000
6000
5000
4000
3000
2000
1000
mitogen-stimulated PBMC
0
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
30000
25000
20000
15000
10000
5000
0
7
Stimulation index
6
5
4
3
2
1
0
Figure 1 Mean viability and proliferation of non- and mitogen-stimulated peripheral blood mononuclear cells
(PBMC) and the stimulation index during the trial. Cows received a low concentrate (∆) diet with a
concentrate:roughage ratio of 20:80 (n = 14) or a high concentrate (●) diet with a concentrate:roughage ratio of 60:40
a.p.. Postpartal concentrate:roughage ratio was changed stepwise from 30:70 to 50:50 within 2 weeks in the low
concentrate group (∆) and 3 weeks in the high concentrate groups (●). Treatment included no supplementation (─) (n
= 13), the administration of a monensin controlled-release capsule (∙∙∙) (n = 14) at day (d) -21 and the addition of 1
g/d/cow of a blend of essential oils (---) (n = 15) from d -21 until d 56 relative to calving.
67
PAPER II
The course of ELISA-antibodies after vaccination with an inactivated BVDV-vaccine is shown in
Figure 2. The BVDV-specific ELISA-reactions of a.p. and p.p. vaccinated cows were dependent
on the number of vaccinations as it increased after booster injection (p < 0.001). For a.p.
vaccinated animals there was a treatment x vaccination interaction detected (p = 0.014) as
vaccinated cows of HC/MO group developed a 103% higher score than vaccinated cows of
HC/EO group (p = 0.018) over the whole trial. An increase in BVDV-specific antibody ELISApercentages after booster injection could only be detected in LC (p < 0.001) and HC/MO group
(p < 0.001). This interaction was also found for p.p. vaccinated cows, but an increase in BVDVELISA-antibodies after booster injection was found in all groups (p < 0.001). A 64% and 58%
higher antibody score was found in vaccinated cows of HC/MO than in vaccinated cows of LC (p
= 0.023) and HC/EO group (p = 0.024), respectively. A positive vaccination reaction in a.p. and
p.p. vaccinated animals could be detected in 86% and 40% of LC, 80% and 75% of HC, 100%
and 86% of HC/MO and 80% and 29% of HC/EO cows, respectively.
68
Percentage of ante partum
vaccinated cows, %
PAPER II
45
40
35
30
25
20
15
10
5
0
Treatment
Vaccination Status
Treatment x Vaccination Status
Percentage of post partum
vaccinated cows, %
-42 -35 -28 -21 -14
45
40
35
30
25
20
15
10
5
0
-7
p = 0.227
p < 0.001
p = 0.014
0
7
Treatment
Vaccination Status
Treatment x Vaccination Status
-42 -35 -28 -21 -14
-7
14
21
28
35
42
49
56
21
28
35
42
49
56
p = 0.065
p < 0.001
p < 0.001
0
7
14
Figure 2 Antibody scores expressed as Bovine Viral Diarrhea virus (BVDV)-specific antibody ELISA-percentages
during the trial. Vaccination (↑) was ante partum (day (d) -42 and -14) or post partum (d +1 and +28). Cows
received a low concentrate (∆) diet with a concentrate:roughage ratio of 20:80 (n = 7 and 5) or a high concentrate (●)
diet with a concentrate:roughage ratio of 60:40 ante partum. Postpartal concentrate:roughage ratio was changed
stepwise from 30:70 to 50:50 within 2 weeks in the low concentrate group (∆) and 3 weeks in the high concentrate
groups (●). Treatment included no supplementation (─) (n = 5 and 8), the administration of a monensin controlledrelease capsule (∙∙∙) (n = 6 and 7) at d -21 and the addition of 1 g/d/cow of a blend of essential oils (---) (n = 6 and 7)
from d -21 until d 56 relative to calving. The statistical analysis included treatment (LC, HC, HC/MO, HC/EO),
vaccination status (before vaccination vs. after boost) and the interaction.
Lymphocyte subpopulations were not influenced by treatment or period but a treatment x period
interaction was found for CD4+ subpopulations (p = 0.044) and the CD4+/CD8+ ratio (p = 0.030,
Table 4). In the prepartal period, percentage of CD4+ was highest in HC/MO (37.5%) with a peak
69
PAPER II
on day -14 and lowest in HC group (33.0%, Figure 3). In the second period it stayed on lowest
level in HC group (31.4%) with a nadir on day +1 while it was nearly at 35% in the other groups
(Figure 3). Over the remaining trial, no clear differences were visible. The CD4+/CD8+ ratio was
highest in HC/EO group with a peak on day +35 (Figure 3).
Table 4 Proportion of CD4+ and CD8+ subpopulations and the ratio CD4+/CD8+ during the trial from day (d) -42
relative to calving until 56 days in milk (DIM).
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period
Trt*Period
CD4+, %
d -42 until calving
35.3
33.0
37.5
34.3
1.6
d 1 until 14 DIM
35.4
31.4
35.0
34.8
1.6
0.403
0.166
0.044
d 15 until 56 DIM
36.9
33.5
34.7
34.9
1.5
CD8+, %
d -42 until calving
14.9
14.3
14.9
13.0
1.3
d 1 until 14 DIM
14.9
13.6
14.3
13.1
1.3
0.462
0.711
0.568
d 15 until 56 DIM
15.4
13.4
15.2
12.0
1.2
Ratio CD4+ : CD8+
d -42 until calving
2.6
2.6
3.0
3.0
0.3
d 1 until 14 DIM
2.6
2.7
3.0
3.0
0.3
0.561
0.958
0.030
d 15 until 56 DIM
2.6
2.7
2.7
3.4
0.3
†
DIM.
All parameters of DHR-assay were influenced by period (p < 0.05) while treatment exerted no
effect (Table 5). Basal and stimulated R123+ population dropped in the second period and
increased thereafter again (p < 0.05, Table 5). The basal R123+ population was on the highest
level on d -42 in all groups with a maximum in HC/EO group (24%) and declined thereafter
gradually to about 2% at day of calving. Thereafter it increased again with the most rapid course
in LC group (Figure 4). Via PMA stimulation it was possible to activate ROS-production in more
than 97.5% of the PMN population (Figure 4). The nadir of stimulated R123+ population was also
on day of calving in all groups but HC and HC/EO group showed a second drop, namely on d
70
PAPER II
+35 and d +28, respectively (Figure 4). The basal MFI of the R123+ population was on one level
within all groups except for HC group on d -42 and increased in the second period before a
similar level was reached in the last period again (Figure 4). Within groups, great variation
occurred between d -7 and d +28 (Figure 4). During this interval, MFI increased up to a peak in
HC group on d -3 with a drop in all treatments next to calving (Figure 4). The course of the
stimulated MFI of the R123+ population developed similarly in all groups while it reached a
maximum on d +7 with highest values in HC/MO and HC group (Figure 4).
Table 5 Mean proportion and fluorescence intensity (MFI) of basal and stimulated R123 + population of
polymorphonuclear leukocytes during the trial from day (d) -42 relative to calving until 56 days in milk (DIM).
Treatment
P-value
HC
Item
LC
Control
MO
EO
SEM*
Trt†
Period
Trt*Period
+
R123 , %
d -42 until calving
12.3
10.4
10.4
11.5
1.3
d 1 until 14 DIM
5.9
3.9
3.2
2.9
1.4
0.264
<0.001
0.778
d 15 until 56 DIM
12.2
10.5
10.2
8.7
1.2
R123+ MFI
d -42 until calving
6038
5992
4823
5459
600
d 1 until 14 DIM
7435
6184
5969
6024
618
0.139
0.030
0.940
d 15 until 56 DIM
5796
5674
4959
5158
531
Stimulated R123+ , %
d -42 until calving
98.9
98.6
98.6
98.5
0.6
d 1 until 14 DIM
96.7
97.6
97.6
97.8
0.7
0.836
0.016
0.807
d 15 until 56 DIM
98.8
97.8
98.8
98.6
0.5
Stimulated R123+ MFI
d -42 until calving
49824
51375
49434
49975
1828
d 1 until 14 DIM
53671
55819
57091
54357
1883
0.845
<0.001
0.888
d 15 until 56 DIM
50817
50475
51438
50638
1583
†
DIM.
71
PAPER II
Discussion
The purpose of this study was to determine biochemical and hematological parameters, immune
cell subsets and function in blood of dairy cows differing in body condition around parturition
and to examine if monensin or essential oils elicit any effect on immune response.
Performance and rumen fermentation parameters as well as serum levels of NFEA, βhydroxybutyrate (BHB) and glucose of this study have previously been reported (DRONG et al.
2015). In summary, the experimental design according to the animal model (SCHULZ et al.
2014) facilitated to generate 2 distinguishing investigational groups. Namely the LC group with
moderate body condition until calving and fat depot mobilization p.p. that served as a positive
control group. Furthermore, the high condition groups with a significant higher net energy
balance (EB) a.p. than LC group culminating in a more than 1.0 point on scale higher BCS at
calving. Dry matter intake was 3.3 kg/d higher in HC/MO than in LC cows in dry period and was
not different between the groups after calving (DRONG et al. 2015). The EB switched to
negative values p.p. especially in HC/MO group in period 2 (-82.5 MJ NEL/d) and HC/EO group
in period 3 (-37.4 MJ NEL/d) which was significantly lower than EB of LC group (-41.1 and 17.0 MJ NEL/d, respectively) (DRONG et al. 2015). It was accompanied by a massive loss in
body condition in early lactation, especially in the high condition cows. In fact, NEFA
concentration was more than twice as high and prevalence of either subclinical or clinical p.p.
ketosis was 36% higher in HC than in LC group. While administration of essential oils did not
accomplish any protective effect, monensin improved the feed efficiency (kg energy corrected
milk/ kg dry matter intake) of high conditioned cows and decreased, although fat mobilization
and NEFA concentrations could not be diminished, the concentration of BHB in blood.
72
CD4+ population, %
PAPER II
45
40
35
30
25
20
15
10
5
0
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
-42 -35 -28 -21 -14
-7
0
7
14
21
28
35
42
49
56
-42 -35 -28 -21 -14 -7
0
7 14 21 28
35
42
49
56
CD8+ population, %
25
20
15
10
5
Ratio CD4+/CD8+
0
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Figure 3 Mean proportion of CD4+ and CD8+ subpopulation and the ratio CD4+/CD8+ during the trial. Cows received
a low concentrate (∆) diet with a concentrate:roughage ratio of 20:80 (n = 14) or a high concentrate (●) diet with a
concentrate:roughage ratio of 60:40 ante partum. Postpartal concentrate:roughage ratio was changed stepwise from
30:70 to 50:50 within 2 weeks in the low concentrate group (∆) and 3 weeks in the high concentrate groups (●).
Treatment included no supplementation (─) (n = 13), the administration of a monensin controlled-release capsule (∙∙∙)
(n = 14) at day (d) -21 and the addition of 1 g/d/cow of a blend of essential oils (---) (n = 15) from d -21 until d 56
relative to calving.
73
PAPER II
Elevated blood concentrations of the fat-derivatives NEFA and ketone bodies in early lactation
together with fatty infiltration of the liver have been postulated as important risk factors for
periparturient diseases in dairy cows (ZERBE et al. 2000; ROCHE et al. 2009; ESPOSITO et al.
2014). Biochemical parameters of the present trial suggest an impairment of liver health and
function in high condition groups in the present trial. While levels of AST, GGT and GLDH were
located within physiological range in LC group (KRAFT 2005), AST was increased in HC group
in the first two weeks of lactation and GGT and GLDH were increased in HC/EO group in period
3.. Bilirubin concentrations exceeded the physiological range in all high condition groups in the
first 2 weeks of lactation (KRAFT 2005) giving evidence for a decreased bile flow in the liver
(BOBE et al. 2004) whereby bilirubin was shown to increase with increased triacylglycerol
accumulation in the liver (REID et al. 1983; WEST 1990). In accordance to that a positive
correlation could be found between liver lipid content of the present trial (DRONG et al. 2015)
and AST (r = 0.42, p < 0.001), GGT (r = 0.30, p = 0.001), GLDH (r = 0.53, p < 0.001) and
bilirubin (r = 0.3357, p < 0.001). Correspondingly, cholesterol concentrations were lower in high
condition groups in late dry period and after calving. As cholesterol is a component of serum
lipoproteins and its concentration in serum is an indicator of overall lipoprotein concentrations
(KANEENE et al. 1997), it gives further evidence for an impaired liver function. Serum activities
of AST, GGT and GLDH of monensin-treated cows did not significantly differ from values of
LC group. This may indicate a protective effect against hepatocyte cell damage although the
bilirubin synthesis and/or excretion was diminished in HC/MO cows in early lactation, too. The
higher blood urea concentration after monensin supplementation together with an increased milk
urea (DRONG et al. 2015) is similar to results of DUFFIELD et al. (2003) and GREEN et al.
(1999) after delivery of a CRC 3 weeks a.p. and is thought to be a result of its protein-sparing
effect in the rumen with subsequent increased intestinal amino acid resorption. Results do not
support the idea of a corresponding protein-sparing effect of essential oils (MCINTOSH et al.
2003).
74
100
Stimulated R123+ population, %
Basal R123+ population, %
30
25
20
15
10
5
99.5
99
98.5
98
97.5
97
96.5
96
95.5
95
0
94.5
0
-7
0
7
14
21
28
35
42
49
-42 -35 -28 -21 -14 -7
MFI of stimulated R123+ population
10000
56
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
-42 -35 -28 -21 -14 -7
0
7
14 21 28 35 42 49 56
0
7
14
21
28
35
42
49
56
70000
PAPER II
75
MFI of basal R123+ population, %
-42 -35 -28 -21 -14
60000
50000
40000
30000
20000
10000
0
-42 -35 -28 -21 -14 -7
0
7
14 21 28 35 42 49 56
Figure 4 Mean basal and stimulated proportion and fluorescence intensity (MFI) of R123+ population of total polymorphonuclear leukocytes during
the trial. Cows received a low concentrate (∆) diet with a concentrate:roughage ratio of 20:80 (n = 14) or a high concentrate (●) diet with a
concentrate:roughage ratio of 60:40 ante partum. Postpartal concentrate:roughage ratio was changed stepwise from 30:70 to 50:50 within 2 weeks in
the low concentrate group (∆) and 3 weeks in the high concentrate groups (●). Treatment included no supplementation (─) (n = 13), the administration
of a monensin controlled-release capsule (∙∙∙) (n = 14) at day (d) -21 and the addition of 1 g/d/cow of a blend of essential oils (---) (n = 15) from d -21
until d 56 relative to calving.
PAPER II
Immune cell populations and function of the present trial were rather influenced by the event of
calving than by treatment. The results from PBMC proliferation assay showed no differences
between groups but the lowest proliferation was found near calving in all groups similar to results
from other studies (ISHIKAWA 1987; DETILLEUX et al. 1994; LESSARD et al. 2004;
RENNER et al. 2012) what supports the thesis of an immunosuppression during that time.
Similarly, a distinctive drop in the proportion of ROS-producing cells relative to the total PMN
population was visible immediately after calving on d +1 as reported previously (DOSOGNE et
al. 1999; RINALDI et al. 2008). The basal proportion of R123+ declined to about 2% irrespective
of treatment, emphasizing the great impact of this event on the oxidative system. This may
indicate a decreased metabolic activity of the cells at this time point. The great variability of basal
MFI in this interval, as a parameter giving evidence for intensity of ROS-production per cell,
may therefore only reflect the disparity of the current population at calving. Regarding the higher
MFI of HC and HC/MO on d +7, it may give evidence for a more efficient oxidative burst in
these groups. Also the stimulated R123+ population showed a nadir on day of parturition and
additionally a second drop in 2 groups, signaling any immunological challenges 4 to 5 weeks
after calving in those groups. This might have impaired host defense mechanisms as an
inadequate ROS-production may restrict elimination of pathogens and the function as messengers
for modulation of and recovery from inflammation may diminish.
High concentrations of NEFA and ketone bodies and liver lipid content were reported to disrupt
immune cell function (ZERBE et al. 2000; SCALIA et al. 2006; CONTRERAS and SORDILLO
2011). Analogous to that, we found a negative correlation between NEFA concentration and SI of
AB assay (r = -0.11, p < 0.05) as well as between NEFA concentration and stimulated R123 +
population (r = -0.15, p < 0.001) and NEFA, BHB concentration and liver lipid content and basal
R123+ population (r = -0.31, r = -0.09 and r = -0.37, respectively, p < 0.05).
An increase in WBC and lymphocytes at calving as found in the present trial has been reported
previously (MOREIRA DA SILVA et al. 1998; ZERBE et al. 2000) although results from
literature are not consistent (QUIROZ-ROCHA et al. 2009; TREVISI et al. 2012). The lower
WBC in monensin-supplemented cows in comparison to HC (P = 0.027) and HC/EO group (P =
0.024) may not have any clinical relevance as WBC was within physiological range in all groups
76
PAPER II
(Kraft 2005). There was reported a depletion of CD4+ (KIMURA et al. 1999) and CD8+
populations (VAN KAMPEN and MALLARD 1997) next to calving. Although the nadir of
CD4+ populations in all groups was located somewhere between d -7 and d 7 in the present trial
(Figure 3), too, no effect of period or treatment were visible.
Antibody titer of cows that were vaccinated immediately after calving could not reach an extent
as did a.p. vaccinated cows, and furthermore the ELISA-percentages seem to diminish steadily in
these groups after a first peak (Figure 2). During parturition, there was additionally a drop in
antibody production of all a.p. vaccinated cows from the dry period to early lactation which may
be most likely due to a redirection of antibodies to mammary gland for colostrogenesis or to an
impaired antibody production. This is in accordance to previous results observed in 52 Holstein
cows that showed a drop of serum concentrations of Immunoglobulin G at late gestation with
lowest values at parturition (ISHIKAWA 1987), similar to results reported by DETILLEUX et al.
(1994) and HERR et al. (2011). The meaning of a second drop of antibody-percentages of a.p.
vaccinated cows on 21 DIM in unclear, whereas after 35 DIM antibody response seems to
generally diminish. Conclusions have to be drawn cautiously as we cannot assess further course
after 56 DIM and the frequency of blood sampling was different between the 2 vaccination
groups. However, it has also to be stated, that the used indirect ELISA detects especially
antibodies against the envelope proteins of BVDV which are the target for the protective
neutralizing activity. Furthermore, the moderate immunogenicity of the used vaccine allowed to
measure in a range of non-saturated ELISA-percentages which enables a direct comparison
without titration.
The influence of supplementation measure on antibody response is difficult to assess as it was
only one week between begin of both supplementations and the boost of the a.p. vaccinated cows.
However, a positive vaccination reaction could be detected in more than 80% of cows of each
group with HC/MO group showing a 100% positive reaction. Regarding the p.p. vaccination titer,
monensin increased the antibody response in comparison to essential oils supplementation (p =
0.024) and monensin-supplemented cows showed the highest percentage of a positive vaccination
reaction with 86% in contrast to only 29% in the HC/EO group. Interestingly, LC cows with one
77
PAPER II
of the highest BVDV-antibody-scores in a.p. vaccinated cows, showed a lessened responsiveness
when vaccinated immediately p.p..
In summary, the provoked large energy deficit in HC/EO cows as seen in a highly ketotic
metabolic status (Peak at d +35; BHB = 2.25 mmol/L) and great liver fat accumulation (Peak at d
+21; liver fat content = 215 mg/g fresh liver weight) (DRONG et al. 2015) coincidences with the
mentioned seemingly impairment of liver health parameters (GGT, GLDH) and a diminished
response to BVD vaccination. Similar circumstances certainly existed in HC group, too, although
parameters were not that prominent. Although actual research attribute essential oils antioxidative
(MIGUEL 2010) and anti-inflammatory properties (FACHINI-QUEIROZ et al. 2012), no effect
of supplementation could be detected under present conditions whereas other combinations or
solitary application of promising substances may achieve better results. The fact that monensinsupplemented cows markedly mobilized fat tissue but somehow did not reach a ketogenic
metabolic status and accumulated liver fat in an extent similar to their high conditionedcounterparts (DRONG et al. 2015), implies a more distinct complete oxidation of NEFA.
Monensin was shown to increase β-oxidation and therefore limit hepatic fat storage, decrease
peroxisomal NEFA oxidation with subsequent ROS-production and lessen accumulation of lipid
metabolites that may impair liver metabolism via influencing abundance of carnitine
palmitoyltransferase (CPT)-1a responsible for uptake of NEFA from cytosol into mitochondria
(MULLINS et al. 2012). As liver plays a key role in lipid metabolism and an optimal liver
function is central to the ability to make a balanced transition period (DRACKLEY et al. 2005)
the idea is that monensin might have attenuated inflammatory conditions in the liver and limit its
effects on immune functions. Effects on concentration of acute phase proteins haptoglobin and
ceruloplasmin are reported in literature (STEPHENSON et al. 1997; CRAWFORD et al. 2005).
However, the only direct or indirect effect on immune cell function in the present trial was found
in the highest percentage of a positive vaccination reaction with 100% and 86% of a.p. and p.p.
vaccinated cows.
Observed changes in red blood cell profile in the course of the trial have not consistently been
reported in literature (GĂVAN et al. 2010). A higher hematocrit in our study around calving may
be due to a haemoconcentration because of a reduced water intake and a subsequent moderate
78
PAPER II
dehydration. Although hematocrit was 2.5% higher in HC than in LC group, it was within range
in all groups (KRAFT 2005) and this effect of treatment may not have any clinical and
immunological relevance.
Conclusion
The event of calving induced massive changes in immune status of the cows as seen in short-term
effects on parameters like ROS production in PMN and antibody titer. Biochemical parameters
give evidence for an impaired liver health and synthesis as displayed in increased bilirubin
concentrations in all high condition cows and increased levels of AST, GGT or GLDH after
calving in HC and HC/EO group.
A monensin CRC led to a reduced concentration of blood metabolites that may indicate in
impaired hepatocyte integrity and generated highest BVDV antibody scores in high condition
cows. These results suggest direct or indirect monensin effects on the liver and the immune
system. The nature of these effects needs to be clarified further.
References
BOBE, G., J. YOUNG, and D. BEITZ (2004):
Invited Review: Pathology, etiology, prevention, and treatment of fatty liver in dairy cows.
J. Dairy Sci. 87, 3105-3124
CONTRERAS, G. A., and L. M. SORDILLO (2011):
Lipid mobilization and inflammatory responses during the transition period of dairy cows.
Comp. Immunol. Microbiol. Infect. Dis. 34, 281-289
CRAWFORD, R. G., K. E. LESLIE, R. BAGG, C. P. DICK, and T. F. DUFFIELD (2005):
The impact of controlled release capsules of monensin on postcalving haptoglobin concentrations
in dairy cattle.
Can. J. Comp. Med. 69, 208
DAHLGREN, C., and A. KARLSSON (1999):
Respiratory burst in human neutrophils.
J. Immunol. Methods 232, 3-14
79
PAPER II
DETILLEUX, J. C., K. J. KOEHLER, A. E. FREEMAN, M. E. KEHRLI Jr, and D. H. KELLEY
(1994):
Immunological parameters of periparturient Holstein cattle: Genetic variation.
J. Dairy Sci. 77, 2640-2650.
DOSOGNE, H., C. BURVENICH, A. FREEMAN, M. KEHRLI JR, J. DETILLEUX, J. SULON,
J.-F. BECKERS, and D. HOEBEN (1999):
Pregnancy-associated glycoprotein and decreased polymorphonuclear leukocyte function in early
post-partum dairy cows.
Vet. Immunol. Immunopathol. 67, 47-54
DRACKLEY, J. K., H. M. DANN, G. N. DOUGLAS, N. A. J. GURETZKY, N. B.
LITHERLAND, J. P. UNDERWOOD, and J. J. LOOR (2005):
Physiological and pathological adaptations in dairy cows that may increase susceptibility to
periparturient diseases and disorders.
Ital. J. Anim. Sci. 4, 323–344
DRONG, C., U. MEYER, D. VON SOOSTEN, J. FRAHM, J. REHAGE, G. BREVES, and S.
DÄNICKE (2015):
Effect of monensin and essential oils on performance and energy metabolism of transition dairy
cows.
DUFFIELD, T., S. LEBLANC, R. BAGG, K. LESLIE, J. TEN HAG, and P. DICK (2003):
Effect of a monensin controlled release capsule on metabolic parameters in transition dairy cows.
J. Dairy Sci. 86, 1171-1176
DUFFIELD, T., A. RABIEE, and I. LEAN (2008):
reproduction.
J. Dairy Sci. 91, 2328-2341
EDMONSON, A. J., I. J. LEAN, L. D. WEAVER, T. FARVER, and G. WEBSTER (1989):
A body condition scoring chart for Holstein dairy cows.
J. Dairy Sci. 72, 68-78
80
PAPER II
EDRIS, A. E. (2007):
Pharmaceutical and therapeutic potentials of essential oils and their individual volatile
constituents: A review.
Phytother. Res. 21, 308-323
ESPOSITO, G., P. C. IRONS, E. C. WEBB, and A. CHAPWANYA (2014):
Interactions between negative energy balance, metabolic diseases, uterine health and immune
response in transition dairy cows.
Anim. Reprod. Sci. 144, 60-71
FACHINI-QUEIROZ, F. C., R. KUMMER, C. F. ESTEVAO-SILVA, M. D. D. B.
CARVALHO, J. M. CUNHA, R. GRESPAN, C. A. BERSANI-AMADO, and R. K. N. CUMAN
(2012):
Effects of thymol and carvacrol, constituents of Thymus vulgaris L. essential oil, on the
inflammatory response.
Evid. Based Compl. Alt. 1, 1-10
GĂVAN, C., C. RETEA, and V. MOTORGA (2010):
Changes in the hematological profile of Holstein primiparous in periparturient period and in early
to mid lactation.
Sci. Pap. Anim. Sci. Biotechnol. 43, 244-246
(1999):
cow.
J. Dairy Sci. 82, 333-342
J. Anim. Sci. 73, 2820-2833
HERR, M., H. BOSTEDT, and K. FAILING (2011):
IgG and IgM levels in dairy cows during the periparturient period.
Theriogenology 75, 377-385
81
PAPER II
HOEBEN, D., R. HEYNEMAN, and C. BURVENICH (1997):
Elevated levels of β-hydroxybutyric acid in periparturient cows and in vitro effect on respiratory
burst activity of bovine neutrophils.
INGVARTSEN, K. L., R. J. DEWHURST, and N. C. FRIGGENS (2003):
On the relationship between lactational performance and health: Is it yield or metabolic
imbalance that cause production diseases in dairy cattle? A position paper.
Livest. Sci. 83, 277-308
ISHIKAWA, H. (1987):
Observation of lymphocyte function in perinatal cows and neonatal calves.
Jpn. J. Vet. Sci. 49, 469-475
KANEENE, J. B., R. MILLER, T. H. HERDT, and J. C. GARDINER (1997):
The association of serum nonesterified fatty acids and cholesterol, management and feeding
practices with peripartum disease in dairy cows
Prev. Vet. Med. 31, 59-72
KIMURA, K., J. GOFF, M. KEHRLI, and J. HARP (1999):
Phenotype analysis of peripheral blood mononuclear cells in periparturient dairy cows.
J. Dairy Sci. 82, 315-319
KRAFT, W. (2005):
Klinische Labordiagnostik in der Tiermedizin.
6. Aufl. Schattauer Verlag, Stuttgart
LACETERA, N., D. SCALIA, O. FRANCI, U. BERNABUCCI, B. RONCHI, and A.
NARDONE (2004):
Short Communication: Effects of nonesterified fatty acids on lymphocyte function in dairy
heifers.
J. Dairy Sci. 87, 1012-1014
LACETERA, N., D. SCALIA, U. BERNABUCCI, B. RONCHI, D. PIRAZZI, and A.
NARDONE (2005):
Lymphocyte functions in overconditioned cows around parturition.
J. Dairy Sci. 88, 2010-2016
82
PAPER II
LESSARD, M., N. GAGNON, D. L. GODSON, and H. V. PETIT (2004):
Influence of parturition and diets enriched in n-3 or n-6 polyunsaturated fatty acids on immune
response of dairy cows during the transition period.
J. Dairy Sci. 87, 2197-2210
LITTELL, R., P. HENRY, and C. AMMERMAN (1998):
Statistical analysis of repeated measures data using SAS procedures.
J. Anim. Sci. 76, 1216-1231
MALLARD, B. A., J. C. DEKKERS, M. J. IRELAND, K. E. LESLIE, S. SHARIF, C. LACEY
VANKAMPEN, L. WAGTER, and B. N. WILKIE (1998):
Alteration in immune responsiveness during the peripartum period and its ramification on dairy
cow and calf health.
J. Dairy Sci. 81, 585-595
(2003):
MICHIELS, J., J. MISSOTTEN, A. VAN HOORICK, A. OVYN, D. FREMAUT, S. DE SMET,
and N. DIERICK (2010):
Effects of dose and formulation of carvacrol and thymol on bacteria and some functional traits of
the gut in piglets after weaning.
Arch. Anim. Nutr. 64, 136-154
MIGUEL, M. G. (2010):
Antioxidant and anti-inflammatory activities of essential oils: A short review.
Molecules 15, 9252-9287
MILLER, J. K., E. BRZEZINSKA-SLEBODZINSKA, and F. C. MADSEN (1993):
Oxidative Stress, antioxidants, and animal function.
J. Dairy Sci. 76, 2812-2823
MOREIRA DA SILVA, F., C. BURVENICH, A. MASSART LEEN, and L. BROSSÉ (1998):
Assessment of blood neutrophil oxidative burst activity in dairy cows during the period of
parturition.
Anim. Sci. 67, 421-426
83
PAPER II
dairy cows.
J. Dairy Sci. 95, 1323-1336
QUIROZ-ROCHA, G. F., S. J. LEBLANC, T. F. DUFFIELD, D. WOOD, K. E. LESLIE, and R.
M. JACOBS (2009):
Reference limits for biochemical and hematological analytes of dairy cows one week before and
one week after parturition.
Can. Vet. J. 50, 383
REID, I., G. ROWLANDS, A. DEW, R. COLLINS, C. ROBERTS, and R. MANSTON (1983):
The relationship between post-parturient fatty liver and blood composition in dairy cows.
J. Agric. Sci. 101, 473-480
RENNER, L., A. SCHWABE, S. DÖLL, M. HÖLTERSHINKEN, and S. DÄNICKE (2011):
Effect of rare earth elements on beef cattle growth performance, blood clinical chemical
parameters and mitogen stimulated proliferation of bovine peripheral blood mononuclear cells in
vitro and ex vivo.
Toxicol. Lett. 201, 277-284
RENNER, L., D. VON SOOSTEN, A. SIPKA, S. DÖLL, A. BEINEKE, H.-J. SCHUBERTH,
and S. DÄNICKE (2012):
Effect of conjugated linoleic acid on proliferation and cytokine expression of bovine peripheral
blood mononuclear cells and splenocytes ex vivo.
RINALDI, M., P. MORONI, M. J. PAAPE, and D. D. BANNERMAN (2008):
Differential alterations in the ability of bovine neutrophils to generate extracellular and
intracellular reactive oxygen species during the periparturient period.
Vet. J. 178, 208-213
BERRY (2009):
welfare.
J. Dairy Sci. 92, 5769-5801
84
PAPER II
SCALIA, D., N. LACETERA, U. BERNABUCCI, K. DEMEYERE, L. DUCHATEAU, and C.
BURVENICH (2006):
In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.
J. Dairy Sci. 89, 147-154
DÄNICKE (2014):
J. Dairy Res. 81, 257–266
Nutr. Res. Rev. 6, 185-208
SORDILLO, L. M., and S. L. AITKEN (2009):
Impact of oxidative stress on the health and immune function of dairy cattle.
STEPHENSON, K. A., I. J. LEAN, and T. J. O'MEARA (1996):
The effect of monensin on the chemotactic function of bovine neutrophils.
Aust. Vet. J. 74, 315-317
STEPHENSON, K. A., I. J. LEAN, M. L. HYDE, M. A. CURTIS, J. K. GARVIN, and L. B.
LOWE (1997):
Effects of monensin on the metabolism of periparturient dairy cows.
J. Dairy Sci. 80, 830-837
SURIYASATHAPORN, W., A. DAEMEN, E. NOORDHUIZEN-STASSEN, S. DIELEMAN,
M. NIELEN, and Y. SCHUKKEN (1999):
β-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media
affect the in vitro chemotaxis of bovine leukocytes.
TIIHONEN, K., H. KETTUNEN, M. H. L. BENTO, M. SAARINEN, S. LAHTINEN, A.
OUWEHAND, H. SCHULZE, and N. RAUTONEN (2010):
The effect of feeding essential oils on broiler performance and gut microbiota.
Br. Poult. Sci. 51, 381-392
85
PAPER II
TREVISI, E., G. BERTONI, I. ARCHETTI, M. AMADORI, and N. LACETERA (2011):
Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases.
InTech, Rijeka
TREVISI, E., M. AMADORI, S. COGROSSI, E. RAZZUOLI, and G. BERTONI (2012):
Metabolic stress and inflammatory response in high-yielding, periparturient dairy cows.
Res. Vet. Sci. 93, 695-704
VAN KAMPEN, C., and B. A. MALLARD (1997):
Effects of peripartum stress and health on circulating bovine lymphocyte subsets.
WEST, H. (1990):
Effect on liver function of acetonaemia and the fat cow syndrome in cattle.
Res. Vet. Sci. 48, 221-227
ZERBE, H., N. SCHNEIDER, W. LEIBOLD, T. WENSING, T. A. M. KRUIP, and H. J.
SCHUBERTH (2000):
Altered functional and immunophenotypical properties of neutrophilic granulocytes in
postpartum cows associated with fatty liver.
86
GENERAL DISCUSSION
GENERAL DISCUSSION
The present study was based on an animal model to investigate subclinical ketosis (SCHULZ et
al. 2014). The combination of the factors high BCS AP, overfeeding in the dry period and an
accelerated energy supply PP in the high body condition groups (HC, HC/MO, HC/EO) were
supposed to evoke a negative EB PP with subsequent body fat mobilization and ketogenesis. In
contrast to that, the LC group represents a positive control group with moderate body condition
AP, an adequate energy supply in dry period and early lactation and therefore less body fat
mobilization PP.
Proportion of concentrate on dry
matter basis, %
LC
70
HC
BCS = 3.95 ± 0.08
60
50
40
30
20
10
BCS = 2.77 ± 0.14
0
Figure 4: The experimental design of the trial consisting of different body condition scores
(BCS, LSMeans ± SEM) 6 weeks before calving and varying concentrate proportions in the total
mixed ration.
The classification of the cows according to their BCS 6 weeks AP resulted in a more than 1.0
point in scale BCS difference between the 2 groups while no cow of LC group exceeded and no
cow of high condition group came below a BCS value of 3.25 on scale. Due to the feeding
strategy AP, it was even possible to further enhance BCS in the high condition groups while it
remained on a same level in LC cows until calving (Figure 5). Energy density of the total mixed
ration (TMR) AP was 6.62 MJ and 7.46 MJ net energy for lactation (NE L)/kg dry matter (20%
87
GENERAL DISCUSSION
and 60% concentrate) for LC and high condition groups in the present trial, respectively, in
comparison to 6.78 MJ and 7.71 MJ NEL/kg dry matter in the study by SCHULZ et al. (2014).
The PP TMR adjusted for lactation requirement showed an energy density of 7.01 MJ NEL/kg dry
matter (30% concentrate) that was raised to 7.55 MJ NEL/kg dry matter (50% concentrate) within
2 weeks in LC and 3 weeks in high condition groups. The distinct loss in body condition in the
high condition cows after calving (Figure 5) indicates a massive mobilization of body reserves as
it can also be seen in increasing levels of serum NEFA concentration (Figure 1, PAPER I). The
NEFA concentration was more than 50% higher and the prevalence of either subclinical or
clinical ketosis (BHB > 1.2 mmol/L) was 36% higher in HC than in LC group (Table 4, PAPER
I).
LC
HC
HC/MO
HC/EO
Body condition score
5
4.5
4
3.5
3
2.5
2
-6
-5
-4
-3
Period 3
Period 2
Period 1
1.5
-2
-1
1
2
3
4
5
6
7
8
Experimental week relative to calving
Figure 5: Body condition scores (Means) of the experimental groups during the trial.
So taken together, the aims of the animal model were successfully achieved and provide adequate
conditions for testing the antiketogenic effect of monensin and essential oils and their effects on
cow performance and health.
The feeding-associated higher net energy intake in the high condition groups is reflected by an
EB that reached higher values in those cows before calving (Figure 6). After calving, the EB was
statistically more negative in HC/MO cows in the first 2 weeks of lactation and in HC/EO cows
88
GENERAL DISCUSSION
from 15 until 56 DIM than in LC group (Table 2, PAPER I). Overall groups did not reach a
positive level within the 8 weeks of lactation in the trial, but considering the individual cows, 8
cows of LC (n = 14, 57%), 6 cows of HC (n = 13, 45%), 3 cows of HC/MO (n = 14, 21%) and 2
cows of HC/EO group (n = 15, 13%) were able to reach a positive value in the observed lactation
period.
LC
HC
HC/MO
HC/EO
150
a
EB, MJ NEL/d
100
ab ab
b
50
0
-50
-100
Period 2
Period 1
-150
-6
-5
-4
-3
-2
-1
1
2
Period 3
3
4
5
6
7
8
Figure 6: Net energy balance (EB, LSMeans) of the experimental groups during the trial.
The main action of the 2 supplements was intended to take place in the rumen of the cows via
modulating the rumen microflora. Considering monensin, the inhibition of Gram-positive
bacteria is reported to lead to an increased propionate and decreased acetate and butyrate
production. Because of the absence of an outer membrane, they are more sensitive to the mode of
action of ionophores than Gram-negative bacteria (RUSSELL and STROBEL 1989). In fact, a
higher proportion of propionate, a lower proportion of butyrate and a diminished acetate to
propionate ratio was found after monensin supplementation in comparison to HC control group
(Table 5, PAPER I) according to results from literature (VAN MAANEN et al. 1978) (Table 5,
PAPER I). Another hint is given by a trend for an increased free LPS concentration in HC/MO
in comparison to HC cows (5,898 vs. 3,616 endotoxin units/ml, p = 0.060, data not shown) over
the whole trial. As LPS are part of the outer membrane of Gram-negative bacteria (RIETSCHEL
89
GENERAL DISCUSSION
et al. 1994) and are released at bacterial cell lysis, this might indicate an increase in these
bacterial populations.
The increase of the major glucogenic precursor propionate (SEAL and REYNOLDS 1993) is
proposed to increase hepatic glucose production. More circulating glucose acts as an important
energy source to the cow in the critical time around calving and is believed to prevent metabolic
derailment like excessive body fat mobilization, ketogenesis and fatty liver infiltration with
subsequent effects on cow performance and health.
Milk and serum urea concentrations of HC/MO group in the present trial (Table 3, PAPER I and
Table 2, PAPER II) support the theory of a protein sparing effect of monensin namely via
inhibition of HAP bacteria species in the rumen that degrade high amounts of dietary protein
(RUSSELL et al. 1988). Therefore, more protein of dietary origin can reach the small intestine
and the increased absorption of AA could also contribute to an increased hepatic glucose
production as its precursor. Besides, GREEN et al. (1999) stated that not only an increased
intestinal AA absorption but also the mobilization of muscle protein resources in transition period
contribute to a higher AA availability. The important role of AA as glucogenic substrates in early
lactation has been reported previously (OVERTON et al. 1998,
BELL et al. 2000). The
subsequent deamination before gluconeogenesis could therefore contribute to the increased
concentrations of serum and milk urea (DUFFIELD et al. 1998 b). Anyway, the ammonia
concentration in rumen fluid was not different between groups in the present trial (Table 5,
PAPER I). Additionally, there was no difference in glucose concentration detected (Table 4 and
Figure 1, PAPER I). Previous literature reported only a moderate increase in glucose
concentration by 3% in a meta-analyses with monensin (DUFFIELD et al. 2008 a) and not all
individual studies could detect this change (SAUER et al. 1998, MULLINS et al. 2012). A
glucose kinetic assay was conducted by ARIELI et al. (2001) after supplementation of 300 mg of
monensin/d and they found an unaffected blood glucose concentration but an increase in glucose
distribution space and pool size by monensin that implies that conclusions about total glucose
pool of the cows can not only be drawn from its blood concentration. Besides, glucose is a wellregulated parameter and its determination is highly dependent on cow-individual parameters,
sampling and analyze procedures.
90
GENERAL DISCUSSION
Considering essential oils, a shift of the ruminal fermentation pattern towards an increased
propionate production could not be verified by our results but the butyrate proportion was found
to be lower (Table 5, PAPER I). Although serum urea was not different, milk urea tended to be
higher in HC/EO than in HC cows (Table 3, PAPER I) and might indicate a similar effect
against HAP species analogous to monensin. This effect was also shown in in vitro investigations
although essential oils were found to be less effective than monensin (MCINTOSH et al. 2003).
The fact that a change of ruminal fermentation pattern is highly dependent on the component
and/or dose of essential oils fed (CALSAMIGLIA et al. 2007, PATRA 2011) might give a
reason for the lack of influence on ruminal propionate production and therefore presumably for
the glucose supply.
The supplements were not able to reduce mobilization of body reserves as seen in BCS loss and
increase of NEFA concentration in all high condition groups PP (Figure 1 and 5, PAPER I). But
monensin and essential oils altered BHB concentration and prevalence of ketosis (Table 4 and
Figure 1, PAPER 1). While the prevalence of subclinical (BHB > 1.2 mmol/L) and clinical
ketosis (BHB > 2.5 mmol/L) was 57% and 7% in LC and 54% and 46% in HC group, it
amounted to 50% and 7% in HC/MO and 33% and 53% in HC/EO (PAPER I).
The increased ketone body formation in HC/EO group continued from the first peak at 7 DIM
nearly until the end of the trial. The first peak is therefore rather a consequence of the induced
body fat mobilization while the second peak at 42 DIM could be linked to a secondary phase of
energy deficiency, e.g. due to health problems.
In the present trial, cows were classified as healthy or diseased according to the following
criteria: Cows with the veterinary diagnosis retained placenta were classified as diseased for the
first 2 weeks of lactation, with the veterinary diagnosis metritis were classified as diseased for the
following 14 days and cows with the veterinary diagnosis mastitis or a somatic cell count
>300.000 cells/ml were classified as diseased until the end of therapy.
Regarding the PP phase of the trial, the prevalence of retained placenta, metritis and mastitis
amounted to 43%, 36% and 64% in LC, 23%, 46% and 54% in HC, 36%, 50% and 14% in
HC/MO and 27%, 27% and 89% in HC/EO group. However, DMI was not noticeably altered in
91
GENERAL DISCUSSION
HC/EO cows. Besides a hepatic production, the increased BHB concentration could have also
been the consequence of a diminished peripheral utilization. As ruminal butyrate production was
decreased, an enhanced conversion of butyrate to BHB in the rumen epithelium is rather unlikely.
The phenomena of a reduced ketogenesis after monensin supplementation is long-established and
verified by meta-analyses (SAUER et al. 1989, DUFFIELD et al. 2008 a). Interestingly, an
increased NEFA concentration was not accompanied by a rise in BHB concentration in cows of
the present trial (Figure 1 and Table 4, PAPER I). Propionate enters the TCA cycle as succinylCoA and can increase both, gluconeogenesis and the oxaloacetate pool, while the latter may
redirect acetyl-CoA from other fuels away from ketogenesis (DUFFIELD et al. 1998 a, ALLEN
et al. 2009). Regarding gluconeogenesis, KARCHER et al. (2007) evaluated the mRNA
expression of PC and cytosolic PEPCK, two potential rate-limiting enzymes for hepatic
gluconeogenesis from propionate, in 34 multiparous Holstein cows after addition of 0 or 300
mg/d monensin. They stated an increased PEPCK mRNA at d -14 and +1 calving with AP
monensin feeding which is earlier than in studies without monensin supplementation reporting an
increase in PEPCK mRNA at d +28 (GREENFIELD et al. 2000, HARTWELL et al. 2001) and
might therefore be a consequence of an enhanced ruminal propionate supply by monensin (VAN
MAANEN et al. 1978, SAUER et al. 1998). Furthermore, the diminished ruminal butyrate
production as shown for monensin in the present study can also contribute to lower
concentrations of ketones in serum because of the reduced availability of butyrate for the
conversion to BHB by the rumen epithelium. An increased glucose concentration leads to the
release of insulin and can therefore limit lipid mobilization (DUFFIELD et al. 1998 a). However,
the lack of effect on NEFA concentration in the present trial indicates that the effect of monensin
takes place in NEFA metabolism, especially ketogenesis. Of course it is also possible that this
effect on ketogenesis might underlie a mechanism independent of or besides an increased ruminal
propionate supply and gluconeogenesis.
While milk yield was not different between the groups, DMI was higher in HC/MO than in LC
group in the dry period but no effects of treatment were visible after calving (Table 2, Paper I).
As DMI decreases by 30% in dairy cows in the weeks before calving (HAYIRLI et al. 2002), a
92
GENERAL DISCUSSION
high and more rapidly increasing DMI after calving is a central approach to improve EB PP, to
provide sufficient available energy and nutrients for the onset of lactation and to ensure health in
this critical period (INGVARTSEN and ANDERSEN 2000). In fact, in the present trial a higher
DMI was positively correlated with a higher milk yield (r = 0.62, p < 0.001). Figure 7 shows a
similar course of DMI in all high condition groups, namely values above LC group AP that are
likely related to the experimental design and in contrast to that, values below LC group PP.
LC DMI
LC ECM
30
HC DMI
HC ECM
HC/MO DMI
HC/MO ECM
HC/EO DMI
HC/EO ECM
DMI, kg/d
20
15
10
5
Period 1
0
-6
-5
-4
-3
Period 2
-2
-1
1
Period 3
2
3
4
5
6
7
Energy-corrected milk, kg/d
25
50
45
40
35
30
25
20
15
10
5
0
8
Figure 7: Dry matter intake (DMI, Means) and energy-corrected milk (ECM, Means) of the
experimental groups during the trial. Both parameters were used to form a feed efficiency
parameter (ECM/DMI).
Although negative EB and body tissue mobilization to some degree are physiological processes
in early lactation (MCART et al. 2013), the observed excessive body fat mobilization in high
condition cows (Fig 1, PAPER I) under present conditions influenced performance parameters of
the cows. The main impacts affect milk fat content and the milk fat to protein ratio (Table 3,
PAPER I).
93
GENERAL DISCUSSION
10
y =y 3.99
– 0.0238
= 3.9975
- 0.0238*x;* x;
p < 0.001;
r² =r20.42
r = -0.6497;
p = 0.0000;
= 0.4221
y = 1.41 – 0.0065 * x;
3.03
y = 1.4161 - 0.0065*x;
2
p < 0.001;
r² = r0.38
r = -0.6190;
p = 0.0000;
= 0.3832
fat:protein ratio
6
4
Fat : protein ratio
Milk fat content, %
2.53
8
2
2
2.0
1.52
1.01
0.51
0
-160 -140 -120 -100 -80
-60
-40
-20
0
20
00
-160 -140 -120 -100 -80
40
-60
-40
-20
0
20
40
Figure 8: Correlation of net energy balance with milk fat content and the fat to protein ratio.
Statistica Software (Version 12).
Sources for milk fat synthesis in the mammary gland include NEFA and also BHB and liver
proteins. Though, the increased availability of the metabolites during negative EB for synthesis is
reflected in a negative correlation of EB with milk fat content and the fat to protein ratio (Figure
8). The higher milk fat content and fat to protein ratio in HC/EO than in LC group over the
lactation period (Table 3, PAPER I) is probably a consequence of the extensive lipolysis in this
group and may be due to the experimental design (PAPER I).
A similar trend for a higher milk fat to protein ratio was also detected for HC/MO cows (Table 3,
PAPER I). That is in contrast to previous literature reporting a decreased milk fat content with
monensin (DUFFIELD et al. 2008 b) that was suggested to be linked to the shifted ruminal
propionate to acetate ratio (VAN DER WERF et al. 1998).
Figure 7 shows a great variation in energy-corrected milk (ECM) yield at the onset of lactation
that is due to a high variability in yield of milk components at that time. Relative to the other
groups, HC/MO cows had the lowest DMI and the highest ECM yield in the first 2 weeks of
lactation that is reflected in a statistically higher feed efficiency than in LC and HC cows (Table
3, PAPER I). Similar results have been reported before, namely a depressed feed intake without
a loss in milk yield due to monensin (SAUER et al. 1989, DUFFIELD et al. 2008 b). A long-
94
GENERAL DISCUSSION
established argumentation is the increased efficiency to convert food energy after monensin
supplementation, namely via a decreased acetate to propionate ratio and via inhibiting the
production of methane and hydrogen in rumen after modulating the fermentation pattern
(RICHARDSON et al. 1976). On the other hand, the hepatic oxidation theory of ALLEN et al.
(2009) proposed that propionate as one of the oxidative fuels for the liver is a regulator of feed
intake in ruminants. So an increased hepatic provision of propionate after monensin
supplementation, as reported for the present trial (Table 5, PAPER I), might be hypophagic and
so unfortunately further decrease DMI in times of negative EB PP. However, no statistical
differences for DMI after calving with monensin were found in the present trial.
The increased availability of propionate for gluconeogenesis and the fact that the amount of
available glucose and its mammary product lactose is linked to the quantity of milk produced
(MEPHAM 1993) may lead to the assumption of an increased milk yield in HC/MO cows. Milk
yield and lactose yield were not statistically different between groups (Table 3, PAPER I), but
they were positively correlated with each other (r = 0.16, p = 0.002).
Taken together, one of the main findings of the present trial is the fact that HC/MO cows
mobilized fat reserves similar to the high condition counterparts HC and HC/EO, but did not
reach a similar ketogenic metabolic status. After the release from fat depots, NEFA can be used
as an energy source of milk fat synthesis in mammary gland, re-esterified to triglycerides or
partially oxidized to ketone bodies in the liver or completely oxidized by skeletal muscle or liver
as an energy source (DRACKLEY 1999, ROCHE et al. 2009). We could not detect a massive
liver lipid accumulation (Figure 1 and Table 4, PAPER I) or use for milk fat synthesis in
HC/MO group (Table 3, PAPER I) what leads to the assumption of a more favorable complete
oxidation of NEFA. MULLINS et al. (2012) proposed a theory of the mode of action of
monensin that is independent of an improvement of energy status but that is related to the
abundance of CPT-1a in liver which controls the entry of NEFA into the mitochondria for betaoxidation to carbon dioxide or ketone bodies. Monensin increased the CPT-1A mRNA
abundance in liver slices of cows fed 400 mg monensin/d as top-dress, which corresponded to a
lower rate of liver TAG accumulation from d -7 to +7 in the mentioned trial (MULLINS et al.
95
GENERAL DISCUSSION
2012). This implies a limited hepatic fat storage in times of great NEFA flow to the liver, but also
less accumulation of fat metabolites that may impair liver metabolism and a decreased
peroxisomal NEFA oxidation. Although oxidation in the peroxisomes is attributed a role as an
overflow pathway in times of increased flow of fatty acids to the liver (GRUM et al. 1994), it is
accompanied by the production of the radical hydrogen peroxide (DRACKLEY 1999) and may
lead to oxidative challenges within the tissue.
So one possibility is that monensin might have attenuated inflammatory condition in the liver
which is known to be involved in periparturient performance and health disorders in dairy cows
(SORDILLO et al. 2009). Regarding parameters of liver integrity and function, serum bilirubin
was similarly increased in all HC groups (Table 2, PAPER II) in comparison to LC group,
whereby it is negatively correlated with EB (r = -0.54, p < 0.001) and positively correlated with
liver lipid content (r = 0.34, p < 0.001) and NEFA and BHB concentrations (r = 0.58 and 0.34,
respectively, p < 0.001). So the increase of bilirubin seems to have been a consequence of the
negative energy status of the cows and may be explained by a decreased bile flow during fatty
infiltration. Similarly, serum activity of γ-glutamyltransferase (GGT), another indicator of
cholestasis reacting more slowly, was found to be higher in HC/EO group in the last period of the
trial (Table 2, PAPER II). Serum activities of aspartate-aminotransferase (AST) and glutamatedehydrogenase (GLDH) indicative for loss of liver integrity were increased in HC and especially
HC/EO group while they were on a similar level in LC and HC/MO group. Also these 3
parameters are negatively correlated with EB and positively correlated with liver lipid content,
NEFA and BHB (all p < 0.05).
To further investigate possible associations between inflammatory conditions in the liver, blood
metabolites and cow performance in the present trial, a liver functionality index (LFI) introduced
by BERTONI et al. (2006) was applied. It is based on serum levels of albumin, cholesterol and
bilirubin standardized according to average values observed in healthy cows (Figure 9) in view of
the fact that the stimulation of acute phase response in liver by proinflammatory cytokines
induces the synthesis of positive acute phase proteins like haptoglobin and ceruloplasmin while
synthesis of negative acute phase proteins like albumin are reduced (FLECK 1989, BERTONI et
96
GENERAL DISCUSSION
al. 2008). The 6 cows of the present trial with highest (HLFI, ranging from +3.7 to +5.3) and
lowest LFI values (LLFI, ranging from -2.6 to -5.7) were selected, representing about 20% of the
present dairy herd similar to sample size in a study by TREVISI et al. (2012).
The HLFI group consists of 3 LC and 3 HC/EO cows while LLFI group is made up of 4 cows of
HC, 1 cow of HC/MO and 1 cow of HC/EO group. Therefore, the HLFI group is characterized by
a more distinct increase of albumin and cholesterol synthesis and a decreased rise of bilirubin
concentration in the first month of lactation giving evidence for a better liver function and a
reduced inflammatory response (TREVISI et al. 2012).
The BCS of HLFI cows in the dry period was 3.3 ± 0.2 and it was significantly lower than BCS
of LLFI cows with a BCS of 4.0 ± 0.2 (LSmean ± SEM). Interestingly, present investigation
(MIXED procedure, Software SAS, Version 9.2) reveals a 7.7 kg/d higher DMI (p < 0.001) as
well as a 10.7 kg/d higher milk yield (p = 0.005) in cows of HLFI in comparison to LLFI group
in the lactation period which matches to results reported by TREVISI et al. (2012) and BERTONI
and TREVISI (2013). Furthermore, the results confirm a higher NEFA (p = 0.016), BHB (p =
0.084) and haptoglobin concentration (p = 0.006) in LLFI group (TREVISI et al. 2012,
BERTONI and TREVISI 2013) which is one of the most sensitive indicator for an acute phase
response in cattle (ECKERSALL and CONNER 1988).
Although inflammatory phenomena are experienced by essentially all cows after calving
(BRADFORD et al. 2015), it is strengthened by, part and cause of clinical and subclinical health
problems (BERTONI and TREVISI 2013). Considering health status of the 12 cows according to
the previously described criteria, 4 cows of HLFI did not show any clinical problems while 2 had
mastitis. In LLFI 1 cow had no clinical problems while 2 suffered from multisystemic diseases
(retained placenta, metritis, mastitis) and 2 cow showed either metritis or mastitis.
There were no distinct differences in investigated immunological parameters between LLFI and
HLFI group concerning lymphocyte proliferation and CD4+/CD8+ phenotyping but the stimulated
R123+ population was lower in cows with a high LFI than in cows with a low LFI (p = 0.013). A
reduced responsiveness to stimuli may imply an impaired host defense mechanism in these cows
as an inadequate ROS-production may restrict elimination of pathogens and the function as
messengers for modulation of and recovery from inflammation may diminish. As expected,
97
GENERAL DISCUSSION
differences between groups were also visible in serum activity of AST and GGT which were
higher in cows with a larger inflammatory response associated to impairment of hepatocyte
integrity.
Overall, results from assessment of LFI imply that it is a useful parameter to evaluate the
manifestation of an inflammatory state in transition cows. It gives evidence that there is an
obvious connection between inflammation and metabolism, performance and health while it
should generally considered that there is certainly an interrelationship between these areas and
the origin is difficult to determine.
98
GENERAL DISCUSSION
Liver Functionality Index (LFI)
(𝐴𝑙𝑏𝐼 − 17.71) (𝐶ℎ𝑜𝑙𝐼 − 2.57) (𝐵𝑖𝑙𝐼 − 6.08)
+
−
1.08
0.43
2.17
Albumin (AlbI) 𝑜𝑟 𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 𝑠𝑢𝑏𝑖𝑛𝑑𝑒𝑥 (𝐶ℎ𝑜𝑙𝐼 ) = 0.5 ∗ 𝑑1 + 0.5 ∗ (𝑑28 − 𝑑1)
● High-LFI
∆ Low-LFI
𝐿𝐹𝐼 =
B𝑖𝑙𝑖𝑟𝑢𝑏𝑖𝑛 𝑠𝑢𝑏𝑖𝑛𝑑𝑒𝑥 (𝐵𝑖𝑙𝐼) = 0.67 ∗ 𝑑1 + 0.33 ∗ (𝑑28 − 𝑑1)
300
60
2.5
250
50
200
40
150
30
100
20
50
10
0
0
2
1.5
1
0.5
─ AST, U/L
3
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
0
b)
25
─ DMI, kg/d
15
10
5
LLFI
HLFI
5
4
4
4
3
3
3
2
2
1
1
0
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
0
c)
--- Milk yield, kg/d
40
35
30
25
20
15
10
5
0
20
--- GGT, U/L
1.4
1.2
1
0.8
0.6
0.4
0.2
0
--- BHB, mmol/L
─ NEFA, mmol/L
a)
0
0
Healthy
Mastitis
Metritis Retained
placenta
e)
100
Haptoglobin, mg/ml
6
95
90
85
5
4
3
2
1
0
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
80
0
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
Stimulated R123+
population, %
d)
Figure 9: Liver functionality index (LFI, Means) according to BERTONI and TREVISI (2013). The 6 cows with
highest and lowest LFI were selected for further analysis of metabolism (a), performance (b), health status (c), ROS
production in polymorphonuclear leukocytes (d) and acute phase reponse (e) representing about 20% of the present
dairy herd. It should be noted that in the present case serum concentrations of the three parameters from sampling
day +1 were used instead of sampling day +3 as proposed by the authors. NEFA = non-esterified fatty acids, BHB =
beta-hydroxybutyrate, AST = aspartateaminotransferase, GGT = gamma-glutamyltransferase, DMI = dry matter
intake.
99
GENERAL DISCUSSION
The mean LFI within the experimental groups amounted up to 1.17 ± 2.04 (Standard Deviation)
in LC, -0.88 ± 2.38 in HC, -0.26 ± 1.45 in HC/MO and 1.23 ± 2.45 in HC/EO cows with values
above zero to be considered favorable (BERTONI and TREVISI 2013). This does commonly not
match the expectations given by results from PAPER I and II as one would rather classify HC/EO
in a more negative range. But it is in accordance to analyzed serum haptoglobin concentrations
(Figure 10a) that were significantly increased in LC and HC/MO group (p < 0.001) and tended to
be higher in HC/MO than in HC/EO cows in the second period (p = 0.088).
Regarding our classification criteria, haptoglobin concentrations were not generally higher in
diseased animals suffering at least from one of the illnesses retained placenta, metritis and
mastitis (n = 45) than in healthy cows (n = 11) in the observed first 8 weeks of lactation (p =
0.195). If we consider each disease separately, namely if a cow suffered either from retained
placenta, metritis, mastitis or not (irrespective of presence of other diseases), significant
differences are visible in the case of retained placenta (p < 0.001) and metritis (p = 0.041) but not
in the case of mastitis (p = 0.466). That is not in accordance to results by SKINNER et al. (1991)
who reported an association of retained placenta, metritis and mastitis with increased haptoglobin
levels.
However, number of cows is low in the present study and the classification as healthy or diseased
does not distinguish between acute and chronical conditions as well as utilize clinical definitions
from literature as they were for example reported for clinical metritis (SHELDON et al. 2006).
Nevertheless, our classification criteria in the present trial suggest that uterine diseases seemed to
induce a more distinct systemic inflammatory reaction with induction of acute phase response
than mastitis. So taking into consideration only cows with the same health condition next to peak
haptoglobin concentration on d +7, namely uterine diseases in the first 2 weeks of lactation (n = 6
in each group), results show that haptoglobin concentrations were almost on the same level
(Figure 10c) in all cows without uterine diseases (p = 0.799) while they significantly increased in
LC (p = 0.008) and HC/MO group (p < 0.001) in uterus-diseased animals in period 2. Thereby,
higher concentrations could be found in HC/MO than in HC/EO cows (p = 0.001, Figure 10c).
100
GENERAL DISCUSSION
8
7
6
5
4
3
2
1
0
HC
Period 1
HC/MO
HC/EO
Period 3
Period 2
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
Haptoglobin, mg/ml
LC
Period 2
Period 3
Haptoglobin, mg/ml
Period 1
Period 2
Period 3
8
7
6
5
4
3
2
1
0
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
-42
-35
-28
-21
-14
-7
0
7
14
21
28
35
42
49
56
Haptoglobin, mg/ml
Period 1
8
7
6
5
4
3
2
1
0
Figure 10: Haptoglobin concentrations (Means) of a) all cows b) cows with uterus disease (n = 6
in each group) c) cows without uterus disease.
There were certainly other diseases that we did not record like lameness and other sources
causing inflammation but these results imply that the lack of response in haptoglobin level in
uterus-diseased animals in HC/EO group may be a main contributing factor to the overall
differences in haptoglobin concentration between monensin and essential oils supplemented
cows. On the one hand, there is evidence that increased haptoglobin levels could cause
immunosuppression as shown for an impaired lymphocyte blastogenesis in calves (MURATA
and MIYAMOTO 1993). On the other hand, the biological functions of haptoglobin include
antioxidative, anti-inflammatory and immunomodulatory effects (CECILIANI et al. 2012) and so
a lower haptoglobin response to an inflammatory stimuli after calving might indicate a
101
GENERAL DISCUSSION
suboptimal response and hence a reduced immune function as suggested by CRAWFORD et al.
(2005). They examined the effects of a monensin CRC administered 3 weeks AP in 1010 dairy
cows and heifers and also reported a higher haptoglobin response in clinically unhealthy heifers
in the first week of lactation after monensin supplementation. Furthermore, monensin increased
postpartal concentrations of plasma ceruloplasmin, an acute phase protein that is important in
controlling oxidative challenge from free radicals (STEPHENSON et al. 1997). In the present
trial, there is no correlation between haptoglobin and serum glucose or liver fat content, but it is
negatively correlated with milk yield and DMI and positively correlated with serum AST,
bilirubin, NEFA and BHB (p < 0.001) in accordance to considerations from LFI. Furthermore, it
is negatively correlated with unstimulated R123+ population and the stimulation index of PBMC
(p < 0.05) underlining the thesis of an immunosuppressive situation during high haptoglobin
concentrations.
To pick up the mentioned idea of an anti-inflammatory effect of monensin again, monensin
supplementation led to a modification of haptoglobin response but it is difficult to assess if the
increased concentration is rather beneficial or signifies an overreaction.
On the contrary, there is the lack of haptoglobin response to uterine diseases in HC/EO cows.
Although essential oils are attributed anti-inflammatory (FACHINI-QUEIROZ et al. 2012) and
antioxidative properties (MIGUEL 2010), these results might indicate an insufficient immune
response and so might have been rather unfavorable for the cows’ health. This idea would match
with other results indicating some immunological imbalances, namely a drop in stimulated R123 +
population 4 to 5 weeks after calving, the mentioned impairment of liver integrity parameters
(GGT, GLDH), changes in lymphocyte subsets, a diminished response to BVDV vaccination (all
PAPER II), a highly ketotic metabolic status (Peak at d +35, BHB = 2.25 mmol/L, PAPER I)
and great liver fat accumulation (Peak at 21 DIM, liver fat content = 215 mg/g fresh liver weight,
PAPER I).
The idea of an involvement of blood NEFA and BHB in immunosuppression next to calving has
been described previously (HOEBEN et al. 1997, SURIYASATHAPORN et al. 1999, ZERBE
et al. 2000, LACETERA et al. 2002, LACETERA et al. 2004, LACETERA et al. 2005,
102
GENERAL DISCUSSION
SCALIA et al. 2006, LACETERA et al. 2010, CONTRERAS and SORDILLO 2011) and an
association of these metabolites with evaluated immune parameters were visible in the present
study, too. Concentration of NEFA during boost BVDV vaccination negatively affected
measured antibody titer 7 days later (r = -0.33, p < 0.031) and besides, it was negatively
correlated with basal (r = -0.31, p < 0.001) and stimulated R123+ population of PMN (r = -0.15, p
< 0.001) and stimulation index of PBMC (r = -0.10, p = 0.041). Similarly, BHB concentration
was negatively correlated with basal R123+ population of PMN (r = -0.09, p = 0.021) and
positively correlated with CD4+ to CD8+ ratio (r = 0.14, p = 0.001, all PAPER II).
Taken together, the animal model induced a persistent ketogenic metabolic status in HC/EO cows
that might have been the underlying reason for observed impairment of liver integrity (Table 2,
PAPER II) and the low antibody response to BVDV vaccination (Figure 2, PAPER II).
Although differences were not statistically significant, data also suggest an influence on ROSproducing PMN species and lymphocyte subsets (Figure 3 and 4, PAPER II) while the effect on
serum haptoglobin level is difficult to interpret.
Cows of HC/MO group were similarly affected by fat mobilization but somehow circumvent
ketone body formation in the liver whereby the mechanism cannot further be specified by our
results. They showed an increased ruminal propionate production that might have been
accompanied by an increased gluconeogenesis whereby serum glucose was not higher in
comparison to the other experimental groups. Besides, the feed efficiency was increased in the
first 2 weeks of lactation. Blood metabolites like AST, GGT and GLDH were generally located
in a range between the LC and HC control groups and might indicate a direct or indirect effect of
monensin on the liver. The effects on evaluated immune parameters were a strong antibody
response to BVDV vaccination and a high haptoglobin production after calving while the latter is
again difficult to rate. Together with reports from literature about effects of monensin on
neutrophil chemotaxis (STEPHENSON et al. 1996), it raises the question if there are direct or
indirect effects of monensin on immune system independent of an improved energy household of
the cow that might be related to NEFA metabolism or inflammatory conditions in the liver.
103
SUMMARY
SUMMARY
Caroline Drong (2016)
Effects of monensin and essential oils on ruminal fermentation, performance, energy
metabolism and immune parameters of dairy cows during the transition period
Genetic and management progress in modern dairy farming allow continuing increasing milk
performance of herds whereas metabolism of individual cows often lags behind. The decreasing
feed intake at calving together with growth of the fetus and the initiated lactation leads to a
negative energy balance of the cow. It is not surprising that the highest incidence of production
diseases can be found in early lactation. Therefore, ruminant nutrition has concentrated for a long
time on measures to increase and improve feed intake, energy status, metabolism and health in
these critical weeks around calving.
One approach is the modulation of fermentation processes in the rumen to increase efficiency to
convert feed energy. The ionophore antimicrobial drug monensin was successfully applied for
this intention as it leads to a shift of the ruminal microflora towards Gram-negative bacteria
increasing propionate and decreasing methane production. Propionate is the main precursor of
hepatic gluconeogenesis and may therefore improve glucose availability at calving and so
antagonize the postpartal energy deficit. After the ban on antibiotics as feed additives in the
European Union, the research in natural alternatives to ionophores was greatly enhanced while
essential oils caught great interest. These secondary metabolites obtained from plant material also
modulate the rumen microflora although changes in short-chain fatty acid profile are not
consistent but dependent on used essential oils component and dose. Recently, monensin was
relaunched in the European Union as a ruminal controlled-release capsule (CRC) indicated for
high condition dairy cows administered just before calving. However, in times of public attention
for possible antibiotic residues in milk and meat and the development of bacterial antibiotic
resistances, the quest for alternatives to monensin still proceeds.
The blood metabolites non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) that
are high during negative energy balance were attributed an etiological involvement in an
impaired immune cell function at calving. This raised the question if monensin and essential oils
104
SUMMARY
are able to influence immune function, either indirectly via an improved energy status or even
directly, and so to contribute to an improved health status in early lactation.
Therefore, a study was conducted with 60 multiparous German Holstein cows (parity 2.3 ± 1.4
(SD)) based on an animal model to investigate subclinical ketosis. They were allocated 6 weeks
before calving according to their body condition score (BCS) to high and low condition group
(LC, n = 14) while an overfeeding in the dry period and an decelerated energy supple in the high
condition group in early lactation was intended to provoke a negative energy balance with
subsequent body fat mobilization and ketogenesis. High condition cows formed a control group
(HC, n = 13), received a monensin CRC 3 weeks before calving (HC/MO, n = 14) or a blend of
essential oils from day -21 until day +56 relative to calving (HC/EO, n = 15).
Feedstuff samples and performance parameters together with samples of blood, rumen fluid and
milk as well as liver biopsies were taken over the trial to assess performance, energy status,
ruminal fermentation pattern, biochemical and hematological parameters, Bovine Viral Diarrhea
virus (BVDV) antibody titer, leukocyte subsets and function.
Results imply that the purposes of the animal model were successfully achieved as all high
condition cows further increased their body condition until calving and showed a massive loss of
condition in early lactation as seen in BCS and also serum NEFA concentration. The prevalence
of either subclinical or clinical ketosis (BHB > 1.2 mmol/L) was 36% higher in HC than in LC
group. Ruminal fermentation pattern showed an increased propionate production in HC/MO but
not in HC/EO cows which was not accompanied by a higher serum glucose concentration in both
groups. Concerning performance, dry matter intake (DMI) and milk yield were not different after
calving whereas monensin increased feed efficiency (DMI/energy-corrected milk). Biochemical
parameters give evidence for an impaired liver function and integrity as displayed in increased
bilirubin concentrations in all high condition cows and increased levels of aspartateaminotransferase (AST), gamma-glutamyltransferase (GGT) or glutamine dehydrogenase
(GLDH) after calving in HC and HC/EO group. Monensin reduced serum BHB concentration
although NEFA mobilization was unaltered and led to a reduced concentration of blood
metabolites that may indicate in impaired hepatocyte integrity. Immune parameters were greatly
105
SUMMARY
influenced by the event of calving as seen in short-term effects on parameters like reactive
oxygen species (ROS) production in polymorphonuclear cells and the BVDV antibody titer.
From the immune parameters investigated, the BVDV antibody response was more pronounced
in HC/MO compared to HC/EO.
Dairy cows with a high condition around calving may profit from a monensin CRC administered
3 weeks before calving, as the energy status was improved likely via an increased supply of
ruminal propionate to hepatic gluconeogenesis exerting an antiketogenic effect. Furthermore, the
feed efficiency was increased in early lactation. Results suggest direct or indirect monensin
effects on the liver and the immune system while nature of these effects needs to be clarified
further. The supplemented dose of a blend of essential oils showed no effect on ruminal
propionate production, energy status, performance and health of transition dairy cows under the
current conditions whereas other solitary components and combinations or doses may achieve
better results.
106
ZUSAMMENFASSUNG
ZUSAMMENFASSUNG
Caroline Drong (2016)
Effekte von Monensin und ätherischen Ölen auf die Pansenfermentation, die Leistung, den
Energiemetabolismus und Immunparameter von Milchkühen während der Transitphase
Durch genetischen Fortschritt und eine Verbesserung des Managements werden in der modernen
Milchviehhaltung immer höhere Milchleistungen erzielt, während der Metabolismus der
einzelnen Kühe so vermehrt vor Herausforderungen gestellt wird. Die abnehmende
Futteraufnahme rund um die Abkalbung führt zusammen mit dem Bedarf des Fetus für das
Wachstum und den Bedarf der einsetzenden Laktation zu einer negativen Energiebilanz der Kuh.
Es ist nicht überraschend, dass in dieser frühen Phase der Laktation die höchste Inzidenz an
Produktionserkrankungen vorliegt. Daher konzentriert sich das Gebiet der Rinderernährung seit
langer Zeit auf die Erforschung von Maßnahmen, welche die Futteraufnahme, den Energiestatus,
den Metabolismus und die Gesundheit der Milchkuh in dieser kritischen Phase unterstützen.
Solche Eigenschaften wurden für das ionophore Antibiotikum Monensin nachgewiesen, da
gezeigt wurde, dass es die mikrobielle Pansenflora hin zu Gram-negativen Bakterien verschiebt
und so zu einer gesteigerten Propionatbildung und einer verminderten Methanbildung führt.
Propionat stellt den Hauptvorläufer der hepatischen Glukoneogenese dar und könnte daher die
Glukoseverfügbarkeit rund um die Abkalbung verbessern und so dem postpartalen
Energiemangel entgegenwirken. Nachdem
Antibiotika zur Leistungsförderung in der
Europäischen Union verboten wurden, hat sich die Suche nach natürlichen Alternativen zu
Monensin stark vermehrt und insbesondere die ätherischen Öle sind in den Fokus gerückt. Denn
für diese sekundären Pflanzenmetaboliten konnte ebenfalls eine Beeinflussung der Pansenflora
nachgewiesen werden, obwohl die Ergebnisse hinsichtlich des Fermentationsprofiles der
kurzkettigen Fettsäuren nicht immer konstant waren sondern abhängig von der verwendeten
Komponente und der Dosis. Vor Kurzem wurde Monensin als intraruminaler Bolus wieder in der
Europäischen Union für hoch konditionierte Kühe als Tierarzneimittel kurz vor der Abkalbung
zugelassen. Doch im Angesicht des öffentlichen Interesses für mögliche Rückstände von
107
ZUSAMMENFASSUNG
Antibiotika in Nahrungsmittel tierischen Ursprunges und der Entwicklung von bakteriellen
Resistenzen, besteht nach wie vor Bedarf nach natürlichen Alternativen zu Monensin.
Den Blutmetaboliten nicht-veresterte Fettsäuren (NEFA) und beta-Hydroxybutyrat (BHB),
welche besonders in Energiemangelsituation stark ansteigen, wird eine zentrale Rolle in der
verminderten Immunfunktion rund um die Abkalbung nachgesagt. Daher stellt sich ebenso die
Frage, ob Monensin und ätherische Öle auch einen Einfluss auf die Immunfunktion haben, zum
einen indirekt, über eine Verbesserung des Energiehaushaltes der Kuh, oder aber direkt und so
den Gesundheitsstatus in der frühen Laktation verbessern können.
Hierfür wurde eine Studie mit 60 multiparen Deutsch Holsteinkühen (mittlere Laktation 2,3 ± 1,4
(Standardabweichung)) durchgeführt, welche auf einem Tiermodell zur Untersuchung der
subklinischen Ketose basierte. Die Kühe wurden 6 Wochen vor der Abkalbung entsprechend
ihrer Körperkondition (BCS) in eine Gruppe geringerer (LC, n = 14) und höherer Kondition
unterteilt. Letztere wurden in der Trockensteherphase überfüttert und in der frühen Laktation
verzögert mit Energie versorgt, um eine negative Energiebilanz mit einhergehender
Körperfettmobilisierung und Ketonkörperbildung hervorzurufen. Diese hoch konditionierten
Tiere wurden entweder einer Kontrollgruppe zugeordnet (HC, n = 13) oder sie erhielten einen
Monensin-freisetzenden intraruminalen Bolus 3 Wochen vor der Abkalbung (HC/MO, n = 14)
oder ein Gemisch von ätherischen Ölen von Tag -21 bis Tag +56 bezogen auf die Abkalbung
(HC/EO, n = 15).
Es wurden Futtermittelproben genommen und Leistungsparameter dokumentiert und des
Weiteren Blut-, Pansensaft-, Milchproben und Leberbiopsien entnommen. Hierdurch wurden die
Leistung und der Energiehaushalt der Kühe erfasst und das ruminale Fermentationsprofil,
biochemische und hämatologische Variablen, Antikörper gegen das Bovine Virale Diarrhö Virus
(BVBV) und die Populationen und Funktionen der Leukozyten ermittelt.
Die Ergebnisse deuten darauf hin, dass die Ziele des Tiermodells erfolgreich erreicht wurden: Die
Kühe konnten durch die Fütterung zur Abkalbung weiter zunehmen und haben nach dieser enorm
in der Frühlaktation an Körpermaße verloren, wie man an den BCS und NEFA Werten erkennen
kann. Die Prävalenz der subklinischen und klinischen Ketose (BHB > 1.2 mmol/L) lag in der HC
108
ZUSAMMENFASSUNG
Gruppe 36% höher als in der LC Gruppe. Im ruminalen Fermentationsprofil konnte eine
gesteigerte Propionatbildung in der Monensingruppe, aber nicht in der ätherischen Öle Gruppe,
nachgewiesen werden, welche in keiner der beiden Gruppen mit einer gesteigerten
Blutglukosekonzentration einherging. Betrachtet man die Leistung der Kühe, konnten die
Futteraufnahme (DMI) in der Laktation und die Milchmenge nicht durch die Supplemente
verändert werden, aber Monensin führte zu einer gesteigerten Futtereffizienz (DMI/Energiekorrigierte Milch). Biochemische Parameter deuten darauf hin, dass in den hoch konditionierten
Tieren eine verminderte Leberfunktion und -integrität vorlag, da die Konzentration im Blut von
Bilirubin in allen hoch konditionieren Tieren und von Aspartat-Aminotransferase (AST), gammaGlutamyltransferase (GGT) und Glutamat Dehydrogenase (GLDH) in der HC und HC/EO
Gruppe erhöht waren. Monensin konnte die Konzentration von BHB im Blut senken, obwohl die
NEFA Freisetzung unverändert war. Es führte zu einer geringeren Konzentration von
Blutmetaboliten, die auf eine verminderte Hepatozytenintegrität hinweisen könnten. Die
Immunparameter zeigten eine deutliche Beeinflussung durch die Abkalbung, wie es besonders an
der Produktion von reaktiven Sauerstoffspezies (ROS) in den neutrophilen Granulozyten und den
BVDV Antikörpertitern sichtbar wurde. Die Antikörperreaktion zeigte sich in der Monensin
Gruppe besonders hoch in Vergleich zu der ätherischen Öle Gruppe.
Milchkühe, die vor der Abkalbung eine hohe Körperkondition aufweisen, scheinen von einem
Monensin-freisetzenden Bolus 3 Wochen vor der Abkalbung zu profitieren, da eine erhöhte
Bereitstellung
von
ruminalem
Propionat
für
die
hepatische
Glukoneogenese
die
Energieverfügbarkeit der Kuh zu verbessern scheint und so vermutlich einen antiketogene
Wirkung ausübt. Des Weiteren konnte so die Futtereffizienz in der Frühlaktation gesteigert
werden. Außerdem lassen die Ergebnisse darauf schließen, dass Monensin direkte oder indirekte
Effekte auf die Leber und das Immunsystem ausübt, welche noch weiterer Abklärung bedürfen.
Für die supplementierte Dosis des Gemisches von ätherischen Ölen konnte kein Einfluss auf die
ruminale Propionatbildung, den Energiehaushalt, die Leistung und die Gesundheit der Milchkühe
nachgewiesen werden. Andere Komponenten der ätherischen Öle, andere Gemische und andere
Dosen könnten möglicherweise bessere Ergebnisse erzielen.
109
REFERENCES
REFERENCES
(cited in Introduction, Background and General Discussion)
AGTARAP, A., J. W. CHAMBERLIN, M. PINKERTON, and L. K. STEINRAUF (1967):
Structure of monensic acid, a new biologically active compound.
ACS Cent Sci. 89, 5737-5739
ALLEN, M., B. BRADFORD, and M. OBA (2009):
Board-invited Review: The hepatic oxidation theory of the control of feed intake and its
application to ruminants.
J. Anim. Sci. 87, 3317-3334
ANASSORI, E., B. DALIR-NAGHADEH, R. PIRMOHAMMADI, and M. HADIAN (2015):
Changes in blood profile in sheep receiving raw garlic, garlic oil or monensin.
ARIELI, A., J.E. VALLIMONT, Y. AHARONI, and G. A. VARGA (2001):
Monensin and growth hormone effects on glucose metabolism in the prepartum cow.
J. Dairy Sci. 84, 2770-2776
BAKKALI, F., S. AVERBECK, D. AVERBECK, and M. IDAOMAR (2008):
Biological effects of essential oils – A review.
Food Chem. Toxicol. 46, 446-475
BASSOLÉ, I. H. N., and H. R. JULIANI (2012):
Essential oils in combination and their antimicrobial properties.
Molecules 17, 3989-4006
BAUMAN, D. E., and W. B. CURRIE (1980):
Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving
homeostasis and homeorhesis.
J. Dairy Sci. 63, 1514-1529
BEAUCHEMIN, K. A., and S. M. MCGINN (2006):
Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
J. Anim. Sci. 84, 1489-1496
110
REFERENCES
BELL, A. W. (1995):
Regulation of organic nutrient metabolism during transition from late pregnancy to early
lactation.
J. Anim. Sci. 73, 2804-2819
BELL, A. W., W. S. BURHANS, and T. R. OVERTON ( 2000):
Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy
cows.
Proc. Nutr. Soc. 59, 119-126
BENCHAAR, C., H. PETIT, R. BERTHIAUME, T. WHYTE, and P. CHOUINARD (2006):
Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation,
milk production, and milk composition in dairy cows.
J. Dairy Sci. 89, 4352-4364
BENCHAAR, C., H. PETIT, R. BERTHIAUME, D. OUELLET, J. CHIQUETTE, and P.
CHOUINARD (2007):
Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk
production, and milk composition in dairy cows fed alfalfa silage or corn silage.
J. Dairy Sci. 90, 886-897
BERGEN, W. G., and D. B. BATES (1984):
Ionophores: Their effect on production efficiency and mode of action.
J. Anim. Sci. 58, 1465-1483
BERGMAN, E., and R. HEITMANN (1978):
Metabolism of amino acids by the gut, liver, kidneys, and peripheral tissues
Fed. Proc. 5, 1228
BERNABUCCI, U., B. RONCHI, N. LACETERA, and A. NARDONE (2002):
Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot
Season.
J. Dairy Sci. 85, 2173-2179
BERNABUCCI, U., B. RONCHI, N. LACETERA, and A. NARDONE (2005):
Influence of body condition score on relationships between metabolic status and oxidative
Stress in periparturient dairy cows.
J. Dairy Sci. 88, 2017-2026
111
REFERENCES
BERTICS, S. J., R. R. GRUMMER, C. CADORNIGA-VALINO, and E. E. STODDARD
(1992):
Effect of prepartum dry matter intake on liver triglyceride concentration and early lactation.
J. Dairy Sci. 75, 1914-1922
BERTONI, G., and E. TREVISI (2013):
Use of the liver activity index and other metabolic variables in the assessment of metabolic health
in dairy herds.
Vet. Clin. North Am. Food Anim. Pract. 29, 413-431
BERTONI, G., E. TREVISI, A. FERRARI, and A. GUBBIOTTI (2006):
The dairy cow performances can be affected by inflammations occurring around calving.
in: 57th Annual Meeting of the European Association for Animal Production, Antalya, Turkey,
2006,
Proc. 57th Ann. Meet. EAAP, p. 325
BERTONI, G., E. TREVISI, X. HAN, and M. BIONAZ (2008):
Effects of inflammatory conditions on liver activity in puerperium period and consequences for
performance in dairy cows.
J. Dairy Sci. 91, 3300-3310
BOBE, G., J. YOUNG, and D. BEITZ (2004):
Invited Review: Pathology, etiology, prevention, and treatment of fatty liver in dairy cows.
J. Dairy Sci. 87, 3105-3124
BRADFORD, B., K. YUAN, J. FARNEY, L. MAMEDOVA, and A. CARPENTER (2015):
Invited review: Inflammation during the transition to lactation: New adventures with an old
flame.
J. Dairy Sci. 98, 6631-6650
BRINDLE, N. P., V. A. ZAMMIT, and C. I. POGSON (1985):
Regulation of carnitine palmitoyltransferase activity by malonyl-CoA in mitochondria from
sheep liver, a tissue with a low capacity for fatty acid synthesis.
Biochem. J. 232, 177-182
112
REFERENCES
BRODERSEN, B. W., and C. L. KELLING (1999):
Alteration of leukocyte populations in calves concurrently infected with bovine respiratory
syncytial virus and bovine viral diarrhea virus.
Viral Immunol. 12, 323-334
BURT, S. (2004):
Essential oils: Their antibacterial properties and potential applications in foods — a review.
BUSQUET, M., S. CALSAMIGLIA, A. FERRET, P. CARDOZO, and C. KAMEL (2005):
Effects of cinnamaldehyde and garlic oil on rumen microbial fermentation in a dual flow
continuous culture.
J. Dairy Sci. 88, 2508-2516
CADÓRNIGA-VALIÑO, C., R. R. GRUMMER, L. E. ARMENTANO, S. S. DONKIN, and S. J.
BERTICS (1997):
Effects of fatty acids and hormones on fatty acid metabolism and gluconeogenesis in bovine
hepatocytes.
J. Dairy Sci. 80, 646-656
CALSAMIGLIA, S., M. BUSQUET, P. CARDOZO, L. CASTILLEJOS, and A. FERRET
(2007):
Invited review: Essential oils as modifiers of rumen microbial fermentation.
J. Dairy Sci. 90, 2580-2595
CASTILLEJOS, L., S. CALSAMIGLIA, A. FERRET, and R. LOSA (2005):
Effects of a specific blend of essential oil compounds and the type of diet on rumen microbial
fermentation and nutrient flow from a continuous culture system.
CASTILLEJOS, L., S. CALSAMIGLIA, and A. FERRET (2006):
Effect of essential oil active compounds on rumen microbial fermentation and nutrient flow in in
vitro systems.
J. Dairy Sci. 89, 2649-2658
113
REFERENCES
CASTILLEJOS, L., S. CALSAMIGLIA, A. FERRET, and R. LOSA (2007):
Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen
fermentation.
CECILIANI, F., J. J. CERON, P. D. ECKERSALL, and H. SAUERWEIN (2012):
Acute phase proteins in ruminants.
J. Proteomics 75, 4207-4231
CHAO, S. C., D. G. YOUNG, and C. J. OBERG (2000):
Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses.
J. Essent. Oil Res. 12, 639-649
CONTRERAS, G. A., and L. M. SORDILLO (2011):
Lipid mobilization and inflammatory responses during the transition period of dairy cows.
Comp. Immunol. Microbiol. Infect. Dis. 34, 281-289
CRAWFORD, R. G., K. E. LESLIE, R. BAGG, C. P. DICK, and T. F. DUFFIELD (2005):
The impact of controlled release capsules of monensin on postcalving haptoglobin concentrations
in dairy cattle.
Can. J. Comp. Med. 69, 208
DAHLGREN, C., and A. KARLSSON (1999):
Respiratory burst in human neutrophils.
J. Immunol. Methods 232, 3-14
DANFÆR, A., V. TETENS, and N. AGERGAARD (1995):
Review and an experimental study on the physiological and quantitative aspects of
gluconeogenesis in lactating ruminants.
Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 111, 201-210
DANN, H., G. DOUGLAS, T. OVERTON, and J. DRACKLEY (2000):
Carnitine palmitoyltransferase activity in liver of periparturient dairy cows.
DEANS, S., and G. RITCHIE (1987):
Antibacterial properties of plant essential oils.
114
REFERENCES
DETILLEUX, J. C., M. E. KEHRLI JR, J. R. STABEL, A. E. FREEMAN, and D. H. KELLEY
(1995):
Study of immunological dysfunction in periparturient Holstein cattle selected for high and
average milk production.
DEVASAGAYAM, T., J. TILAK, K. BOLOOR, K. S. SANE, S. S. GHASKADBI, and R. LELE
(2004):
Free radicals and antioxidants in human health: current status and future prospects.
J. Assoc. Physicians India 52, 4
DRACKLEY, J. K. (1999):
Biology of dairy cows during the transition period: The final frontier?
J. Dairy Sci. 82, 2259-2273
DRACKLEY, J. K., T. R. OVERTON, and G. N. Douglas (2001):
Adaptations of glucose and long-chain fatty acid metabolism in liver of dairy cows during the
periparturient period.
DRACKLEY, J. K., H. M. DANN, G. N. DOUGLAS, N. A. J. GURETZKY, N. B.
LITHERLAND, J. P. UNDERWOOD, and J. J. LOOR (2005):
Physiological and pathological adaptations in dairy cows that may increase susceptibility to
periparturient diseases and disorders.
Ital. J. Anim. Sci. 4, 323–344
DICK, and R. BAGG (1998 a):
Efficiency of monensin for the prevention of subclinical ketosis in lactating dairy cows.
J. Dairy Sci. 81, 2866-2873
DICK, and R. BAGG (1998 b):
Effect of prepartum administration of monensin in a controlled-release capsule on postpartum
energy indicators in lactating dairy cows.
J. Dairy Sci. 81, 2354-2361
115
REFERENCES
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 a):
A meta-analysis of the impact of monensin in lactating dairy cattle. Part 1: Metabolic effects.
J. Dairy Sci. 91, 1334-1346
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 b):
A meta-analysis of the impact of monensin in lactating dairy cattle. Part 2: Production effects.
J. Dairy Sci. 91, 1347-1360
DUFFIELD, T., A. RABIEE, and I. LEAN (2008 c):
reproduction.
J. Dairy Sci. 91, 2328-2341
ECKERSALL, P., and J. CONNER (1988):
Bovine and canine acute phase proteins.
Vet. Res. Commun. 12, 169-178
EDRIS, A. E. (2007):
Pharmaceutical and therapeutic potentials of essential oils and their individual volatile
constituents: A review.
Phytother. Res. 21, 308-323
EVANS, J. D., and S. A. MARTIN (2000):
Effects of thymol on ruminal microorganisms.
Curr. Microbiol. 41, 336-340
FACHINI-QUEIROZ, F. C., R. KUMMER, C. F. ESTEVAO-SILVA, M. D. D. B.
CARVALHO, J. M. CUNHA, R. GRESPAN, C. A. BERSANI-AMADO, and R. K. N. CUMAN
(2012):
Effects of thymol and carvacrol, constituents of Thymus vulgaris L. essential oil, on the
inflammatory response.
Evid. Based Compl. Alt. 1, 1-10
FELDBERG, R., S. CHANG, A. KOTIK, M. NADLER, Z. NEUWIRTH, D. SUNDSTROM,
and N. THOMPSON (1988):
In vitro mechanism of inhibition of bacterial cell growth by allicin.
Antimicrob. Agents Chemother. 32, 1763-1768
116
REFERENCES
FLECK, A. (1989):
Clinical and nutritional aspects of changes in acute-phase proteins during inflammation.
Proc. Nutr. Soc. 48, 347-354
FRONK, T. J., L. H. SCHULTZ, and A. R. HARDIE (1980):
Effect of dry period overconditioning on subsequent metabolic disorders and performance of
dairy cows.
J. Dairy Sci. 63, 1080-1090
GILLUND, P., O. REKSEN, Y. T. GRÖHN, and K. KARLBERG (2001):
Body condition related to ketosis and reproductive performance in norwegian dairy cows.
J. Dairy Sci. 84, 1390-1396
GOFF, J. P., and R. L. HORST (1997):
Physiological changes at parturition and their relationship to metabolic disorders.
J. Dairy Sci. 80, 1260-1268
(1999):
cow.
J. Dairy Sci. 82, 333-342
GREENFIELD, R. B., M. J. CECAVA, and S. S. DONKIN (2000):
Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the
transition to lactation.
J. Dairy Sci. 83, 1228-1236
GRIFFIN, S. G., S. G. WYLLIE, J. L. MARKHAM, and D. N. LEACH (1999):
The role of structure and molecular properties of terpenoids in determining their antimicrobial
activity.
Flavour Fragr. J. 14, 322-332
GRÖHN, Y., J. R. THOMPSON, and M. L. BRUSS (1984):
Epidemiology and genetic basis of ketosis in Finnish Ayrshire cattle.
Prev. Vet. Med. 3, 65-77
117
REFERENCES
GRUM, D., L. HANSEN, and J. DRACKLEY (1994):
Peroxisomal β-oxidation of fatty acids in bovine and rat liver.
Comp. Biochem. Physiol., B 109, 281-292
J. Anim. Sci. 73, 2820-2833
HARP, J., M. KEHRLI, D. HURLEY, R. WILSON, and T. BOONE (1991):
Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient
period.
HARTWELL, J. R., M. J. CECAVA, and S. S. DONKIN (2001):
Rumen undegradable protein, rumen-protected choline and mRNA expression for enzymes in
gluconeogenesis and ureagenesis in periparturient dairy cows.
J. Dairy Sci. 84, 490-497
HAYIRLI, A., R. GRUMMER, E. NORDHEIM, and P. CRUMP (2002):
Animal and dietary factors affecting feed intake during the prefresh transition period in Holsteins.
J. Dairy Sci. 85, 3430-3443
HEGARDT, F. (1999):
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: A control enzyme in ketogenesis.
Biochem. J. 338, 569-582
HEITMANN, R. N., D. J. DAWES, and S. C. SENSENIG (1987):
Hepatic ketogenesis and peripheral ketone body utilization in the ruminant.
J. Nutr. 117, 1174-1180
HELANDER, I. M., H.-L. ALAKOMI, K. LATVA-KALA, T. MATTILA-SANDHOLM, I.
POL, E. J. SMID, L. G. GORRIS, and A. VON WRIGHT (1998):
Characterization of the action of selected essential oil components on Gram-negative bacteria.
J. Agric. Food Chem. 46, 3590-3595
118
REFERENCES
HOEBEN, D., R. HEYNEMAN, and C. BURVENICH (1997):
Elevated levels of β-hydroxybutyric acid in periparturient cows and in vitro effect on respiratory
burst activity of bovine neutrophils.
HOEBEN, D., C. BURVENICH, A.-M. MASSART-LEËN, M. LENJOU, G. NIJS, D. VAN
BOCKSTAELE, and J.-F. BECKERS (1999):
In vitro effect of ketone bodies, glucocorticosteroids and bovine pregnancy-associated
glycoprotein on cultures of bone marrow progenitor cells of cows and calves.
HOLTENIUS, P., and K. HOLTENIUS (1996):
New aspects of ketone bodies in energy metabolism of dairy cows: A review.
J. Vet. Med. A Physiol. Pathol. Clin. Med. 43, 579-587
HUNTINGTON, G. B. (1997):
Starch utilization by ruminants: From basics to the bunk.
J. Anim. Sci. 75, 852-867
INGVARTSEN, K. L., and J. B. ANDERSEN (2000):
Integration of metabolism and intake regulation: A review focusing on periparturient animals.
J. Dairy Sci. 83, 1573-1597
INGVARTSEN, K. L., R. J. DEWHURST, and N. C. FRIGGENS (2003):
On the relationship between lactational performance and health: Is it yield or metabolic
imbalance that cause production diseases in dairy cattle? A position paper.
Livest. Sci. 83, 277-308
INGVARTSEN, K. L. (2006):
Feeding- and management-related diseases in the transition cow: Physiological adaptations
around calving and strategies to reduce feeding-related diseases.
ISHIKAWA, H. (1987):
Observation of lymphocyte function in perinatal cows and neonatal calves.
Jpn. J. Vet. Sci. 49, 469-475
119
REFERENCES
JESSE, B. W., R. S. EMERY, and J. W. THOMAS (1986):
Control of bovine hepatic fatty acid oxidation.
J. Dairy Sci. 69, 2290-2297
JORRITSMA, R., H. JORRITSMA, Y. SCHUKKEN, and G. WENTINK (2000):
Relationships between fatty liver and fertility and some periparturient diseases in commercial
Dutch dairy herds.
KARCHER, E., M. PICKETT, G. VARGA, and S. DONKIN (2007):
Effect of dietary carbohydrate and monensin on expression of gluconeogenic enzymes in liver of
transition dairy cows.
J. Anim. Sci. 85, 690-699
KIMURA, K., J. GOFF, M. KEHRLI, and J. HARP (1999):
Phenotype analysis of peripheral blood mononuclear cells in periparturient dairy cows.
J. Dairy Sci. 82, 315-319
KIMURA, K., J. GOFF, M. KEHRLI, J. HARP, and B. J. NONNECKE (2002):
Effects of mastectomy on composition of peripheral blood mononuclear cell populations in
periparturient dairy cows.
J. Dairy Sci. 85, 1437-1444
KLEPPE, B. B., R. J. AIELLO, R. R. GRUMMER, and L. E. ARMENTANO (1988):
Triglyceride accumulation and very low density lipoprotein secretion by rat and goat hepatocytes
in vitro.
J. Dairy Sci. 71, 1813-1822
KVIETYS, P. R., and D. N. GRANGER (2012):
Role of reactive oxygen and nitrogen species in the vascular responses to inflammation.
Free Radical Biol. Med. 52, 556-592
LACETERA, N., O. FRANCI, D. SCALIA, U. BERNABUCCI, B. RONCHI, and A.
NARDONE (2002):
Effects of nonesterified fatty acids and β-hydroxybutyrate on functions of mononuclear cells
obtained from ewes.
Am. J. Vet. Res. 63, 414-418
120
REFERENCES
LACETERA, N., D. SCALIA, O. FRANCI, U. BERNABUCCI, B. RONCHI, and A.
NARDONE (2004):
Short Communication: Effects of nonesterified fatty acids on lymphocyte function in dairy
heifers.
J. Dairy Sci. 87, 1012-1014
LACETERA, N., D. SCALIA, U. BERNABUCCI, B. RONCHI, D. PIRAZZI, and A.
NARDONE (2005):
Lymphocyte functions in overconditioned cows around parturition.
J. Dairy Sci. 88, 2010-2016
LACETERA, N., G. KUZMINSKY, P. MORERA, and L. BASIRICÒ (2010):
Fatty acids affect proliferation of peripheral blood mononuclear cells in dairy cows.
Ital. J. Anim. Sci. 6, 434-436
LOOR, J. J., H. M. DANN, R. E EVERTS, R. OLIVEIRA, C. A. GREEN, N. A. J. GURETZKY,
S. L. RODRIGUEZ-ZAS, H. A. LEWIN, and J. K. DRACKLEY (2005):
Temporal gene expression profiling of liver from periparturient dairy cows reveals complex
adaptive mechanisms in hepatic function.
Physiol. Genomics 23, 217-226
LOWICKI, D., and A. HUCZYSKI (2013):
Structure and antimicrobial properties of monensin a and its derivatives: Summary of the
achievements.
Biomed. Res. Int. 1, 1-14.
MALEKKHAHI, M., A. M. TAHMASBI, A. A. NASERIAN, M. DANESH MESGARAN, J. L.
KLEEN, and A. A. PARAND (2015):
Effects of essential oils, yeast culture and malate on rumen fermentation, blood metabolites,
growth performance and nutrient digestibility of Baluchi lambs fed high-concentrate diets.
MALLARD, B. A., J. C. DEKKERS, M. J. IRELAND, K. E. LESLIE, S. SHARIF, C. LACEY
VANKAMPEN, L. WAGTER, and B. N. WILKIE (1998):
Alteration in immune responsiveness during the peripartum period and its ramification on dairy
cow and calf health.
J. Dairy Sci. 81, 585-595
121
REFERENCES
MASSILLON, D., I. J. ARINZE, C. XU, and F. BONE (2003):
Regulation of glucose-6-phosphatase gene expression in cultured hepatocytes and H4IIE cells by
short-chain fatty acids role of hepatic nuclear factor-4α.
J. Biol. Chem. 278, 40694-40701
MCART, J. A., D. V. NYDAM, and G. R. OETZEL (2012):
Epidemiology of subclinical ketosis in early lactation dairy cattle.
J. Dairy Sci. 95, 5056-5066
MCART, J. A., D. V. NYDAM, G. R. OETZEL, T. R. OVERTON, and P. A. OSPINA (2013):
Elevated non-esterified fatty acids and β-hydroxybutyrate and their association with transition
dairy cow performance.
Vet. J. 198, 560-570
MCGUFFEY, R., L. RICHARDSON, and J. WILKINSON (2001):
Ionophores for dairy cattle: Current status and future outlook.
(2003):
MEPHAM, T (1993):
The development of ideas on the role of glucose in regulating milk secretion.
Crop Pasture Sci. 44, 509-522
MICHIELS, J., J. MISSOTTEN, A. VAN HOORICK, A. OVYN, D. FREMAUT, S. DE SMET,
and N. DIERICK (2010):
Effects of dose and formulation of carvacrol and thymol on bacteria and some functional traits of
the gut in piglets after weaning.
MIGUEL, M. G. (2010):
Antioxidant and anti-inflammatory activities of essential oils: A short review.
Molecules 15, 9252-9287
122
REFERENCES
MILLER, J. K., E. BRZEZINSKA-SLEBODZINSKA, and F. C. MADSEN (1993):
Oxidative Stress, antioxidants, and animal function.
J. Dairy Sci. 76, 2812-2823
dairy cows.
J. Dairy Sci. 95, 1323-1336
MURATA, H., and T. MIYAMOTO (1993):
Bovine haptoglobin as a possible immunomodulator in the sera of transported calves.
Br. Vet. J. 149, 277-283
NEWBOLD, C., F. MCINTOSH, P. WILLIAMS, R. LOSA, and R. WALLACE (2004):
Effects of a specific blend of essential oil compounds on rumen fermentation.
NEWBOULD, F (1976):
Phagocytic acitivity of bovine leukocytes during pregnancy.
Canadian Comp. Med. 40, 111
NIKAIDO, H., and M. VAARA (1985):
Molecular basis of bacterial outer membrane permeability.
Microbiol. Rev. 49, 1
ODONGO, N. E., R. BAGG, G. VESSIE, P. DICK, M. M. OR-RASHID, S. E. HOOK, J. T.
GRAY, E. KEBREAB, J. FRANCE, and B. W. MCBRIDE (2007):
Long-term effects of feeding monensin on methane production in lactating dairy cows.
J. Dairy Sci. 90, 1781-1788
OVERTON, T., J. DRACKLEY, G. DOUGLAS, L. EMMERT, and L. CLARK (1998):
Hepatic gluconeogenesis and whole-body protein metabolism of periparturient dairy cows as
affected by source of energy and intake of the prepartum diet.
123
REFERENCES
OVERTON, T. R., J. K. DRACKLEY, C. J. OTTEMANN-ABBAMONTE, A. D. BEAULIEU,
L. S. EMMERT, and J. H. CLARK (1999):
Substrate utilization for hepatic gluconeogenesis is altered by increased glucose demand in
ruminants.
J. Anim. Sci. 77, 1940-1951
PASTER, B. J., J. B. RUSSELL, C. YANG, J. CHOW, C. R. WOESE, and R. TANNER (1993):
Phylogeny of the ammonia-producing ruminal bacteria Peptobacillucoccus anaerobius,
Clostridium sticklandii, and Clostridium aminophilum sp. .
Int. J. Syst. Bacteriol. 43, 107-110
PATRA, A. K. (2011):
Effects of essential oils on rumen fermentation, microbial ecology and ruminant production.
Asian J. Anim. Vet. Adv. 6, 416–428
PILKIS, S. J., and D. GRANNER (1992):
Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.
Annu. Rev. Physiol. 54, 885-909
PRESSMAN, B. C. (1976):
Biological applications of ionophores.
Annu. Rev. Biochem. 45, 501-530
PRESSMAN, B. C., and M. FAHIM (1982):
Pharmacology and toxicology of the monovalent carboxylic ionophores.
Annu. Rev. Pharmacol. Toxicol. 22, 465-490
RHOADS, R. P., J. W. KIM, B. J. LEURY, L. H. BAUMGARD, N. SEGOALE, S. J. FRANK,
D. E. BAUMAN, and Y. R. BOISCLAIR (2004):
Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of
periparturient dairy cows.
J. Nutr. 134, 1020-1027
RICHARDSON, L., A. RAUN, E. POTTER, C. COOLEY, and R. RATHMACHER (1976):
Effect of monensin on rumen fermentation in vitro and in vivo.
J. Anim. Sci. 43, 657-664
124
REFERENCES
RIETSCHEL, E. T., T. KIRIKAE, F. U. SCHADE, U. MAMAT, G. SCHMIDT, H. LOPPNOW,
A. J. ULMER, U. ZÄHRINGER, U. SEYDEL, and F. DI PADOVA (1994):
Bacterial endotoxin: Molecular relationships of structure to activity and function.
FASEB J. 8, 217-225
BERRY (2009):
welfare.
J. Dairy Sci. 92, 5769-5801
RUSSELL, J. B. (1987):
A proposed mechanism of monensin action in inhibiting ruminal bacterial growth: Effects on ion
flux and protonmotive force.
J. Anim. Sci. 64, 1519-1525
RUSSELL, J., H. STROBEL, and G. CHEN (1988):
Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia
production.
RUSSELL, J. B., and H. STROBEL (1989):
Effect of ionophores on ruminal fermentation.
SAAD, A., C. CONCHA, and G. ÅSTRÖM (1989):
Alterations in neutrophil phagocytosis and lymphocyte blastogenesis in dairy cows around
parturition.
J. Vet. Med. B Infect. Dis. Vet. Public Health 36, 337-345
SAUER, F., J. KRAMER, and W. CANTWELL (1989):
Antiketogenic effects of monensin in early lactation.
J. Dairy Sci. 72, 436-442
125
REFERENCES
SAUER, F., V. FELLNER, R. KINSMAN, J. KRAMER, H. JACKSON, A. LEE, and S. CHEN
(1998):
Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added
to the diet.
J. Anim. Sci. 76, 906-914
SCALIA, D., N. LACETERA, U. BERNABUCCI, K. DEMEYERE, L. DUCHATEAU, and C.
BURVENICH (2006):
In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.
J. Dairy Sci. 89, 147-154
SCHÖNFELD, P., and L. WOJTCZAK (2008):
Fatty acids as modulators of the cellular production of reactive oxygen species.
Free Radical Biol. Med. 45, 231-241
DÄNICKE (2014):
J. Dairy Res. 81, 257–266
Nutr. Res. Rev. 6, 185-208
SHAFER-WEAVER, K. A., G. M. PIGHETTI, and L. M. SORDILLO (1996):
Diminished mammary gland lymphocyte functions parallel shifts in trafficking patterns during
the postpartum period.
Exp. Biol. Med. 212, 271-279
SHELDON, I. M., G. S. LEWIS, S. LEBLANC, and R. O. GILBERT (2006):
Defining postpartum uterine disease in cattle.
SIES, H. (1991):
Oxidative stress: From basic research to clinical application.
Am. J. Med. 91, 31-38
126
REFERENCES
SIKKEMA, J., J. DE BONT, and B. POOLMAN (1994):
Interactions of cyclic hydrocarbons with biological membranes.
J. Biol. Chem. 269, 8022-8028
SKINNER, J., R. BROWN, and L. ROBERTS (1991):
Bovine haptoglobin response in clinically defined field conditions.
Vet. Rec. 128, 147-149
SMITH-PALMER, A., J. STEWART, and L. FYFE (1998):
Antimicrobial properties of plant essential oils and essences against five important food-borne
pathogens.
Lett. Appl. Microbiol. 26, 118-122
SORDILLO, L. M., and S. L. AITKEN (2009):
Impact of oxidative stress on the health and immune function of dairy cattle.
SORDILLO, L. M., G. CONTRERAS, and S. L. AITKEN (2009):
Metabolic factors affecting the inflammatory response of periparturient dairy cows.
Anim. Health Res. Rev. 10, 53-63
SPEARS, J. W., and W. P. WEISS (2008):
Role of antioxidants and trace elements in health and immunity of transition dairy cows.
Vet. J. 176, 70-76
STEPHENSON, K. A., I. J. LEAN, and T. J. O'MEARA (1996):
The effect of monensin on the chemotactic function of bovine neutrophils.
Aust. Vet. J. 74, 315-317
STEPHENSON, K. A., I. J. LEAN, M. L. HYDE, M. A. CURTIS, J. K. GARVIN, and L. B.
LOWE (1997):
Effects of monensin on the metabolism of periparturient dairy cows.
J. Dairy Sci. 80, 830-837
127
REFERENCES
SURIYASATHAPORN, W., A. DAEMEN, E. NOORDHUIZEN-STASSEN, S. DIELEMAN,
M. NIELEN, and Y. SCHUKKEN (1999):
β-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media
affect the in vitro chemotaxis of bovine leukocytes.
TASSOUL, M., and R. SHAVER (2009):
Effect of a mixture of supplemental dietary plant essential oils on performance of periparturient
and early lactation dairy cows.
J. Dairy Sci. 92, 1734-1740
TAYLOR, G., L. THOMAS, S. WYLD, J. FURZE, P. SOPP, and C. HOWARD (1995):
Role of T-lymphocyte subsets in recovery from respiratory syncytial virus infection in calves.
J. Virol. 69, 6658-6664
THEILGAARD, P., N. FRIGGENS, K. H. SLOTH, and K. L. INGVARTSEN (2002):
The effect of breed, parity and body fatness on the lipolytic response of dairy cows.
Anim. Sci. 75, 209-219
THORNTON, J., and F. OWENS (1981):
Monensin supplementation and methane production by steers.
J. Anim. Sci. 52, 628-634
TIIHONEN, K., H. KETTUNEN, M. H. L. BENTO, M. SAARINEN, S. LAHTINEN, A.
OUWEHAND, H. SCHULZE, and N. RAUTONEN (2010):
The effect of feeding essential oils on broiler performance and gut microbiota.
Br. Poult. Sci. 51, 381-392
TOMKINS, N. W., S. E. DENMAN, R. PILAJUN, M. WANAPAT, C. S. MCSWEENEY, and
R. ELLIOTT (2015):
Manipulating rumen fermentation and methanogenesis using an essential oil and monensin in
beef cattle fed a tropical grass hay.
TREVISI, E., M. AMADORI, S. COGROSSI, E. RAZZUOLI, and G. BERTONI (2012):
Metabolic stress and inflammatory response in high-yielding, periparturient dairy cows.
Res. Vet. Sci. 93, 695-704
128
REFERENCES
ULTEE, A., E. KETS, and E. SMID (1999):
Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus.
ULTEE, A., M. BENNIK, and R. MOEZELAAR (2002):
The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen
Bacillus cereus.
VAN DER WERF, J., L. JONKER, and J. OLDENBROEK (1998):
Effect of monensin on milk production by Holstein and Jersey cows.
J. Dairy Sci. 81, 427-433
VAN KAMPEN, C., and B. A. MALLARD (1997):
Effects of peripartum stress and health on circulating bovine lymphocyte subsets.
VAN MAANEN, R. W., J. H. HERBEIN, A. D. MCGILLIARD, and J. W. YOUNG (1978):
Effects of monensin on in vivo rumen propionate production and blood glucose kinetics in cattle.
J. Nutr. 108, 1002-1007
Control of rumen methanogenesis.
Environ. Monit. Assess. 42, 73-97
Effect of monensin on rumen metabolism in vitro.
VERNON, R. G., and C. M. POND (1997):
Adaptations of maternal adipose tissue to lactation.
J. Mammary Gland Biol. Neoplasia 2, 231-241
WALLACE, R. J., N. R. MCEWAN, F. M. MCINTOSH, B. TEFEREDEGNE, and C. J.
NEWBOLD (2002):
Natural products as manipulators of rumen fermentation.
Asian-australas. J. Anim. Sci. 15, 1458-1468
129
REFERENCES
WALLACE, R. J. (2004):
Antimicrobial properties of plant secondary metabolites.
Proc. Nutr. Soc. 63, 621-629
WENSING, T., T. KRUIP, M. GEELEN, G. WENTINK, and A. VAN DEN TOP (1997):
Postpartum fatty liver in high-producing dairy cows in practice and in animal studies: The
connection with health, production and reproduction problems.
Comp. Haematol. Int. 7, 167-171
ZAMMIT, V. (1990):
Ketogenesis in the liver of ruminants – adaptations to a challenge.
The Journal of Agricultural Science 115, 155-162
ZAMMIT, V. A. (1996):
Role of insulin in hepatic fatty acid partitioning: Emerging concepts.
Biochem. J. 314, 1-14
ZERBE, H., N. SCHNEIDER, W. LEIBOLD, T. WENSING, T. A. M. KRUIP, and H. J.
SCHUBERTH (2000):
Altered functional and immunophenotypical properties of neutrophilic granulocytes in
postpartum cows associated with fatty liver.
ZHU, L., L. ARMENTANO, D. BREMMER, R. GRUMMER, and S. BERTICS (2000):
Plasma concentration of urea, ammonia, glutamine around calving, and the relation of hepatic
triglyceride, to plasma ammonia removal and blood acid-base balance.
J. Dairy Sci. 83, 734-740
130
DANKSAGUNG
DANKSAGUNG
Mein Dank geht an all die selbstlosen und fleißigen Helfer, die diesen aufwändigen Versuch,
trotz aller Widrigkeiten, so erfolgreich zu Ende gebracht haben. Insbesondere an Reka Tienken,
Melanie Schären, Marc-Alexander Liebold und Dirk von Soosten. Des Weiteren gilt mein
besonderer Dank:
Herrn Prof. Dr. Dr. Sven Dänicke, Leiter des Institutes für Tierernährung, Friedrich-LoefflerInstitut Braunschweig, für die Überlassung der Auswertung dieses hochinteressanten Versuches.
Sein Vertrauen, die vielen wissenschaftlichen Diskussionen und inspirierende Ideen bilden das
Grundgerüst für diese Arbeit und werden mich auch weiterhin in meinem Werdegang begleiten.
Herrn Prof. Dr. Jürgen Rehage, Klinik für Rinder, Tierärztliche Hochschule Hannover, für die
Übernahme meiner Betreuung und seine Unterstützung.
Herrn Dr. Ulrich Meyer und Herrn Dr. Dirk von Soosten, Friedrich-Loeffler-Institut
Braunschweig, die jeder Zeit ein Ohr für meine Anliegen hatten und durch viele hilfreiche
wissenschaftliche Diskussionen, besonders im Bereich der Rinderernährung, meine Arbeit in
großem Maße unterstützt haben.
Der Arbeitsgruppe „Immunonutrition“, Friedrich-Loeffler-Institut Braunschweig, insbesondere
Frau Dr. Jana Frahm, Nicola Mickenautsch, Lara Lindner und Elenia Scholz für die vielen
Probenaufbereitungen und Analysen. Und für die tapfere Beantwortung unendlich vieler Fragen
und dieses stets mit einem Lächeln.
Allen weiteren Mitarbeitern des Friedrich-Loeffler-Institutes, insbesondere der Versuchsstation
für das Management und die Pflege des Milchkuhbestandes und der Hammelhaltung und der
Basisanalytik für die Probenanalysen.
Insbesondere gilt mein Dank all meinen Mitstreitern, ob Mitdoktoranden, Masteranden,
Bacheloranden oder Praktikanten, für viele angenehme Gespräche, Pausen und Aktivitäten, die
mir oftmals eine willkommene Ablenkung waren.
Schließlich gilt mein Dank meiner Familie und Vincent für die grenzenlose Unterstützung, den
Rückhalt, die vielen tollen Momente und die Liebe, die mich während und rundum diesen
Lebensabschnitt bestärkt und motiviert haben.
131

University of Veterinary Medicine Hannover Effects of monensin and

Transcription

Similar documents

SNU Rally at the Fallon Cantaloupe Festival September 2012

uro-Star Indexes

presentation here

Health Tech - Dairy Nutrition Plus

Dr. Emilio de León Ponce de León, Director of Feeding and

Life in Palm Beach County - Digital Collection Center

Factors associated with lameness in dairy cattle