Content uploaded by Vesna Ilić
Author content
All content in this area was uploaded by Vesna Ilić on May 11, 2022
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tjas20
Italian Journal of Animal Science
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tjas20
Oral supplementation with organically modified
clinoptilolite during prepartum period influences
the redox status of peripheral blood and
colostrum of primiparous dairy cows
Ivana Drvenica, Milica Stojić, Natalija Fratrić, Marijana Kovačić, Jelica Grujić-
Milanović, Branislav Vejnović, Dragana Marković, Dragan Gvozdić & Vesna
Ilić
To cite this article: Ivana Drvenica, Milica Stojić, Natalija Fratrić, Marijana Kovačić, Jelica Grujić-
Milanović, Branislav Vejnović, Dragana Marković, Dragan Gvozdić & Vesna Ilić (2022) Oral
supplementation with organically modified clinoptilolite during prepartum period influences the
redox status of peripheral blood and colostrum of primiparous dairy cows, Italian Journal of Animal
Science, 21:1, 847-858, DOI: 10.1080/1828051X.2022.2070034
To link to this article: https://doi.org/10.1080/1828051X.2022.2070034
© 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Published online: 04 May 2022. Submit your article to this journal
View related articles View Crossmark data
PAPER
Oral supplementation with organically modified clinoptilolite during
prepartum period influences the redox status of peripheral blood and
colostrum of primiparous dairy cows
Ivana Drvenica
a
, Milica Stoji
c
b
, Natalija Fratri
c
b
, Marijana Kova
ci
c
a
, Jelica Gruji
c-Milanovi
c
c
,
Branislav Vejnovi
c
d
, Dragana Markovi
c
a
, Dragan Gvozdi
c
e
and Vesna Ili
c
a
a
Grupa za imunologiju, Institute for Medical Research, National Institute of Republic of Serbia University of Belgrade, Beograd, Serbia;
b
Katedra za fiziologiju i biohemiju, Faculty of Veterinary Medicine, University of Belgrade, Beograd, Serbia;
c
Grupa za
kardiovaskularnu fiziologiju, Institute for Medical Research, National Institute of Republic of Serbia University of Belgrade, Beograd,
Serbia;
d
Katedra za ekonomiku i statistiku, Faculty of Veterinary Medicine, University of Belgrade, Beograd, Serbia;
e
Katedra za
patofiziologiju, Faculty of Veterinary Medicine, University of Belgrade, Beograd, Serbia
ABSTRACT
Redox imbalance in peripartum period influences health of dairy cows and their foetus and,
through the colostrum, health of new-born calves. Oxidative stress in cattle can be suppressed
by dietary supplementation with natural minerals, and we investigated the effect of supplemen-
tation with organically modified clinoptilolite on redox status parameters in healthy pregnant
primiparous dairy cows. Holstein cows were randomly assigned to receive daily oral drenching,
with 1 L of water containing either 0 g/L (n¼14; control group) or 150 g/L of clinoptilolite
(n¼17; supplemented group). Treatment lasted from 24 ± 4 days prior to parturition until 2 days
postpartum (pp). Blood samples were collected on days 24 ± 4 (–24 D) and 4± 2 (–4 D) prior to
parturition and on days 1 (þ1 D), 2 (þ2 D), and 7 (þ7 D) pp, and colostrum were collected at 2,
12, 24 and 36 h pp. Total antioxidant capacity, lipid peroxides, and advanced oxidation protein
products (AOPP) levels were determined in peripheral blood plasma, erythrocytes, and colostrum
whey. The concentration of antioxidants in the peripheral blood of supplemented cows was
increased by 41% and 19% on (þ2D) and (þ7 D), respectively, while the concentration of lipid
peroxides on (þ7 D) was lowered by 57% compared with the control group. In addition, this
supplementation increased erythrocyte AOPP level on (–4 D) 61%) and colostral lipid peroxides
level (90%) at 24 h pp. The results of this study showed that applied short-term supplementation
with clinoptilolite influences redox homeostasis and may contribute to effective adaptation of
primiparous cows to redox imbalance in the peripartum period.
HIGHLIGHTS
Short-term dietary supplementation with clinoptilolite in the prepartum period modulates
redox homeostasis of the dairy cows’blood plasma.
Short-term dietary supplementation with clinoptilolite contributes to adaptation of dairy
cows to redox imbalance in the peripartum period.
Short-term dietary supplementation with clinoptilolite increase the level of lipid peroxides in
colostrum of dairy cows.
ARTICLE HISTORY
Received 17 January 2022
Revised 24 March 2022
Accepted 20 April 2022
KEYWORDS
Advanced oxidation protein
products; antioxidants;
colostral whey; erythrocytes;
lipid peroxidation
Introduction
Various environmental, physiological, and dietary con-
ditions can contribute to the exacerbation of oxida-
tive stress in high-producing dairy cows, already
intrinsically susceptible to oxidative stress (V
azquez-
A~
n
on et al. 2008). Among known physiological fac-
tors, the periparturient or transition period (defined
as the period from 3 weeks before calving until
3 weeks after calving) is considered critical for dairy
cows’health (Gitto et al. 2002, Sordillo and Aitken,
2009; Sharma et al. 2011; Abuelo et al. 2019). In this
period, dairy cows are more prone to infection and
metabolic diseases (mastitis, metritis, ketosis, digest-
ive disorders, displaced abomasum, lameness) than in
the period of peak or late lactation (Sordillo et al.
CONTACT Vesna Ili
cvesnai@imi.bg.ac.rs
Supplemental data for this article can be accessed here.
ß2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ITALIAN JOURNAL OF ANIMAL SCIENCE
2022, VOL. 21, NO. 1, 847–858
https://doi.org/10.1080/1828051X.2022.2070034
2007; Sordillo and Aitken, 2009), due to impaired
redox balance, or the so-called ‘Oxygen Paradox’
(Gitto et al. 2002; Sordillo and Aitken, 2009; Sharma
et al. 2011; Abuelo et al. 2019). Namely, during this
transition phase cows have substantial energy
requirements due to foetal growth, rapid differenti-
ation of secretory parenchyma and mammary gland
growth, and colostrum and milk synthesis and secre-
tion (Gitto et al. 2002). To meet the energy demands,
cows mobilise reserves predominately from adipose
tissues, and the increased lipid mobilisation increases
the generation of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) (Gitto et al. 2002).
ROS and RNS are essential signalling molecules
involved in the regulation of normal, physiological,
immune response. However, an imbalance in their
productions might change antioxidative capacity and
allow pro-oxidative mediators to induce an inflamma-
tory reaction, tissue damage, and immune dysfunc-
tions responsible for numerous diseases occurring
within 1 month after calving (Abuelo et al. 2019).
The calves experiencing oxidative stress in utero
are shown to have compromised immune response
and higher level of basal inflammation (Abuelo et al.
2019). Calves also produce ROS intrinsically, and even
before the ingestion of colostrum, their serum ROS
level is higher than in their dams (birth-associated
oxidative stress phenomenon; Ga
al et al, 2006;
Abuelo et al. 2014). The colostrum contains numer-
ous enzymatic and non-enzymatic antioxidants
(Przybylska et al. 2007), and the colostrum antioxi-
dant capacity, lipid peroxidation, and protein
SH-group level increase in time (Kankofer and Lipko-
Przybylska, 2008; Albera and Kankofer, 2011). The col-
ostrum antioxidant activity correlated negatively with
ROS level, but a positive correlation is found with
serum IgG in new-born calves 2 h after colostrum
ingestion (Abuelo et al. 2014). Besides antioxidants,
colostrum also contains prooxidants. Namely, colostral
ROS is an essential component of anti-bacterial
defence in the intestine of calves (Przybylska et al.
2007). However, in some pathological conditions, a
high ROS concentration (ROS originating from colos-
trum as well as intrinsic calves’ROS) outbalance
calves antioxidant capacity leading to oxidative stress,
immune dysfunctions, and onset of diarrhoea and
pneumonia (Abuelo 2019).
According to EU Regulation No 651/2013 (2013)
natural zeolite, clinoptilolite has been authorised for
the use as an additive in feedstuffs for farm animals.
Clinoptilolite improves production performance, act-
ing as ammonia-, heavy metal-, and mycotoxin-
binding adsorbent, as an antioxidant, and anti-diar-
rheic and growth-promoting agent (Papaioannou
et al. 2004; Katsoulos et al. 2006; Dakovi
c et al. 2007;
Safameher, 2008; Pourliotis et al. 2012; Karatzia et al.
2013; Wu et al. 2015). There are also data indicating
its role in the prevention of metabolic diseases in
dairy cows (milk fever, ketosis) and positive effects
on general health (Papaioannou et al. 2005; Katsoulos
et al. 2006) and milk production (Karatzia et al.
2013). Although zeolites might act as antioxidants,
limited and contradictory data are available on the
effect of zeolites on the redox status in calves.
Yarovan (2008) measured the concentration of malon-
dialdehyde (MDA), conjugated dienes, keto dienes
and ceruloplasmin (CP) in the plasma of dairy cows
and showed that natural zeolite (Khotynets) has a
positive effect on the oxidant status. However, Ipek
et al. (2012) showed that although supplementation
with natural zeolite (Nat Min 9000
V
R
) in the period
from 2 to 4 months after calving decreased the lipid
hydroperoxides concentration, it did not further
strengthen the antioxidant defence system in healthy
dairy cows. Besides, Kerwin et al. (2019) showed that
3-week-long prepartum supplementation of cows in
the second and greater lactation with synthetic zeo-
lite A (X-Zelit) did not modulate the concentration of
reactive oxygen and nitrogen species and antioxidant
capacity of peripheral blood plasma. Although all
these studies showed that zeolite/clinoptilolite influ-
ence cows’redox status, the results are difficult to
compare directly because of differences in (1) zeolite/
clinoptilolite forms and concentrations, (2) age of
cows (3) timing and duration of treatment, (4) ana-
lysed biomarkers, and (5) applied analytical methods.
We have already shown that supplementation with
organically modified clinoptilolite (patent of Milosevic
and Tomasevic-Canovic 2003,2009) improved the
quality of colostrum of primiparous dairy cows by
increasing the percentage of fat and proteins and con-
centration and mass of IgG, with no adverse effects on
the cows’energy status, protein, lipid, and mineral
metabolism (Stoji
c et al. 2020). Since the colostrum
redox balance affects passive immune transfer, Abuelo
et al. (2014) proposed an examination of the oxidative
profile of colostrum as a parameter of colos-
trum quality.
This study assessed whether the supplementation
affects the redox status of primiparous cows’periph-
eral blood and colostrum by measuring total antioxi-
dant capacity, lipid peroxidation levels, and advanced
oxidation protein products.
848 I. DRVENICA ET AL.
Material and methods
Experimental design
The total number of 31 healthy pregnant Holstein
primiparous dairy cows, grown at a tie stall barn farm
(Padinska Skela, AL Dahra, Belgrade, Serbia), were ran-
domly selected 30 days before the expected calving
term and placed in a separate facility in which they
were kept under the same hygienic and dietetic condi-
tions. According to the National Regulation on Animal
Welfare, the Ethical Committee of the Faculty of
Veterinary Medicine, University of Belgrade, approved
the study on animals (the ethical approval code num-
ber: 01-19/10). Investigations on cows were enrolled at
the same season, from September to November.
On the farm where the study was performed cows
were artificially synchronised and inseminated.
Deworming program was performed at least 60 days
before calving, and vaccination against clostridial dis-
eases with Bravoxin (MSD Animal Health, EU) was also
performed. All cows were, on regular basis, underwent
a general clinical examination (determination of body
temperature, pulse rate, respiration, and rumen con-
traction) by an authorised veterinarian on the farm.
Before the start of the experiment, 25 days before the
expected calving term, cows included in the study
were randomly selected from the farm ‘pool’of
healthy primiparous cows, and then placed in a separ-
ate facility where they kept under the same hygienic
and dietetic conditions. These cows were in good gen-
eral health condition without any obvious clinical
signs of disease, in optimal body condition score, and
aged 23 ± 2 months. During the experiment it was per-
formed regular clinical examination and taking the
blood samples for determination the parameters of
metabolic profile. The results are given as
Supplementary Material Table S11. Routine mastitis
diagnostics was completed following calving and cows
included in this study had no mastitis.
Cows received identical diets as TMR to allow ad
libitum feed intake throughout the study. Twenty-five
days before the expected time of parturition, cows
were assigned to the close-up diet (NE
L
¼1.60 Mcal/
kg; Table S1; Supplementary material). The lactation
diet (NE
L
¼1.71 Mcal/kg; Table S1) was provided for
all animals postpartum until day 30 of lactation. All
offered diets were initially formulated to either meet
or exceed the NRC (2001) requirements. The feed was
offered in two equal portions at 7 and 18 h, and cows
had free access to water throughout the study. All ani-
mals were randomly assigned to two experimental
groups: (1) treated group, T; (n¼17) received oral
supplementation of organically modified clinoptilolite
(Minazel Plus
V
R
, Patent Co., Serbia; Milosevic and
Tomasevic-Canovic 2003,2009) 150 g/animal/day in 1 L
of drinking water, starting at 24 ± 4 days before the
expected calving term up to 2 days after calving, and
(2) control group, C; (n¼14) received, instead of the
clinoptilolite suspension, only 1 L water. Clinoptilolite
suspension was administered by a glass bottle and
given orally in one portion, 2 h after the morn-
ing meal.
Blood samples collection and isolation of blood
plasma and erythrocytes
Blood samples were collected on days 24 ± 4 (–24 D)
and 4 ± 2 (–4 D) before calving, and on days 1 (þ1 D),
2(þ2 D), and 7 (þ7 D) postpartum (pp) by jugular
venepuncture into sterile, plastic, vacutainer tubes
with sodium citrate as an anticoagulant. The first
blood sample (–24 D) was taken before adding the
supplement. Blood components were separated by
centrifugation at 2450 g for 25 min within 2 h of
blood collection. Plasma was collected, aliquoted, and
stored at 20 C until the use. Leucocytes were dis-
carded by vacuum aspiration, and the remaining pel-
leted erythrocytes were resuspended in isotonic
(0.9% w/v) saline solution, three times washed via
centrifugation, aliquoted, and stored at 20 C until
the use.
Collection of colostrum and isolation of
colostral whey
Colostrum was collected 2–3, 12, 24 and 36 h pp and
labelled as (2 h), (12 h), (24 h), and (36 h) representing
1st, 2nd, 3rd, and 4th milking, respectively. Colostrum
samples (200 mL) were immediately frozen and stored
at 20 C until use. Just before the analysis the colos-
trum samples were thawed in a warm water bath
(37 C), brought up to room temperature (20 C), cen-
trifuged (800 g, 20 min), and the fatty layer (formed
as a result the freezing induced partial deemulsifica-
tion of colostrum) was removed by vacuum aspiration.
Casein was precipitated by adding seven drops
(approximately 175 lL) of calf rennet to 10 ml of the
supernatant. After the 30 min incubation at 37 C, sam-
ples were centrifuged (15 min, 3000 g), and super-
natant (colostral whey) was aliquoted and stored at
20 C until the use for biochemical analysis.
ITALIAN JOURNAL OF ANIMAL SCIENCE 849
Analysis of colostral whey, blood plasma and
erythrocyte lysates
Colostral whey and blood plasma total protein con-
centration was determined by the biuret method
(Doumas et al. 1981). Colostral whey proteins were
separated by agarose gel electrophoresis and the c
globulin level was determined as described in Stoji
c
et al. (2017). The cyanmethemoglobin method (Zwart
et al. 1996) was used to determine haemoglobin con-
centration in erythrocyte lysates after thawing.
The total antioxidant capacity of colostral whey,
blood plasma, and erythrocyte was measured by fer-
ric reducing antioxidant power (FRAP) colorimetric
assay (Benzie and Strain, 1999). The essay was per-
formed with undiluted blood plasma and colostral
whey and 48 times diluted (in 0.9% NaCl) erythro-
cyte lysates.
Lipid peroxidation in blood plasma, erythrocytes,
and colostral whey was estimated by thiobarbituric
acid reactive species (TBARS) assay (Ohkawa et al.
1979). This assay is based on the reaction of malon-
dialdehyde (MDA), as a lipid peroxidation marker, with
thiobarbituric acid (TBA). TBA reacts with MDA to form
a pink chromogen, which can be detected spectro-
photometrically at 530 nm. The modification was
made in sample/TCA volume ratio. For plasma: the
sample in a volume of 300 mL was mixed with 100 mL
28% TCA. For colostral sera: the sample in a volume of
100 mL was mixed with 1000 mL 28% TCA. For erythro-
cyte: erythrocyte lysates were 48 times diluted in 0.9%
NaCl, and 100 mL of the diluted sample was mixed
with 500 mL 28% TCA. The absorption of TBARS was
measured at 530 nm (OD
530
). A standard curve was
constructed by plotting the MDA standards against
OD
530
. MDA standard was prepared by the acid
hydrolysis of 1,1,3,3-tetramethoxypropane (malonalde-
hyde bis(dimethyl acetal)), purchased from
Sigma Aldrich.
Oxidative damage of proteins was determined by
measuring advanced oxidation protein products
(AOPP) in an acidic condition in the presence of potas-
sium iodide, following the method of Selmeci et al.
(2005), with minor modifications. Briefly, 20 ll sample
was transferred (in duplicate) to wells in a 96-well
microplate, followed by the addition of 180 ll 0.2 M
citric acid, and potassium iodide (10 ll 1.19 M). After
10 min on a microplate shaker, the absorbance of
AOPP was read at 340 nm. The calibration curve was
constructed with chloramine - T.
The values of examined parameters in erythrocytes
were normalised to the haemoglobin content (Figure
S1, Supplementary Material).
Statistical analysis
Statistical analysis of the results obtained in the
experiment was carried out using statistical software
GraphPad Prism version 6 (GraphPad, San Diego, CA,
USA). Untransformed data are presented in Figures
and Supplementary material’s Tables. Given that some
data were not normally distributed (Shapiro–Wilk nor-
mality test p<0.05), several transformations of data
have been attempted until adequate transformation
was found. For the data of colostral whey following
transformations were applied: y¼xþ2 for FRAP,
TBARS, and AOPP/proteins; y¼xþ50 for AAOP and
Proteins; In blood plasma: y¼xþ2 for TBARS and
AOPP/proteins, and y¼xþ50 for AOPP; In RBC:
y¼xþ2 for TBARS/gHb and AOPP/gHb. After transfor-
mations all data where normally distributed
(Shapiro–Wilk normality test p>0.05). Groups were
compared using two-way ANOVA with repeated meas-
ures in one factor (time of sampling) followed by
Tukey’s test within groups over sampling time and
Sidak’s test between groups through each sam-
pling time.
Correlation between analysed redox status parame-
ters was tested, and Pearson product–moment correl-
ation coefficients (r) were calculated using the above-
mentioned software. Differences with p-values of <
0.05 were considered significant.
Results
Effect of the oral supplementation with
organically modified clinoptilolite on redox status
parameters of peripheral blood plasma and
erythrocytes of primiparous dairy cows
Time-course changes in the blood plasma total anti-
oxidant capacity, lipid peroxides, and AOPP in control
and the clinoptilolite supplemented dairy cows, from
(–24 D) to (þ7 D) is shown in Figure 1and Tables S2
and S3 (Supplementary Material).
The concentration of total antioxidant capacity
detected by FRAP method in the blood plasma of con-
trol animals fluctuated, reaching their maximum and
minimum levels on (þ1 D) and (þ2 D), respectively. In
the blood plasma of cows supplemented with clinopti-
lolite, total antioxidant capacity gradually increased
until the (þ2 D) and then remained unchanged until
the end of the experiment. A significant increase in
the total antioxidant capacity in blood plasma of sup-
plemented cows, compared to healthy ones, was
detected on (þ2 D) and (þ7 D) (41% and 19%,
respectively).
850 I. DRVENICA ET AL.
In the blood plasma of control group of cows, the
TBARS concentration remained unchanged from
(–24 D) to (þ2 D), but increased on (þ7 D). In the sup-
plemented group, the TBARS level was unchanged
during the experimental period. The significant differ-
ence in blood plasma TBARS level between groups
was detected on (þ7 D), when the TBARS level in the
treated group was 57% lower than in the con-
trol group.
The concentration of proteins in the blood plasma
of control cows varied over time. It stayed unchanged
from (–24 D) to (þ2 D), after which it grew until
(þ7 D). The concentration of proteins in the blood
plasma of the clinoptilolite supplemented cows
remained unchanged throughout the experimental
period. The statistical analysis did not reveal a signifi-
cant effect of this supplementation on the concentra-
tion of blood plasma protein.
Figure 1. Effect of the oral supplementation with organically modified clinoptilolite during prepartum period on the level of redox
status parameters of peripheral blood plasma of primiparous dairy cows. C –Control cows (solid line); T –Clinoptilolite supple-
mented cows (dashed line); (–24 D) and (–4D)–days 24 ± 4 and 4 ± 2 prior to parturition; (þ1 D), (þ2 D), (þ7D)–days 1, 2,
and 7 postpartum. The concentration of antioxidants expressed as mM equivalent to FeSO
4
7H
2
O; TBARS: thiobarbituric acid
reactive species; the concentration of lipid peroxides expressed as nM equivalent to malondialdehyde (MDA); AOPP: advanced oxi-
dation protein products; ()–p<0.001.
ITALIAN JOURNAL OF ANIMAL SCIENCE 851
The AOPP level (expressed either as mmol/L or
mmol/g protein) in blood plasma of both, control and
supplemented group of cows was constantly decreas-
ing from (–24 D) to (þ1 D), stayed unchanged from
(þ1D) to (þ2 D), and increased on (þ7 D). A significant
difference in the AOPP level between control and sup-
plemented groups was not found.
The total antioxidant capacity, lipid peroxides and
AOPP in erythrocyte of clinoptilolite treated and con-
trol cows in the period from (–24 D) to (þ7 D) was
shown in Figure 2and Tables S4 and S5
(Supplementary Material).
The total antioxidant capacity in the erythrocyte
lysates of the control group did not change from
(–24 D) to (–4 D), increased on (þ1 D), stayed
unchanged till (þ2 D), and then decreased on (þ7 D).
In the erythrocytes of clinoptilolite supplemented
cows, the increase in the antioxidant level was already
detected on (–4 D) and the upward trend continued
until (þ2 D). As in the control group, in the treated
group a decrease in the total antioxidant capacity was
registered on (þ7 D). A significant difference in the
erythrocytes’antioxidants status of the control and
the supplemented group was not found.
The TBARS level stayed unchanged during the
experimental period in both clinoptilolite supple-
mented and control group. Also, TBARS assay did not
reveal any difference in the erythrocytes’lipid perox-
ides level between the groups.
In the control group, the erythrocyte AOPP level
stayed unchanged during the examination period. In
the clinoptilolite supplemented group the erythrocyte
AOPP level increased on (–4 D), and at this point it
was 61% higher than in the control group.
A significant correlation between total antioxidant
capacity and lipid peroxides or AOPP level in periph-
eral blood plasma or erythrocytes was not found in
any group of cows (Tables S6,
Supplementary Material).
Effect of the supplementation with organically
modified clinoptilolite on redox status parameters
of colostral whey of primiparous dairy cows
First milking colostrum (2 h) of control groups of cows
contained 129 ± 30 g/L IgG and the colostrum of sup-
plemented group contained 152 ± 33 g/L IgG (Figure
S2, Supplementary Material). Both groups of cows pro-
duced high quality colostrum (>50 g/L IgG; McGuirk
and Collins 2004) and the effect of difference in the
colostrum quality on results of our study was not
expected. Redox status indices were analysed in the
colostrum whey (Figure S2, Supplementary Material)
whose concentration of cglobulin (predominately
IgG) almost perfectly correlated with the concentration
IgG in whole colostrum IgG (Table S7,
Supplementary Material).
The results of the analysis of the effect of the cli-
noptilolite oral supplementation on redox status
Figure 2. Effect of the oral supplementation with organically
modified clinoptilolite during prepartum period on the level of
redox status parameters of erythrocytes of primiparous dairy
cows. C –Control cows (solid line); T –clinoptilolite supple-
mented cows (dashed line); (–24 D) and (–4D)–days 24 ± 4
and 4 ± 2 prior to parturition; (þ1 D), (þ2D),(þ7D)–days
1, 2, and 7 postpartum. The concentration of antioxidants
expressed as mM equivalent to FeSO
4
7H
2
O; TBARS: thio-
barbituric acid reactive species; the concentration of lipid per-
oxides expressed as nM equivalent to malondialdehyde (MDA);
AOPP: advanced oxidation protein products; ()–p<0.01.
852 I. DRVENICA ET AL.
parameters of colostral whey of dairy cows are given
in Figure 3, and Tables S8 and S9
(Supplementary Material).
The results show that colostral sera of the control
cows revealed stable total antioxidant capacity during
the experimental period. In colostral sera of clinoptilo-
lite supplemented cows, total antioxidant capacity
increased significantly from second milking (12 h) to
third milking (24 h) and then decreased reaching at
fourth milking (36 h) the same concentration as in the
second milking. A difference in the colostral whey
total antioxidant capacity, between control and
treated cows was not significant.
In both groups of cows, the TBARS level (nmol/L)
was the highest at first milking and continuously
decreased up to fourth milking. In addition, it was
found that the (24 h) colostrum of supplemented cows
contained significantly more (90%) lipid peroxides
then the control colostrum. The decrease in the colos-
tral whey TBARS level followed the trend of
Figure 3. Effect of the oral supplementation with organically modified clinoptilolite during prepartum period on the level of redox
status parameters of colostral whey of primiparous dairy cows. C –Control cows (solid line); T –clinoptilolite supplemented cows
(dashed line); Colostrum was collected 2–3, 12, 24 and 36 h pp and labelled as (2 h), (12h), (24 h), and (36 h). The concentration
of antioxidants expressed as mM equivalent to FeSO
4
7H
2
O; TBARS: thiobarbituric acid reactive species; The concentration of
lipid peroxides expressed as nM equivalent to malondialdehyde (MDA); AOPP: advanced oxidation protein products; ()
–p<0.05.
ITALIAN JOURNAL OF ANIMAL SCIENCE 853
decreasing in the colostrum fat content (Figure S3,
Supplementary Material), and to avoid a misinterpret-
ation of TBARS level measurement, TBARS to colos-
trum lipid ratio was calculated. The data also showed
that colostrum of supplemented cows contained sig-
nificantly more lipid peroxides then the control colos-
trum (65%, as expressed as mmol TBARS/% fat).
The concentration of proteins in colostral whey of
both groups of cows decreased from first to fourth
milking, but the protein concentration in the second
colostrum of supplemented cows was 43% higher
than in control cows. In the colostral whey of control
cows, the concentration of AOPP (lmol/L) decreased
from first (2 h) to second (24 h) milking and stayed
unchanged till forth milking (36 h). On the other hand,
in the control group AOPP level appeared to increase
at (36 h). When expressed as AOPP/protein ratio, it
was shown that AOPP level was significantly higher in
the fourth colostrum of both examined groups of
cows. A significant disparity in AOPP level between
these two groups of cows was not found.
A significant correlation between analysed colostral
whey redox indices was not found in either the con-
trol or the supplemented group Table S10
(Supplementary Material).
Discussion
It has been shown that dietary mineral supplementa-
tion has a vital role in the oxidative/antioxidant bal-
ance of dairy cows and their productive and
reproductive performances (V
azquez-A~
n
on et al. 2008).
In the last few decades, natural zeolite (clinoptilolite),
as an additive to feed, has been applied successfully
in animal breeding for different purposes. The study
conducted by Karatzia et al. (2013) has shown that the
dietary administration of 200 g per day of a natural
zeolite, clinoptilolite, during the last 2 months of preg-
nancy and the subsequent lactation improves the
energy status and the reproductive performance of
dairy cows in their first lactation (Karatzia et al. 2013).
We have recently shown that organically modified oral
clinoptilolite supplementation at 150 g/day signifi-
cantly increases the IgG concentration in colostrum
and has no adverse effects on the energy status, pro-
tein, lipid, and mineral metabolism in primiparous
dairy cattle during the prepartum period (Stoji
c et al.
2020). Although there are several reports on natural
zeolites’antioxidant properties in vitro and in vivo
(Zarkovic et al. 2003; Dogliotti et al. 2012; Montinaro
et al. 2013), data on such effects on peripheral blood
and colostrum of dairy cows during prepartum period
are still scarce.
In this work, we have studied the effect of orally
administrated organically modified clinoptilolite on the
total antioxidative capacity of blood plasma, erythro-
cytes and colostral whey of primiparous dairy cows by
FRAP method, starting from 24 ± 4 days before the
expected calving term up to 2 days pp. Since correctly
evaluating oxidative stress needed determination of
both pro-oxidants and antioxidants (Kerwin et al.
2019), we have also examined the level of lipid perox-
ides by the TBARS method and level of AOPP during
the same investigation period. Calving itself also
causes temporary but significant changes in the anti-
oxidant system of cow blood (Albera and Kankofer,
2011). Thus, it was meaningful to investigate whether
clinoptilolite possesses antioxidant effects on periph-
eral blood and colostrum before and after calving
since antioxidant supplementation only had an out-
come when the nutritional/oxidant status is deficient
(Ipek et al. 2012). Otherwise, healthy cows’biological
antioxidant system is effective enough to combat
these transient changes of redox homeostasis, as dem-
onstrated in the study of Ipek et al. (2012). Ipek et al.
(2012) examined 60 days’influence of 2.5% zeolite
supplementation on oxidant/antioxidant status in
3–4 years old healthy non-pregnant Holstein dairy
cows in the first period of lactation (2 months after
calving), and showed no changes of oxidative and
antioxidant indicators in blood plasma, except for lipid
hydroperoxides that were lowered. Recently, Kerwin
et al. (2019) reported that feeding synthetic zeolite
during the prepartum period of multiparous Holstein
cows effectively improves serum calcium status during
the postpartum period.
Our results obtained in the control group of ani-
mals indicated changes of antioxidant status in blood
plasma by calving itself. Our findings demonstrated
significantly higher FRAP activities in plasma samples
7 days after calving than in samples obtained 20 days
before calving. These results followed study of
Mudro
n et al. (1999) but were opposed to the results
of Ga
al et al. (2006), who found no changes in FRAP
activities before and several days after calving.
Furthermore, our results were in contrast to Albera
and Kankofer (2011) findings, showing that during the
first 24 h after parturition, the antioxidant capacity of
colostrum measured by FRAP (correspond to 3rd milk-
ing in our study) was significantly lower in comparison
to the antioxidant capacity value in blood plasma. Our
study showed that supplementation with organically
modified clinoptilolite induced changes in antioxidant
854 I. DRVENICA ET AL.
capacity of blood plasma of primiparous dairy cows.
The differences in supplemented animals were
revealed on days 2 and 7 after parturition, with an
increase in FRAP activities of the blood plasma of 41%
and 14%, respectively, compared to control animals.
The concentration of specific antioxidants, total anti-
oxidant capacity, the levels of oxidative damages of
lipid and proteins in peripheral blood, colostrum, and
milk, are significantly dependent on the age of cows
(Albera and Kankofer, 2010; Puppel et al. 2012). We
believe that the discrepancy between the results
obtained in our study and some other reports could
be at least partially explained by variations in the
experimental setup: use of different zeolites, distinct
duration time of supplementation, and research on
cows in different stage of lactation or age.
AOPP are well-known biomarkers of the oxidative
modification of proteins, where the plasma proteins
are the key targets (Witko-Sarsat et al. 1996).
Nevertheless, this study revealed no differences
between AOPP levels in the blood plasma of the con-
trol and treated animals, while the AOPP level in the
erythrocytes of supplemented animals was increased 5
days before calving (on the twentieth day after the
start of the supplementation with clinoptilolite). Such
results could be explained by the relatively short half-
life of proteins in the peripheral blood (half-life of
serum albumin: 2–3 weeks, transferrin: 9 days; prealbu-
min 2: days; C reactive protein: 4–6 h, etc. (Tariq and
Morley, 2004); half-life of bovine IgG is up to 4 weeks;
Murphy et al. 2014) as opposed to the 130 days
erythrocyte lifespan (Wood and Quiroz-Rocha, 2010)
and their cell properties. Namely, in the circulation
there are several populations of erythrocytes of differ-
ent ages, and as the cells without a nucleus, erythro-
cytes are not able to respond to some environmental
changes by protein synthesis de novo.
An increase of thiobarbituric acid reactive substan-
ces (TBARS) plasma levels may be considered as a sign
of cellular lipid oxidation and used as a marker of oxi-
dative status (Da Silva et al. 2013). The result of TBARS
analysis of blood plasma of primiparous dairy cows
supplemented with organically modified clinoptilolite
in our study indicated its positive effect on lipid per-
oxidation level 7 days after calving, since the level of
measured MDA was 40% lower in the treated group in
comparison to the control group. Our results are con-
sistent with a study of Yarovan (2008), who measured
the level of MDA and some others oxidative markers
and reported that zeolite has a positive effect on the
oxidant status in the blood plasma of dairy cows.
The physiological settings of periparturient period
also reflect on the colostrum’s redox balance, the neo-
nates’first source of nutrients and immunity. Apart
from nutrient and immunological components, colos-
trum possesses antioxidative systems necessary for the
neonate’s protection and ROS originating from easily
oxidised macromolecules, lipids, or proteins. This study
demonstrated that only at the third milking (24 h pp)
difference in colostral whey antioxidant capacity
between control and zeolite treated animals (33%
higher) was shown to be close to statistical significance
(p¼0.06) Although the literature provides data that
suggest an increase of antioxidants in bovine colostrum
over time, with the highest increase at 36 h pp (4rd
milking in our study) (Kankofer and Lipko-Przybylska,
2008; Albera and Kankofer, 2011), our results did not
show such trend in colostrum samples of control ani-
mals. This discrepancy could be due to the different
approaches in results expression. Proteins, which con-
centration indisputably decreases in colostrum over
time (Stoji
c et al. 2020), contribute to the total antioxi-
dant capacity. Although other authors expressed total
antioxidant capacity as mM per gram of proteins, we
did not. We think that such expression of the results
does not show the proper physiological state but falsely
displays colostrum’s timely unchanged antioxidant cap-
acity. The discrepancy detected in studies on the use of
natural clinoptilolite and other zeolites as antioxidant
agents in diverse animals (dogs, cats, horses, pigs,
poultry, small and large ruminants) might be attributed
to the various factors such the type of zeolite tested,
particle size, pre-treatment and the amounts that were
used in these studies (Valpoti
c et al. 2017). MDA levels
in colostral sera of both groups of cows were continu-
ously decreasing from first to fourth milking. The
decreasing MDA level followed the decreasing trend of
the fat level in colostrum, and we assumed that the
time-dependent change of MDA levels in colostral sera
was a consequence of the changes in the content of fat
in colostrum. Interestingly, lipid peroxide analysis of
colostral whey revealed MDA level in zeolite treated
group 90% higher than MDA level in control animals on
third milking. Although these results indicate a higher
level of lipid peroxides in the third milking colostrum of
treated animals, results of increased total antioxidant
capacity measured by FRAP in the same colostrum sam-
ples indicate a positive effect of zeolite on the redox
status of colostrum. The lipid peroxides act as immuno-
modulators capable to activate neutrophils, dendritic
cells and macrophages (Girotti, 1998; Mushenkova et al.
2021; Zhivaki and Kagan, 2021) and in this way to acti-
vate both innate and adaptive immune response. Their
ITALIAN JOURNAL OF ANIMAL SCIENCE 855
presence in colostrum might be beneficial because acti-
vated macrophages and neutrophils use ROS generat-
ing systems to kill bacteria (Albera and Kankofer, 2011).
In this way, colostral lipid peroxides may help overcome
poorly developed antioxidative defence mechanisms of
neonate’s calves (Przybylska et al. 2007). If the observed
redox status of colostrum in zeolite-treated animals
could be of importance for IgG absorption in new-born
calves (as proposed by Abuelo et al. 2014) remained to
be elucidated.
The exact mechanisms of zeolite’s antioxidant
effects are not well known. Some evidence suggests
that zeolite act via (1) removal of toxins from the gut,
(2) improvement of the immune system through the
mucosal-related intestinal lymphoid tissue, (3) increase
in the bioavailability of minerals that are essential co-
factors for some enzymes, and (4) arrest of generation
of peroxides and free radicals (Hossein Nia et al. 2018).
In addition, it has been proposed that antioxidant
properties of zeolites are attributed to their effect on
macrophages’phagocytic function triggered after
phagocytosis of zeolite particles, subsequently leading
to the production of cytokines such as tumour necro-
sis factor-a, which stimulates immunologic responses
and also increases the expression of superoxide dis-
mutase (Zarkovic et al. 2003).
While speculation on the action mechanism of used
organically modified clinoptilolite merits further investi-
gation, it is indicative that the impacts on total antioxi-
dative capacity and lipid peroxidation of blood plasma
and colostrum by this agent exist, even after short-
term supplementation. In this study, we do not
observe significant effects on erythrocyte redox status
after short-term supplementation with zeolite. To
examine the clinoptilolite’s effect on the erythrocyte
redox status, in future studies it will be necessary to
include longer (several months) supplementation.
Overall, this study showed that organically modified cli-
noptilolite positively modulates the redox status of col-
ostrum and blood plasma of primiparous dairy cows
during adaptation for parturition. These data allow the
recommendation for use of organically modified cli-
noptilolite as an agent for preventing the development
of oxidative stress during some physiological or
unfavourable housing and feeding conditions.
Conclusions
In this study, we showed that short-term oral supple-
mentation of primiparous dairy cows with 150 g per
day of organically modified clinoptilolite during the
prepartum period modulate their redox homeostasis
by increasing total antioxidants and decreasing lipid
peroxides level in the peripheral blood plasma. This
result, together with result of our previous study
showing that this supplementation had no adverse
effects on the cows’energy status, protein, lipid, and
mineral metabolism, indicates that this clinoptilolite
has positive effect on adaptation of primiparous cows
to redox imbalance in the peripartum period.
This study also showed that organically modified cli-
noptilolite supplementation resulted in the secretion of
colostrum with increased level of lipid peroxides. This
result, together with result of our previous study show-
ing that organically modified clinoptilolite increase con-
centration of colostral IgG, indicates that this mode of
supplementation of cows might lead to more efficient
passive immune protection of new-born calf.
Author contributions
The study conception and design: N.F., V.I. and I.D.
Material preparation, data collection and analysis: M.S.,
I.D., M.K., and J.G.M. Statistical analysis: BV. The first
draft of the manuscript was written by V.I. and I.D.
Critical analysis of the entire study and manuscript:
D.M., J.G.M. and D.G. All authors have read and agreed
to the published version of the manuscript.
Disclosure statement
None of the authors has a financial or personal relationship
with other people or organisations that could inappropri-
ately influence this publication.
Ethical approval statement
This study has been approved by Ethical Committee of the
Faculty of Veterinary Medicine, University of Belgrade [01-
19/10].
Funding
This work was supported by the Ministry of Education,
Science and Technological Development of Republic of
Serbia [contract number 451-03-68/2022-14/200015 with
Institute for Medical Research, University of Belgrade; and
contract number 451-03-68/2022-14/200143 with Faculty of
Veterinary Medicine, University of Belgrade].
ORCID
Ivana Drvenica http://orcid.org/0000-0003-4985-1642
Branislav Vejnovi
chttp://orcid.org/0000-0002-6328-7446
Vesna Ili
chttp://orcid.org/0000-0003-1119-1343
856 I. DRVENICA ET AL.
Data availability statement
The data that support the findings of this study are available
on request from the corresponding author [V.I.].
References
Abuelo A, Hern
andez J, Benedito JL, Castillo C. 2019. Redox
biology in transition periods of dairy cattle: role in the
health of periparturient and neonatal animals.
Antioxidants. 8(1):20.
Abuelo
A, P
erez-Santos M, Hern
andez J, Castillo C. 2014.
Effect of colostrum redox balance on the oxidative status
of calves during the first 3 months of life and the relation-
ship with passive immune acquisition. Vet J. 199(2):
295–299.
Albera E, Kankofer M. 2011. The comparison of antioxidative/
oxidative profile in blood, colostrum and milk of early
post-partum cows and their newborns. Reprod Domest
Anim. 46(5):763–769.
Albera E, Kankofer M. 2010. The comparison of antioxidative/
oxidative profile in colostrum, milk and blood of early
post-partum cows during their first and second lactation.
Reprod Domest Anim. 45(6):e417–e425.
Benzie IF, Strain JJ. 1999. Ferric reducing/antioxidant power
assay: direct measure of total antioxidant activity of bio-
logical fluids and modified version for simultaneous meas-
urement of total antioxidant power and ascorbic acid
concentration. Methods Enzymol. 299:15–27.
Da Silva AS, Munhoz TD, Faria JL, Vargas-H
ernandez G,
Machado RZ, Almeida TC, Moresco RN, Stefani LM,
Tinucci-Costa M. 2013. Increase nitric oxide and oxidative
stress in dogs experimentally infected by Ehrlichia canis:
effect on the pathogenesis of the disease. Vet Microbiol.
164(3–4):366–369.
Dakovi
c A, Toma
sevi
c-
Canovi
c M, Rottinghaus GE,
Matija
sevi
c S, Sekuli
c
Z. 2007. Fumonisin B1 adsorption to
octadecyldimethylbenzyl ammonium-modified clinoptilo-
lite-rich zeolitic tuff. Microporous Mesoporous Mater.
105(3):285–290.
Dogliotti G, Malavazos AE, Giacometti S, Solimene U, Fanelli
M, Corsi MM, Dozio E. 2012. Natural zeolites chabazite/
phillipsite/analcime increase blood levels of antioxidant
enzymes. J Clin Biochem Nutr. 50(3):195–198.
Doumas BT, Bayse DD, Carter RJ, Peters T, Schaffer R. 1981.
A candidate reference method for determination of total
protein in serum. I. Development and validation. Clin
Chem. 27(10):1642–1650.
EU Regulation No 651/2013. 2013. Commission
Implementing Regulation (EU) No 651/2013 of 9 July 2013
concerning the authorisation of clinoptilolite of sediment-
ary origin as a feed additive for all animal species and
amending Regulation (EC) No 1810/2005.
Ga
al T, Ribiczeyn
e-Szab
o P, Stadler K, Jakus J, Reiczigel J,
K€
ov
er P, M
ezes M, S€
umeghy L. 2006. 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. 143(4):391–396.
Girotti AW. 1998. Lipid hydroperoxide generation, turnover,
and effector action in biological systems. J Lipid Res.
39(8):1529–1542.
Gitto E, Reiter RJ, Karbownik M, Tan DX, Gitto P, Barberi S,
Barberi I. 2002. Causes of oxidative stress in the pre- and
perinatal period. Biol Neonate. 81(3):146–157.
Hossein Nia B, Khorram S, Rezazadeh H, Safaiyan A, Tarighat-
Esfanjani A. 2018. The effects of natural clinoptilolite and
nano-sized clinoptilolite supplementation on glucose lev-
els and oxidative stress in rats with type 1 diabetes. Can J
Diabetes. 42(1):31–35.
Ipek H, Avci M, Aydilek N, Yerturk M. 2012. The effect of zeo-
lite on oxidant/antioxidant status in healthy dairy cows.
Acta Vet Brno. 81(1):43–47.
Kankofer M, Lipko-Przybylska J. 2008. Physiological antioxida-
tive/oxidative status in bovine colostrum and mature milk.
Acta Vet Beograd. 58(2–3):231–239.
Karatzia MA, Katsoulos PD, Karatzias H. 2013. Diet supple-
mentation with clinoptilolite improves energy status,
reproductive efficiency and increases milk yield in dairy
heifers. Anim Prod Sci. 53(3):234–239.
Katsoulos PD, Panousis N, Roubies N, Christaki E, Arsenos G,
Karatzias H. 2006. Effects of long-term feeding of a diet
supplemented with clinoptilolite to dairy cows on the
incidence of ketosis, milk yield and liver function. Vet Rec.
159(13):415–418.
Kerwin AL, Ryan CM, Leno BM, Jakobsen M, Theilgaard P,
Barbano DM, Overton TR. 2019. Effects of feeding syn-
thetic zeolite A during the prepartum period on serum
mineral concentration, oxidant status, and performance of
multiparous Holstein cows. J Dairy Sci. 102(6):5191–5207.
McGuirk SM, Collins M. 2004. Managing the production, stor-
age, and delivery of colostrum. Vet Clin North Am Food
Anim Pract. 20(3):593–603.
Milosevic S, Tomasevic-Canovic M. 2003. Processes of trybo-
chemical obtaining organozeolite adsorbent of mycotox-
ins, procedure for production and application. EP Patent
No. 1363854A1.
Milosevic S, Tomasevic-Canovic M. 2009. Tribochemical pro-
cess for obtaining organozeolite adsorbent of mycotoxins.
EP Patent No. 1363854B1.
Montinaro M, Uberti D, Maccarinelli G, Bonini SA, Ferrari-
Toninelli G, Memo M. 2013. Dietary zeolite supplementa-
tion reduces oxidative damage and plaque generation in
the brain of an Alzheimer’s disease mouse model. Life Sci.
92(17-19):903–910.
Mudro
n P, Rehage J, Qualmann K, Sallmann H-P, Scholz H.
1999. A study of lipid peroxidation and vitamin E in dairy
cows with hepatic insufficiency. Zentralbl Veterinarmed A.
46(4):219–224.
Murphy JM, Hagey JV, Chigerwe M. 2014. Comparison of
serum immunoglobulin G half-life in dairy calves fed col-
ostrum, colostrum replacer or administered with intraven-
ous bovine plasma. Vet Immunol Immunopathol. 158(3-4):
233–237.
Mushenkova NV, Bezsonov EE, Orekhova VA, Popkova TV,
Starodubova AV, Orekhov AN. 2021. Recognition of oxi-
dized lipids by macrophages and its role in atherosclerosis
development. Biomedicines. 9(8):915.
Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides
in animal tissues by thiobarbituric acid reaction. Anal
Biochem. 95(2):351–358.
Papaioannou D, Katsoulos PD, Panousis N, Karatzias H. 2005.
The role of natural and synthetic zeolites as feed additives
on the prevention and/or the treatment of certain farm
ITALIAN JOURNAL OF ANIMAL SCIENCE 857
animal diseases: a review. Microporous Mesoporous
Mater. 84(1):161–170.
Papaioannou DS, Kyriakis CS, Alexopoulos C, Tzika ED,
Polizopoulou ZS, Kyriakis SC. 2004. A field study on the
effect of the dietary use of a clinoptilolite-rich tuff, alone
or in combination with certain antimicrobials, on the
health status and performance of weaned, growing and
finishing pigs. Res Vet Sci. 76(1):19–29.
Pourliotis K, Karatzia MA, Florou-Paneri P, Katsoulos PD,
Karatzias H. 2012. Effects of dietary inclusion of clinoptilo-
lite in colostrum and milk of dairy calves on absorption of
antibodies against Escherichia coli and the incidence of
diarrhea. Anim Feed Sci Technol. 172(3-4):136–140.
Przybylska J, Albera E, Kankofer M. 2007. Antioxidants in
bovine colostrum. Reprod Domest Anim. 42(4):402–409.
Puppel K, Nałecz-Tarwacka T, Kuczy
nska B, Gołebiewski M,
Kordyasz M, Grodzki H. 2012. The age of cows as a factor
shaping the antioxidant level during a nutritional experi-
ment with fish oil and linseed supplementation for
increasing the antioxidant value of milk. J Sci Food Agric.
92(12):2494–2499.
Safameher A. 2008. Effects of clinoptilolite on performance,
biochemical parameters and hepatic lesions in broiler
chickens during aflatoxosis. J Anim Vet Adv. 7(4):381–388.
Selmeci L, Seres L, Antal M, Lukacs J, Regoly-Merei A, Acsady
G. 2005. Advanced oxidation protein products (AOPP) for
monitoring oxidative stress in critically ill patients: a sim-
ple, fast and inexpensive automated technique. Clin Chem
Lab Med. 43(3):294–297.
Sharma N, Singh NK, Singh OP, Pandey V, Verma PK. 2011.
Oxidative stress and antioxidant status during transition
period in dairy cows. Asian Australas J Anim Sci. 24(4):
479–484.
Sordillo LM, Aitken SL. 2009. Impact of oxidative stress on
the health and immune function of dairy cattle. Vet
Immunol Immunopathol. 128(1–3):104–109.
Sordillo LM, O’Boyle N, Gandy JC, Corl CM, Hamilton E. 2007.
Shifts in thioredoxin reductase activity and oxidant status
in mononuclear cells obtained from transition dairy cattle.
J Dairy Sci. 90(3):1186–1192.
Stoji
c M, Fratri
c N, Kova
ci
c M, Ili
c V, Gvozdi
c D, Savi
cO,
-
Dokovi
c R. 2017. Brix refractometry of colostrum from
primiparous dairy cows and new-born calf blood serum in
the evaluation of failure of passive transfer. Acta Vet
Beograd. 67(4):508–524.
Stoji
c M, Ili
c V, Kova
ci
c M, Gvozdi
c D, Stajkovi
c S, Vejnovi
cB,
Savi
c O, Fratri
c N. 2020. Effects of oral supplementation
with organically modified clinoptilolite during prepartum
period on colostrum quality in primiparous dairy cows. J
Dairy Res. 87(4):429–435.
Tariq SH, Morley JE. 2004. Protein-calorie deficiency—
“Kwashiorkor”. In Johnson LR, editor. Encyclopedia of
Gastroenterology. 1st ed. Amsterdam, The Netherlands:
Academic Press; p. 242–246.
Valpoti
c H, Gra
cner D, Turk R, -
Duri
ci
c D, Vince S, Folno
zi
cI,
Lojki
cM
Z,
Zaja I, Lj B, Ma
ce
si
c N. 2017. Zeolite clinoptilo-
lite nanoporous feed additive for animals of veterinary
importance: potentials and limitations. Period Biol. 119(3):
159–172.
V
azquez-A~
n
on M, Nocek J, Bowman G, Hampton T, Atwell C,
V
azquez P, Jenkins T. 2008. Effects of feeding a dietary
antioxidant in diets with oxidized fat on lactation per-
formance and antioxidant status of the cow. J Dairy Sci.
91(8):3165–3172.
Witko-Sarsat V, Friedlander M, Capeill
ere-Blandin C, Nguyen-
Khoa T, Nguyen AT, Zingraff J, Jungers P, Descamps-
Latscha B. 1996. Advanced oxidation protein products as
a novel marker of oxidative stress in uremia. Kidney Int.
49(5):1304–1313.
Wood D, Quiroz-Rocha GF. 2010. Normal hematology of cat-
tle. In Weiss DJ, Wardrop KJ, editors. Schalm’s veterinary
hematology. 6th ed. Ames (IA): Wiley-Blackwell; p.
829–835.
Wu QJ, Wang YQ, Zhou YM, Wang T. 2015. Dietary clinoptilo-
lite influences antioxidant capability and oxidative status
of broilers. J Appl Poult Res. 24(2):99–104.
Yarovan NI. 2008. Effect of zeolites on adaptation processes
in cows. Russ Agri Sci. 34(2):120–122.
Zarkovic N, Zarkovic K, Kralj M, Borovic S, Sabolovic S, Blazi
MP, Cipak A, Pavelic K. 2003. Anticancer and antioxidative
effects of micronized zeolite clinoptilolite. Anticancer Res.
23(2B):1589–1595.
Zhivaki D, Kagan JC. 2021. Innate immune detection of lipid
oxidation as a threat assessment strategy. Nat Rev
Immunol. [accessed 2021 December 1]:[9 p.] https://doi.
org/10.1038/s41577-021-00618-8.
Zwart A, van Assendelft OW, Bull BS, England JM, Lewis SM,
Zijlstra WG. 1996. Recommendations for reference method
for haemoglobinometry in human blood (ICSH standard
1995) and specifications for international haemiglobino-
cyanide standard (4th edition). J Clin Pathol. 49(4):
271–274.
858 I. DRVENICA ET AL.
