Immunopharmacology and Immunotoxicology, 2011; 33(2): 233–240

© 2011 Informa Healthcare USA, Inc.

ISSN 0892-3973 print/ISSN 1532-2513 online
DOI: 10.3109/08923973.2010.514917

REVIEW ARTICLE

Biomarkers of oxidative stress in ruminant medicine

Pietro Celi
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Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia

Abstract
The study of oxidative stress is a relatively young field of research in ruminant medicine. Oxidative stress results from
increased exposure to or production of oxidants, or from decreased dietary intake, de novo synthesis or increased
turnover of antioxidants. The understanding of the role of oxidants and antioxidants in physiological and pathological
conditions is rapidly increasing. Oxidative stress is an active field of research in veterinary medicine and has been
implicated in numerous disease processes including sepsis, mastitis, acidosis, ketosis, enteritis, pneumonia, respiratory,
and joint diseases. Compared to human medicine, only a limited number of conditions have been investigated in
regard to the effects of oxidative stress in ruminants. Studies in cattle have been sporadic and mainly with mastitis,
pneumonia, and retained placenta. More recently, studies have been focused on metabolic diseases that affect dairy
cows during the peripartum period. Numerous and rapidly evolving methodologies for evaluating oxidative stress
are available to researchers and clinicians, each with their own distinct advantages and disadvantages. Differences
in models and methodologies make it difficult to make meaningful comparisons, even for studies that seem quite
similar superficially. With this in mind, it is the goal of this review to discuss the advantages and shortfalls of different
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methodologies commonly used to measure oxidative stress and damage in ruminants. Clarity of understanding of
the pathophysiology of oxidative stress in ruminants will allow the design of specific antioxidant therapies. Future
research should focus on the establishment of a reference panel of biomarker of oxidative stress to be used in
ruminant medicine. To help accelerate practical applications, we propose the development of an oxidative stress
index as an approach in ruminant and veterinary medicine.
Keywords:  Antioxidants, pro-oxidants, free radicals, sheep, cattle

Introduction
Oxidative stress is the consequence of an imbalance
between oxidants and antioxidants in which oxidant
activity exceeds the neutralizing capacity of antioxi-
dants. In ruminants, oxidative stress may be involved
in several pathological conditions, including conditions
that are relevant for animal production and the general
welfare of the individuals. In dairy cows oxidative stress
has been associated with diseases(1) and reproductive
problems(2). A free radical is defined as any species capa-
ble of independent existence that contains one or more
unpaired electrons(3). Reactive oxygen species (ROS) are
capable of attacking all of the major classes of biomole-
cules, although lipids are particularly susceptible.(2) ROS
have several normal physiological functions,(4) but oxi-
dative stress will occur when excess production cannot
be counteracted by antioxidant mechanisms, potentially
leading to pathological changes.(1) However, ROS are
also involved in several physiological processes,(5)
therefore a certain level is desirable. Unfortunately it is
not known what this level is for ruminants at different
physiological stages. Oxidative stress can be particularly
dangerous because no clinical symptoms are shown
and the condition is diagnosed by means of dedicated
analytical methods. It is important thus to establish the
physiological ranges for biomarkers of oxidative stress in
ruminant medicine so that an oxidative stress threshold
can be determined. For example, given the lack of refer-
ence values for reactive oxygen metabolites (ROMs) and
biological antioxidant potential (BAP) in dairy cows the
causes of oxidative stress are difficult to identify. For this
reason, in a study that was aimed to determine the effect
of diet, energy balance and milk production on oxidative
stress in early-lactating, Holstein–Friesian dairy cows

Address for Correspondence:  Pietro Celi, Faculty of Veterinary Science, University of Sydney, PMB 4003, Narellan, 2567, NSW, Australia.
E-mail: pietro.celi@sydney.edu.au
(Received 08 June 2010; accepted 07 August 2010)

233
 234  Pietro Celi
fed to produce either low or high levels of milk(6) we
developed low, medium, and high energy balance and
milk yield categories to further investigate the relation-
ship between these variables and oxidative stress. This
approach reduced the effect of individual cow variation
and also took into account the feeding system effect
and showed that the information on oxidative stress is
more accurate when combining ROMs and BAP data
than using them separately.(6) However, in order to study
oxidative stress in ruminants, a normal range needs to
be established for the different biomarkers of this pro-
cess. Such an approach is well established in human
medicine and would allow a more evidence-based inter-
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pretation of the results of various studies in ruminants
that currently appear contradictory.
In dairy cows, pregnancy and lactation are physiologi-
cal stages considered to induce metabolic stress.(7) For
example, dairy cows can experience oxidative stress,(8,9)
which may be associated with metabolic diseases dur-
ing the peripartum period.(2) A number of recent studies
have reported variable levels of oxidative stress during
the periparturient period in sheep,(10) dairy cows,(8,11,12)
and dairy goats.(13,14) However, it is not clear whether or
not the level of oxidative stress during this period could
compromise animal performance and health. It seems,
however, that ROS and antioxidants may be involved
in some relevant physiological functions such as milk
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yield(15) and therefore it might be beneficial to supple-
ment cows with antioxidants.(6) In ruminants, the activity
of oxidative stress biomarkers can be influenced by nutri-
tion and season.(16,17) For example, feeding high levels of
starch in the diet to cows at 80 days lactation increased
oxidative stress, possibly due to cellular changes related
to oxidative phosphorylation.(15)
Increasing interest in the role of oxidative stress in
ruminant medicine has increased the need to develop
reliable methods to quantify the markers of oxidative
stress. The methods involve direct or indirect assessment
of oxidant and antioxidant concentrations and, given
the highly reactive nature of the compounds, specialized
equipment is frequently required.(1) With this in mind, it
is the goal of this review to discuss the advantages and
shortfalls of different methodologies commonly used
to measure oxidative stress and damage in ruminants.
Clarity of understanding of the pathophysiology of oxi-
dative stress in ruminants will allow the design of specific
antioxidant therapies.
Measuring oxidative stress in ruminants
Oxidative stress in veterinary medicine and particularly
in ruminant health is a relatively young field of research.
Numerous and rapidly evolving methodologies for
evaluating oxidative stress, each with their own distinct
advantages and disadvantages, are available to clinician
and scientists (Table 1). However, differences in models
and methodologies make it difficult to make meaningful
comparisons, even for studies that seem quite similar.
Methods for quantifying oxidative stress mostly include
 Immunopharmacology and Immunotoxicology
direct or indirect measures of oxidants and antioxidants.
Given their often high reactivity, quantification of oxi-
dants and antioxidants often requires specialized equip-
ment and considerable experience. As oxidative stress
is indicative of an imbalance between oxidants and
antioxidants, methods for quantifying oxidative stress
mostly include direct or indirect measures of oxidants
and antioxidants.(1,2) In the following sections, some prin-
ciples and commonly used measures of oxidative stress
and damage will be briefly outlined.
Pro-oxidants
The most abundant free radicals in biological systems are
the oxygen-centered free radicals and their metabolites,
usually referred to as ROS.(2) ROS are formed continuously
as normal by-products of cellular metabolism and, in low
concentrations, they are essential for several physiologi-
cal processes, including protein phosphorylation, tran-
scription factors activation, cell differentiation, apoptosis,
oocyte maturation, steroidogenesis, cell immunity, and
cellular defense against microorganisms.(2,4,18) However,
when produced in excess, ROS can damage cell func-
tionality as they can harm cellular lipids, proteins, and
DNA.(2,5)
Plasma level of ROMs is considered an indicator of
free radical production.(2) ROMs is a collective term that
includes not only oxygen-centered free radicals such as
superoxide anion and hydroxyl radical, but also some
non-radical derivates of oxygen, such as hydrogen perox-
ide (H2O2), and hypochlorous acid.(19) A ROMs kit has been
developed to assess oxidant levels in plasma and other
biological fluids. The ROMs test has been validated using
electron spin resonance,(20) which is considered the “gold
standard” for measuring total oxidative status. Electron
spin resonance is not suitable for routine analysis as the
method is complex and requires specific technical assis-
tance not available in most laboratories. The utility of the
ROMs assay in monitoring oxidative stress in goats,(13,14,17)
sheep,(10) and dairy cows(8,6,16) has been reported.
The concentrations of individual oxidant components
can be measured separately in the laboratory, but such
measurements are time-consuming, labor intensive, and
costly. It has been shown that free radical analytical sys-
tem 4 technology offers a quick, simple, precise, and reli-
able method of assessing oxidative status in dairy cows(21)
and in horses,(22) which is particularly useful in the field
where it is not always practical or possible to get samples
to a laboratory immediately. The possibility of assessing
oxidative stress directly in blood provides veterinarians
with a simple and reliable method of measuring oxida-
tive stress in clinical situations such as the monitoring
of therapy and in the antioxidant supplementation of
domestic animals. However, given the lack of reference
values for ROMs in ruminants it is difficult to establish
if and when these animals are experiencing oxidative
stress. Therefore, it is important to calculate the specific
referral ranges because a correct biochemical evaluation
of oxidative status is an essential premise to prevent and
 Oxidative stress in ruminants  235

Table 1.  Advantages and disadvantages of various biomarkers of oxidative stress.

Biomarker Assay procedure Advantages Disadvantages
MDA Colirometry, luminometry, Sensitive and reproducible Non-specific product of lipid
chemiluminescence, HPLC, peroxidation; interferes with
GC/MS TBARS assay
TBARS Spectrometry, luminometry Rapid, popular, easy, and economical Non-specific, non-reproducible,
chemiluminescence no quantitative relationship with
lipid peroxidation
F2- EIA, ELISA, HPLC, GC/MS Specific, reproducible, sensitive Expensive, auto-oxidation of
Isoprostane samples, sample derivitisation
required
ORAC Fluorescence Sensitive and covers a wide variety of Requires spectro-fluorimeter;
antioxidants AAPH used a free radical source
undergoes spontaneous decay
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and it is sensitive to temperature

FRAP Spectrometry Serum dilution effect not seen Fe may generate free radicals; not
every free radical reduces Fe; GSH
is not measured
TEAC Spectrometry Extremely fast and simple Results vary with sample dilution;
antioxidant used may interact
with solvent molecules; specificity
varies
TRAP Chemiluminescence Gives an idea of the rate of free radical Antioxidant employed may not
formation trap all types of free radicals
ROMs Spectrometry Extremely fast, simple; only 5–20 µL of Inhibited by sodium azide
plasma/serum required; can be performed
directly in whole blood, inflammatory fluids,
cell extracts and respiratory condensate
BAP Spectrometry Extremely fast, simple and covers a wide Can be performed only in plasma
variety of antioxidants; only 5–20 µL of and serum samples; hyperlipemic
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plasma/serum required samples can underestimate

results
AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; BAP, biological antioxidant potential; EIA, enzyme immunoassay; ELISA,
enzyme-linked immunosorbent assay; FRAP, ferric reducing ability of plasma; GC, gas chromatography; HPLC, high-pressure liquid
chromatography; MDA, malonildialdehyde; MS, mass spectrometry; ORAC, oxygen radical absorbance capacity; ROMs, reactive
oxygen metabolites; TBARS, thiobarbituric acid reactive substances; TEAC, trolox equivalent antioxidant capacity; TRAP, total radical
antioxidant potential.

eventually to treat the effects of oxidative stress in rumi-
nant medicine.
Advanced oxidation protein products (AOPPs) are
terminal products of proteins exposed to free radicals
and arise from the reaction between plasma proteins and
chlorinated oxidants mediated by a neutrophil enzyme
myeloperoxidase.(23,24) In humans, AOPP have been
linked to several diseases like chronic renal failure,(25)
diabetes mellitus,(26) diabetic nephropathy,(27) coronary
artery diseases,(28) and obesity.(29) Chronic accumulation
of AOPP has been demonstrated to promote inflamma-
tory processes in diabetic kidney(27) and in chronic renal
failure(25) indicating that AOPP might be a by-product of
neutrophil activation during infections. Studies in rumi-
nants have reported higher levels of AOPP in lambs(30)
and dairy cows(31) supplemented with Yerba Mate (Ilex
Paraguanensis). More information about the role of pro-
tein oxidation in ruminants’ health and production could
be obtained by the comparison of AOPP with other indi-
cators of protein oxidation, such as advanced glycation
end products (AGE). However, although a correlation
between AGE and inflammatory parameters is usually
not found or is only weak, the induction of proinflam-
matory activities caused by AOPP seems to be more
intense.(32,33) This suggests that oxidative stress is closely
linked to inflammation and acute phase reactions than
advanced glycation process and its end products. AOPP
could thus better describe acute inflammation, whereas
AGE might serve more as a marker of chronic long-last-
ing damage.(32) These observations are highly relevant as
increased levels of AOPP could indicate the presence of
inflammatory process that can compromise the correct
embryonic development in dairy cows.(21,34)
Lipids, in particular those that are polyunsaturated,
are prone to oxidation. Lipids are one of the most suscep-
tible substrates to free radicals damage and biomarkers
of lipid peroxidation are considered the best indicators of
oxidative stress.(35) Malondialdehyde (MDA) is one of the
several low-molecular-weight end products formed dur-
ing the radical induced decomposition of polyunsatu-
rated fatty acid.(36) MDA readily reacts with thiobarbituric
acid producing a red pigment that can be easily mea-
sured by spectrophotometry in the form of thiobarbituric
acid reactive substances (TBARS).(36) It is worth noting
that the MDA assays have been criticized for low speci-
ficity and artifact formation, since only a fraction of the
MDA measured is generated in vivo. Furthermore, the
TBARS assay, a common method used to measure MDA,

© 2011 Informa Healthcare USA, Inc.

 236  Pietro Celi
is considered inaccurate, and returns results that differ
according to the assay conditions used.(37) For example,
studies in dairy cows have yielded contrasting results
with some reports failing to show any significant changes
in plasma MDA concentrations during the peripartum
period,(9,10) whereas in other studies MDA or TBARS con-
centrations increased around calving.(8,16,38) This apparent
discrepancy could also have been mainly due to the great
individual variations observed in MDA concentrations
measured in the studies by Castillo et  al.(9,11) Similarly,
studies in transported cattle have failed to report a con-
sistent change in MDA concentration. It seems that this
discrepancy is partly due to the different methodologies
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employed to assess MDA. TBARS detect a wide range of
lipid peroxidation products, and are rather unspecific for
MDA.(39) High-pressure liquid chromatography would be
expected to be highly specific, and perhaps, more accu-
rate than the spectrophotometric procedures.(40) More
recently an ELISA based isoprostane, which are consid-
ered to be the most reliable markers of lipid oxidation,(41)
assay has become commercially available and might be
able to shed more light on the role of lipid peroxidation
during the peripartum period in ruminants.
Antioxidants
Endogenous antioxidants can be divided into three
major groups.(2) The first group comprises enzymatic
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antioxidants including superoxide dismutase (SOD) and
glutathione-peroxidase (GSH-Px), and represents the
main form of intracellular antioxidant defense. Plasma
GSH-Px activity contributes to the oxidative defense of
animal tissues by catalyzing the reduction of hydrogen
and lipid peroxides(37) and is also considered an indica-
tor of oxidative stress.(42) GSH-Px functions in cellular
oxidation-reduction reactions to protect the cell mem-
brane from oxidative damage caused by free radicals.(43)
SOD catalyses the dismutation of superoxide to H2O2 and
it is considered the first defense against pro-oxidants.(37)
Studies in grazing sheep have shown that GSH-Px activity
is influenced by soil and pasture characteristics(44) and by
season.(45) In dairy goats SOD activity is decreased during
the postpartum period probably a consequence of lower
peroxide generation as testified by the decrease in ROMs
concentrations.(14) Since SOD activity increases H2O2 pro-
duction, protection from reactive oxygen would only be
given by a simultaneous increase in catalase and GSH-Px
activities and availability of glutathione.(46,47) Studies in
dairy goats have shown that blood GSH-Px activity is
decreased during the postpartum period, suggesting
that goats may have experienced some degree of oxida-
tive stress and lipid peroxidation.(13,14) Since GSH-Px is
directly targeted at removing H2O2 generated during the
dismutation of free radicals,(4) it would reasonable to see
a parallel decrease in ROMs levels. ROMs levels indeed,
decreased on week 4 postpartum, however its concen-
trations on week 2 were significantly higher,(14) which
further indicated that the goats experienced oxidative
stress during the early postpartum period. Even if blood
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GSH-Px activity is inhibited,(13,14) the organism could have
been defended against oxidative stress by other alterna-
tive routes. For example, catalase is another antioxidant
enzyme that can catabolise H2O2.(4)
The second group includes non-enzymatic protein
antioxidants that are primarily found in plasma. They are
mainly represented by sulfhydryl (SH) groups of albu-
min and are considered a significant element of extra-
cellular antioxidant defense system. Protein SH groups
are considered a significant element of the extracellular
antioxidant defense system against oxidative stress.(48)
The reducing properties of SH residue are known to be
oxidized under oxidative stress and other physiological
conditions.(49,50) Total thiol groups of plasma represent
the sulfhydryl groups of albumin, l-cysteine, and homo-
cysteine. Under physiological conditions, SH groups are
the most chemically reactive sites and have strong reduc-
ing properties.(51,52) Studies in dairy goats have shown that
plasma albumin concentrations are significantly reduced
during summer.(17) This finding is quite relevant consider-
ing that albumin is part of the antioxidant pool because it is
a free radical scavenger.(53) Plasma albumin levels are also
decreased during the peripartum period in dairy goats,
which further indicates that goats were exposed to oxida-
tive stress during the peripartum period.(13,14) Albumin
is exclusively synthesized by the liver, and is the main
source of plasma SH. The reduction in liver function that
is usually observed in the early postpartum period might
explain lower plasma SH and albumin levels. Studies in
dairy cows have confirmed the antioxidant role played by
albumin particularly near calving when animals usually
do not receive any vitamin/mineral supplementation.(11)
The third group is represented by the non-enzymatic
low-molecular weight antioxidants, and it is found mainly
in plasma but also in other extracellular and intracellu-
lar fluids. The primary antioxidant capacity of serum is
derived from non-enzymatic antioxidants, such as gluta-
thione, α-tocopherol, β-carotene, and uric acid.(3) In par-
ticular, GSH plays an important role in protecting cells
against oxidative stress and toxic agents. It acts as sub-
strate or co-substrate in enzymatic reactions and it also
reacts directly with free radicals and lipid peroxides.(54)
For example, in a selenium deficiency situation,
hepatic glutathione (GSH) synthesis is increased and
this depletes cellular cysteine,(55) so it may impair
physiological processes (growth and wool production)
where cysteine is required for protein synthesis. Indeed,
GSH synthesis seems to compete with wool growth for
cysteine.(56) Cysteine is vital for wool growth and is usually
the first limiting amino acid for wool fiber synthesis.(57)
Sheep selected for high wool production tend to have
lower blood cysteine and GSH concentrations than those
selected for low wool production.(58,59) Low concentrations
of GSH can be associated with impaired animal health as
cysteine and GSH play a key role in the regulation of the
immune response.(60) Therefore, considering that GSH is
a reservoir of cysteine, selecting for both GSH concentra-
tions and wool growth rate might result in improvements

in both wool production and health status. In the mean
(56)
time, when selenium deficiency is diagnosed, its supple-
mentation is recommended to improve the development
of resistance and resilience of sheep to gastro intestinal
parasites.(61)
Because of the difficulty in measuring each anti-
oxidant component separately and their interaction in
plasma, several methods have been developed to assess
total antioxidant capacity (TAC). The measure of anti-
oxidant capacity considers the cumulative action of all
the antioxidants present in plasma and body fluids, thus
providing an integrated parameter rather than the simple
sum of measurable antioxidants.(62) Antioxidant capacity
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can be measured by means of several methods (Table 2),
such as trolox equivalent antioxidant capacity,(63) total
radical-trapping antioxidant parameter,(64) oxygen radi-
cal absorbance capacity,(65) or the ferric reducing ability
of plasma, and BAP.(66) The BAP test provides a global
measurement of many antioxidants, including uric acid,
ascorbic acid, proteins, α-tocopherol and bilirubin.(66) In
dairy cows BAP levels have been measured during early
lactation(6) and mid lactation,(31) however, comparisons
can be hardly done since those studies involved cows
in different physiological phases. Also, the rearing sys-
tem may determine important differences in terms of
the oxidative status of animals. Pasture-based systems
could improve the oxidative status of animals due to the
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elevated antioxidants content of green grass(67,68) and the
physical exercise related with grazing system, have bet-
ter antioxidant status.(67) Grazing may provide dairy cows
with health benefits from certain vitamins and minerals.
Vitamins A and E, and selenium are known antioxidants
that play important roles in animal health and produc-
tion. Though fresh forages are typically considered capa-
ble of supplying adequate levels of antioxidants for dairy
cattle, the availability of these compounds for lactating
grazing cows is diminished when pasture availability
is not adequate to meet their energy requirements. In
this situation the gap between energy required for milk
production and energy intake is often met by supple-
menting cows with conserved forages like silage. Silage
is known for its poor content in antioxidants(69) and thus
this might expose cows to oxidative stress. A decrease in
Table 2.  Key criteria of the validation process of a suitable
biomarker of oxidative stress.
•  Selection of biomarker of oxidative stress
•  Directly implicated in the onset and progression of disease
•  Development of method for measuring the biomarker
•  Verification of potential for pitfalls and artefacts
•  Verification of biomarker in suitable animal models
•  Identification of modifying factor of the biomarker (nutrition,
physiological status, photoperiod)
•  Establishment of reference intervals and values
•  Adaptation of the method to field condition (sensitivity,
simplicity, throughput)
•  Demonstration of the efficacy of antioxidant therapy in animal
models
© 2011 Informa Healthcare USA, Inc.
Oxidative stress in ruminants  237
BAP levels was observed when dairy cows were fed corn
silage and concentrate supplementation.(31) Concentrate
supplementation with the diet has been proved to either
reduce pasture intake(70) and to increase the degrada-
tion of some antioxidants at ruminal level.(71) Plasma
BAP might have decreased also as a direct consequence
of ROMs increase. Indeed, changes in the components
of antioxidants systems are often not the cause, but the
consequence of the oxidative stress induced by higher
free radical activity.(72)
Plasma antioxidant status is the result of the interac-
tion of many different compounds and systemic meta-
bolic interactions.(62) As a single measure, TAC provides
relevant information that may effectively describe the
dynamic equilibrium between pro-oxidants and anti-
oxidant in the plasma compartment.(62,73) In absence
of reference values for ruminants, TAC can be used to
evaluate the effect of treatments when the results are
expressed as change with respect to the basal value.(62)
Indeed, TAC is a useful tool for measuring stress in
transported calves.(74) In conclusion, the evaluation of
TAC on a herd basis may be a useful indicator of animal
welfare and may be more sensitive and reliable than the
measure of a single parameter, which could reveal indi-
vidual variations. Moreover, TAC could be used as a tool
to evaluate the nutritional status of animals fed different
diets or to evaluate the general nutritional status of ani-
mals throughout the year.
Current and future developments
The field of oxidative stress in ruminant medicine is still
in the early stages of development. Despite that oxida-
tive stress has been associated with numerous condi-
tions, there is a great deal to be discovered about its
role in ruminant health and production. For example, if
oxidative stress is a primary cause of pathologic change
or a consequence of disease processes still remains to be
determined. Clarity of understanding of the pathophysi-
ology of oxidative stress in ruminants will allow the
design of specific antioxidant therapies. Future research
should focus on the establishment of a reference panel
of biomarkers of oxidative stress to be used in ruminant
medicine. Future research should also address the issue
of standardization of techniques and methodologies to
study oxidative stress. For example, tissue biopsy sam-
pling techniques can be utilized only in particular cases
in veterinary medicine, therefore a major challenge in
veterinary oxidative stress research is to develop a set
of blood biomarkers that can reliably reflect the tis-
sue oxidative status in the individual animal. There
are numerous techniques that can be used to measure
oxidative damage,(1) however veterinary scientists and
clinicians need to establish whether oxidative stress
biomarkers measured in blood and/or serum provide a
reasonable index of the general oxidative stress status.
Considering that oxidative damage is likely to occur in
only few tissues at the same time, high levels of serum
oxidative stress biomarkers can be the consequence
 238  Pietro Celi
of: (1) a generalized increase in oxidative stress in most
of the tissues, resulting in an increase of the biomarker
concentration in blood proportional to the extent of
the tissues damage; (2) the increase of oxidative stress
just in a particular tissue that is the origin of the patho-
logical dysfunction; (3) an increase of oxidative damage
produced specifically in the circulatory system.(75) Even
if several animal studies seem to suggest that oxidative
stress biomarkers change toward the same direction
in blood and tissues this needs to be fully evaluated
in ruminant species. Therefore, several questions still
remain to be answered in the field of both human and
veterinary oxidative stress research. For example, what
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are the most appropriate biomarkers when investigating
oxidative stress in relation to disease state? What is the
best methodology to measure them? A good biomarker
should satisfy a series of specific criteria (Table 2). Some
biomarkers of oxidative stress might be of limited value
in vivo because they lack sensitivity and/or specificity
or require invasive methods. Other biomarkers could be
useful, however the “ideal” biomarker seems not to exist
in veterinary medicine. Furthermore, in order to func-
tion as suitable biomarker of oxidative stress, it is critical
that the biomarker is a chemically stable product, not
susceptible to artifactual induction, oxidation, or loss
during sample handling, processing, analysis, and stor-
age. It is also important that the biomarker can be easily
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accessed in a target tissue or sample that quantitatively
reflects the oxidative modification of the target tissue.
Finally, it needs to be present at concentrations high
enough for the biomarker to be detected. Currently, the
biomarkers of oxidative stress commonly used in rumi-
nant medicine and the methods used to measure them
vary between studies therefore it is important to perform
a thorough comparison of the different methodologies
that have been employed so that the findings obtained
from different research groups can be compared.
It is clear then that each biomarker of oxidative stress
has its advantages and disadvantages (Table 2), which is
the reason why no single measurement can adequately
describe oxidative stress. It is therefore suggested that
the use of a panel of measurements is important to reli-
ably monitor changes in oxidative stress. We propose
that oxidative damage, as indicated by an individual
animal’s oxidative stress (pro-oxidants/antioxidants)
index, is indicative of an animal’s risk to develop disease.
This hypothesis needs to be fully tested in specifically
designed trials. Thus, when the oxidative stress is low
(pro-oxidants < antioxidants) it would be reasonable to
expect improved health and wellbeing conditions for the
individual animal that will result in better productive per-
formances. This argument is made stronger by the use of
assays that have already shown correlations with health
status. For example, when oxidative stress was monitored
in dairy cows by means of ROMs and BAP, the informa-
tion on oxidative stress level is more accurate when com-
bining ROMs and BAP data than using them separately.(6)
In particular, only restricted production cows in severe
 Immunopharmacology and Immunotoxicology
negative energy balance had a higher level of oxidative
stress.(6) Indeed, when the degree of oxidative stress is
estimated by the calculation of an oxidative stress index
the relationship between the level of oxidative stress and
pathology is higher when ROMs and BAP measurements
are so combined.(76)
Information is steadily growing in the field of oxida-
tive stress in veterinary medicine, indicating the general
importance of oxidative stress and related inflammation
disorders to ruminant health and production. There is an
increasing need to translate this knowledge to practical
applications for animal and veterinary scientists. To help
accelerate practical application, we propose the develop-
ment of an oxidative stress index as an approach in rumi-
nant and veterinary medicine.
Declaration of interest
The author reports no conflicts of interest. The author
alone is responsible for the content and writing of the
paper.
References
1. Lykkesfeldt, J., Svendsen, O. Oxidants and antioxidants in disease:
oxidative stress in farm animals. Vet. J. 2007, 173, 502–511.
2. Miller, J.K., Brzezinska-Slebodzinska, E., Madsen, F.C. Oxidative
stress, antioxidants, and animal function. J. Dairy Sci. 1993, 76,
2812–2823.
3. Halliwell, B., Gutteridge, J.M.C. Free radicals in biology and
medicine. 2nd ed. Oxford, UK: Oxford University Press; 1989.
4. Dröge, W. Free radicals in the physiological control of cell function.
Physiol. Rev. 2002, 82, 47–95.
5. Sugino, N. Roles of reactive oxygen species in the corpus luteum.
Anim Sci J. 2006, 77, 556–565.
6. Pedernera, M., Celi, P., García, S.C., Salvin, H.E., Barchia, I.,
Fulkerson, W.J.Effect of diet, energy balance and milk production
on oxidative stress in early-lactating dairy cows grazing pasture.
Vet. J. 2009, In Press.
7. Drackley, J.K. ADSA Foundation Scholar Award. Biology of dairy
cows during the transition period: the final frontier? J. Dairy Sci.
1999, 82, 2259–2273.
8. Bernabucci, U., Ronchi, B., Lacetera, N., Nardone, A. Influence of
body condition score on relationships between metabolic status
and oxidative stress in periparturient dairy cows. J. Dairy Sci. 2005,
88, 2017–2026.
9. Castillo, C., Hernández, J., Valverde, I., Pereira, V., Sotillo, J.,
Alonso, M.L., Benedito, J.L. Plasma malonaldehyde (MDA) and
total antioxidant status (TAS) during lactation in dairy cows. Res.
Vet. Sci. 2006, 80, 133–139.
10. Rizzo, A., Mutinati, M., Spedicato, M., Minoia, G., Trisolini, C.,
Jirillo, F., Sciorsci, R.L. First demonstration of an increased serum
level of reactive oxygen species during the peripartal period in the
ewes. Immunopharmacol. Immunotoxicol. 2008, 30, 741–746.
11. Castillo, C., Hernandez, J., Bravo, A., Lopez-Alonso, M.,
Pereira, V., Benedito, J.L. Oxidative status during late pregnancy
and early lactation in dairy cows. Vet. J. 2005, 169, 286–292.
12. Gaál, T., Ribiczeyné-Szabó, P., Stadler, K., Jakus, J., Reiczigel, J.,
Kövér, P., Mézes, M., Sümeghy, L. Free radicals, lipid peroxidation
and the antioxidant system in the blood of cows and newborn
calves around calving. Comp. Biochem. Physiol. B, Biochem. Mol.
Biol. 2006, 143, 391–396.
13. Celi, P., Di Trana, A., Quaranta, A. Metabolic profile and oxidative
status in goats during the peripartum period. Aust J Exp Agric.
2008, 48, 1004–1008.

14. Celi, P., Di Trana, A., Claps, S. Effects of plane of nutrition on
oxidative stress in goats during the peripartum period. Vet. J. 2010,
184, 95–99.
15. Gabai, G., Testoni, S., Piccinini, R., Marinelli, L., Stradaioli, G.
Oxidative stress in primiparous cows in relation to dietary starch
and the progress of lactation. Anim Sci. 2004, 79, 99–108.
16. Bernabucci, U., Ronchi, B., Lacetera, N., Nardone, A. Markers of
oxidative status in plasma and erythrocytes of transition dairy
cows during hot season. J. Dairy Sci. 2002, 85, 2173–2179.
17. Di Trana, A., Celi, P., Claps, S., Fedele, V., Rubino, R. The effect of
hot season and nutrition on the oxidative status and metabolic
profile in dairy goats during mid lactation. Anim Sci. 2006, 82,
717–722.
18. Agarwal, A., Gupta, S., Sharma, R.K. Role of oxidative stress in
female reproduction. Reprod. Biol. Endocrinol. 2005, 3, 28.
19. Reilly, P.M., Schiller, H.J., Bulkley, G.B. Pharmacologic approach
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by RMIT University on 09/03/13
to tissue injury mediated by free radicals and other reactive oxygen
metabolites. Am. J. Surg. 1991, 161, 488–503.
20. Alberti, A., Bolognini, L., Macciantelli, D., Caratelli, M. The Radical
Cation of N,N-Diethyl-para-Phenylendiamine: a Possible Indicator
of Oxidative Stress in Biological Samples. Res Chem Interm. 2000,
26, 253–267.
21. Merlo, M., Celi, P., Barbato, O., Gabai, G. Relationships Between
Oxidative Status and Pregnancy Outcome in Dairy Cows. Animal
Production in Australia. 2008, 27, 84.
22. Celi, P., Sullivan, M., Evans, D. The stability of the reactive oxygen
metabolites (d-ROMs) and biological antioxidant potential (BAP)
tests on stored horse blood. Vet. J. 2010, 183, 217–218.
23. Fialová, L., Malbohan, I., Kalousová, M., Soukupová, J., Krofta, L.,
Stípek, S., Zima, T. Oxidative stress and inflammation in pregnancy.
Scand. J. Clin. Lab. Invest. 2006, 66, 121–127.
24. Noyan, T., Güler, A., Sekeroglu, M.R., Kamaci, M. Serum advanced
For personal use only.
oxidation protein products, myeloperoxidase and ascorbic acid
in pre-eclampsia and eclampsia. Aust. N. Z. J. Obstet. Gynaecol.
2006, 46, 486–491.
25. Witko-Sarsat, V., Friedlander, M., Nguyen Khoa, T., Capeillère-
Blandin, C., Nguyen, A.T., Canteloup, S., Dayer, J.M., Jungers, P.,
Drüeke, T., Descamps-Latscha, B. Advanced oxidation protein
products as novel mediators of inflammation and monocyte
activation in chronic renal failure. J. Immunol. 1998, 161,
2524–2532.
26. Kalousová, M., Skrha, J., Zima, T. Advanced glycation end-products
and advanced oxidation protein products in patients with diabetes
mellitus. Physiol. Res. 2002, 51, 597–604.
27. Shi, X.Y., Hou, F.F., Niu, H.X., Wang, G.B., Xie, D., Guo, Z.J., Zhou,
Z.M., Yang, F., Tian, J.W., Zhang, X. Advanced oxidation protein
products promote inflammation in diabetic kidney through
activation of renal nicotinamide adenine dinucleotide phosphate
oxidase. Endocrinology 2008, 149, 1829–1839.
28. Kaneda, H., Taguchi, J., Ogasawara, K., Aizawa, T., Ohno, M.
Increased level of advanced oxidation protein products in patients
with coronary artery disease. Atherosclerosis 2002, 162, 221–225.
29. Atabek, M.E., Keskin, M., Yazici, C., Kendirci, M., Hatipoglu, N.,
Koklu, E., Kurtoglu, S. Protein oxidation in obesity and insulin
resistance. Eur. J. Pediatr. 2006, 165, 753–756.
30. Celi, P., Raadsma, H.W. The effects of Yerba Mate (Ilex paraguarensis)
supplementation on the productive performance of lambs. In:
Ruminant Physiology Digestion, metabolism, and effects of nutrition
on reproduction and welfare. Proceeding of the XIth International
Symposium in Ruminant Physiology. Clermont-Ferrand, France,
2009, 804–805.
31. Celi, P., Raadsma, H.W. Effects of Yerba Mate (Ilex paraguariensis)
supplementation on the productive performance of dairy cows
during mid-lactation. Anim Prod Sci. 2010, 50, 339–344.
32. Kalousová, M., Zima, T., Tesar, V., Dusilová-Sulková, S., Skrha, J.
Advanced glycoxidation end products in chronic diseases-
clinical chemistry and genetic background. Mutat. Res. 2005, 579,
37–46.
33. Witko-Sarsat, V., Nguyen-Khoa, T., Jungers, P., Drüeke, T.B.,
Descamps-Latscha, B. Advanced oxidation protein products as a
© 2011 Informa Healthcare USA, Inc.
Oxidative stress in ruminants  239
novel molecular basis of oxidative stress in uraemia. Nephrol. Dial.
Transplant. 1999, 14 Suppl 1, 76–78.
34. Merlo, M., Barbato, O., Stefani, A., Celi, P., Da Dalt, L., Gabai, G.
Embryonic mortality and plasma advanced oxidation protein
products (AOPP) increase in dairy cows. In: Current Topics in
Dairy Production, Proceedings of the Dairy Research Foundation
Symposium. Camden, Australia, 2008, 49–53.
35. Georgieva, N.V. Oxidative stress as a factor of disrupted ecological
oxidative balance in biological systems – a review. Bulg J Vet Med.
2005, 8, 1–11.
36. Janero, D.R. Malondialdehyde and thiobarbituric acid-reactivity
as diagnostic indices of lipid peroxidation and peroxidative tissue
injury. Free Radic. Biol. Med. 1990, 9, 515–540.
37. Halliwell, B., Chirico, S. Lipid peroxidation: its mechanism,
measurement, and significance. Am. J. Clin. Nutr. 1993, 57, 715S–
724S; discussion 724S.
38. Bouwstra, R.J., Goselink, R.M., Dobbelaar, P., Nielen, M.,
Newbold, J.R., van Werven, T. The relationship between oxidative
damage and vitamin E concentration in blood, milk, and liver
tissue from vitamin E supplemented and nonsupplemented
periparturient heifers. J. Dairy Sci. 2008, 91, 977–987.
39. Griffiths, H.R., Møller, L., Bartosz, G., Bast, A., Bertoni-
Freddari, C., Collins, A., Cooke, M., Coolen, S., Haenen, G.,
Hoberg, A.M., Loft, S., Lunec, J., Olinski, R., Parry, J., Pompella, A.,
Poulsen, H., Verhagen, H., Astley, S.B. Biomarkers. Mol. Aspects
Med. 2002, 23, 101–208.
40. Lykkesfeldt, J. Determination of malondialdehyde as
dithiobarbituric acid adduct in biological samples by HPLC
with fluorescence detection: comparison with ultraviolet-visible
spectrophotometry. Clin. Chem. 2001, 47, 1725–1727.
41. Milne, G.L., Musiek, E.S., Morrow, J.D. F2-Isoprostanes as markers
of oxidative stress in vivo: An overview. Biomarkers. 2005, 10,
10–23.
42. Tüzün, A., Erdil, A., Inal, V., Aydin, A., Bagci, S., Yesilova, Z.,
Sayal, A., Karaeren, N., Dagalp, K. Oxidative stress and antioxidant
capacity in patients with inflammatory bowel disease. Clin.
Biochem. 2002, 35, 569–572.
43. Flohe, L., Günzler, W.A., Schock, H.H. Glutathione peroxidase: a
selenoenzyme. FEBS Lett. 1973, 32, 132–134.
44. Andrés, S., Jiménez, A., Mañé, M.C., Sánchez, J., Barrera, R.
Relationships between some soil parameters and the blood
glutathione peroxidase activity of grazing sheep. Vet. Rec. 1997,
141, 267–268.
45. Andres, S., Mañe, M.C., Sanchez, J., Barrera, R., Jimenez, A.
Temporal variations in blood glutathione peroxidase (GSHPx)
activity in sheep at pasture in a Mediterranean area. Vet. J. 1999,
157, 186–188.
46. Frei, B. Natural Antioxidants in Human Health and Disease. San
Diego, CA: Academic Press; 1994.
47. Kehrer, J.P., Smith, C.V. Free radicals in biology: sources,
reactivities, and roles in the etiology of human dieseases. In:
Frei B (ed). Natural Antioxidants in Human Health and Diseases.
San Diego, CA: Academic Press, 1994, 25–62.
48. Ueland, P.M., Mansoor, M.A., Guttormsen, A.B., Muller, F.,
Aukrust, P., Refsum, H., Svardal, A.M. Reduced, Oxidized and
Protein-Bound Forms of Homocysteine and Other Aminothiols
in Plasma Comprise the Redox Thiol Status-A Possible Element
of the Extracellular Antioxidant Defense System. J Nutr. 1996, 126,
1281S–4.
49. Di Giuseppe, D., Di Simplicio, P., Capecchi, P.L.,
Lazzerini, P.E., Pasini, F.L. Alteration in the redox state of plasma in
heart-transplant patients with moderate hyperhomocysteinemia.
J. Lab. Clin. Med. 2003, 142, 21–28.
50. Di Giuseppe, D., Frosali, S., Priora, R., Di Simplicio, F.C.,
Buonocore, G., Cellesi, C., Capecchi, P.L., Pasini, F.L.,
Lazzerini, P.E., Jakubowski, H., Di Simplicio, P. The effects of age
and hyperhomocysteinemia on the redox forms of plasma thiols.
J. Lab. Clin. Med. 2004, 144, 235–245.
51. Moran, L.K., Gutteridge, J.M., Quinlan, G.J. Thiols in cellular redox
signalling and control. Curr. Med. Chem. 2001, 8, 763–772.
 240  Pietro Celi
52. Soriani, M., Pietraforte, D., Minetti, M. Antioxidant potential of
anaerobic human plasma: role of serum albumin and thiols as
scavengers of carbon radicals. Arch. Biochem. Biophys. 1994, 312,
180–188.
53. Halliwell, B. Albumin–an important extracellular antioxidant?
Biochem. Pharmacol. 1988, 37, 569–571.
54. Briviba, K., Sies, H. Non-enzymatic antioxidants defence system.
In: B. Frei (ed). Natural antioxidants in human health and disease.
San Diego, CA: Academic Press Ltd., 1994, 107–128.
55. Burk, R.F. Biological activity of selenium. Annu. Rev. Nutr. 1983, 3,
53–70.
56. Liu, S.M., Eady, S.J. Glutathione: its implications for animal health,
meat quality, and health benefits of consumers. Aust J Agric Res.
2005, 56, 775–780.
57. Liu, S.M., Masters, D.G. Quantitative analysis of methionine and
cysteine requirements for wool production of sheep. Anim Sci.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by RMIT University on 09/03/13
2000, 71, 175–185.
58. Hopkins, G.J., Roberts, J., Evans, J.V. Red blood cell concentrations
of reduced glutathione and potassium as biochemical
markers of wool growth in Merino sheep. J Agric Sci. 1975, 84,
481–486.
59. Williams, A.J. Metabolism of cystine by Merino sheep genetically
different in wool production IV. Rates of entry of cystine into
plasma, measured with a single intravenous injection of L-[35S]
cystine, and the subsequent incorporation of 35S into wool fibres.
Aust. J. Biol. Sci. 1976, 29, 513–524.
60. Dröge, W., Schulze-Osthoff, K., Mihm, S., Galter, D., Schenk, H.,
Eck, H.P., Roth, S., Gmünder, H. Functions of glutathione and
glutathione disulfide in immunology and immunopathology.
FASEB J. 1994, 8, 1131–1138.
61. Celi, P., Eppleston, J., Armstrong, A., Watt, B., Selenium
supplementation increases wool growth and reduce faecal egg
For personal use only.
counts of Merino weaners in a selenium deficient area. Anim Prod
Sci. 2010, 50, 688–692.
62. Ghiselli, A., Serafini, M., Natella, F., Scaccini, C. Total antioxidant
capacity as a tool to assess redox status: critical view and
experimental data. Free Radic. Biol. Med. 2000, 29, 1106–1114.
63. Miller, N.J., Rice-Evans, C., Davies, M.J., Gopinathan, V.,
Milner, A. A novel method for measuring antioxidant capacity and
its application to monitoring the antioxidant status in premature
neonates. Clin. Sci. 1993, 84, 407–412.
 Immunopharmacology and Immunotoxicology
64. Ghiselli, A., Serafini, M., Maiani, G., Azzini, E., Ferro-Luzzi, A.
A fluorescence-based method for measuring total plasma
antioxidant capability. Free Radic. Biol. Med. 1995, 18, 29–36.
65. Cao, G., Alessio, H.M., Cutler, R.G. Oxygen-radical absorbance
capacity assay for antioxidants. Free Radic. Biol. Med. 1993, 14,
303–311.
66. Benzie, I.F., Strain, J.J. The ferric reducing ability of plasma (FRAP)
as a measure of “antioxidant power”: the FRAP assay. Anal.
Biochem. 1996, 239, 70–76.
67. Gatellier, P., Mercier, Y., Renerre, M. Effect of diet finishing mode
(pasture or mixed diet) on antioxidant status of Charolais bovine
meat. Meat Sci. 2004, 67, 385–394.
68. Descalzo, A.M., Insani, E.M., Biolatto, A., Sancho, A.M.,
García, P.T., Pensel, N.A., Josifovich, J.A. Influence of pasture or
grain-based diets supplemented with vitamin E on antioxidant/
oxidative balance of Argentine beef. Meat Sci. 2005, 70, 35–44.
69. Ballet, N., Robert, J.C., Williams, P.E.V. Vitamins in forages. In: D.I.
Givens EO, R.F.E. Axford and H.M. Omed (ed). Forage Evaluation in
Ruminant Nutrition. Oxon, UK: CABI Publishing, 2000, 399–431.
70. Bargo, F., Muller, L.D., Delahoy, J.E., Cassidy, T.W. Milk response to
concentrate supplementation of high producing dairy cows grazing
at two pasture allowances. J. Dairy Sci. 2002, 85, 1777–1792.
71. Weiss, W.P., Smith, K.L., Hogan, J.S., Steiner, T.E. Effect of forage
to concentrate ratio on disappearance of vitamins A and E during
in vitro ruminal fermentation. J. Dairy Sci. 1995, 78, 1837–1842.
72. Venditti, P., Di Meo, S. Thyroid hormone-induced oxidative stress.
Cell. Mol. Life Sci. 2006, 63, 414–434.
73. Cao, G., Prior, R.L. Comparison of different analytical methods for
assessing total antioxidant capacity of human serum. Clin. Chem.
1998, 44, 1309–1315.
74. Pregel, P., Bollo, E., Cannizzo, F.T., Biolatti, B., Contato, E.,
Biolatti, P.G. Antioxidant capacity as a reliable marker of stress in
dairy calves transported by road. Vet. Rec. 2005, 156, 53–54.
75. Argüelles, S., García, S., Maldonado, M., Machado, A., Ayala, A. Do
the serum oxidative stress biomarkers provide a reasonable index
of the general oxidative stress status? Biochim. Biophys. Acta 2004,
1674, 251–259.
76. Sharma, R.K., Pasqualotto, F.F., Nelson, D.R., Thomas, A.J. Jr,
Agarwal, A. The reactive oxygen species-total antioxidant capacity
score is a new measure of oxidative stress to predict male infertility.
Hum. Reprod. 1999, 14, 2801–2807.