Effect of Sodium Humate on the Content of Trace Elements in Organs of Weaned Piglets


Zralý, Zěk; Písaříková, B

Acta Veterinaria Brno 79(1): 73-79

2010


ACTA
VET.
BRNO
2010,
79:
73-79;
doi:10.275
4
/
a
vb201079010073
Effect
of
Sodium
Humate
on
the
Content
of
Trace
Elements
in
Organs
of
Weaned
Piglets
Zdenek
ZralST,
Bohumila
Pisafikova
Veterinary
Research
Institute,
Brno,
Czech
Republic
Received
February
13,
2009
Accepted
June
30,
2009
Abstract
The
purpose
of
the
present
study
was
to
assess
the
effect
of
a
21-day
feeding
of
a
diet
with
1%
inclusion
of
sodium
humate
(IlNa)
on
the
content
of
trace
elements
(copper,
zinc,
selenium,
manganese,
cobalt)
in
tissues
of
weaned
piglets,
their
efficiency
and
biochemical
indicators.
Significantly
lower
content
of
manganese
in
livers
(P
<
0.01)
and
kidneys
(P
<
0.05)
was
detected
in
piglets
fed
the
1%
HNa
supplemented
diet
in
comparison
with
control,
non-treated
animals.
The
concentrations
of
selenium
in
blood
serum
(P
<
0.05)
and
muscular
tissue
(P
<
0.01)
were
significantly
lower
in
experimental
animals.
Significantly
higher
daily
body
weight
gain
was
detected
in
experimental
piglets
compared
to
control
animals
(0.303
vs.
0.258
kg,
P
<
0.05)
and
the
feed
conversion
rate
was
increased
by
6.4%
(1.60
vs.
1.71
kg).
Increased
concentrations
of
blood
serum
glucose,
triacylglycerols
(P
<
0.01),
calcium
and
iron
(P
<
0.05)
were
detected
in
experimental
vs.
control
piglets.
The
results
of
the
present
study
indicated
that
despite
a
positive
effect,
sodium
humate
caused
a
decrease
in
concentration
of
physiologically
important
manganese
and
selenium
in
the
tissues.
Adsorbents,
additives,
liver,
kidney,
muscle
tissue,
selenium,
manganese
Good quality
feeds
and
rational
nutrition
are
essential
preconditions
for
obtaining
high
producing
healthy
animals
and
economic
prosperity
in
a
herd.
The
issues
associated
with
the
food
chain
protection
and
guarantee
of
food
safety
assurance
require
a
steadily
increased
attention
concentrated
on
the
investigation
of
effective
substances
that
might
mitigate
the
adverse
effects
of
xenobiotics
in
the
organisms
of
food
animals.
Due
to
the
above
mentioned
facts,
animal
diets
are
supplemented
with
different
types
of
additives
including
adsorbents.
Humic
substances
are
major
constituents
of
soil
organic
matter.
They
can
be
found
in
potable
and
ocean
water
and
are
a
natural
component
of
the
food
chain.
The
major
components
of
humic
compounds
are
humic
acids
(HA),
fulvic
acids
and
ulmic
acids
that
are
essential
for
plant
growth
(Stevenson
1994).
HA
have
a
tridimensional
reticulate
structure
and
contain
multiple
functional
groups
such
as
hydroxyl,
carboxylic,
carbonyl,
amino,
amido
and
sulphhydryl
groups
(MacCarthy
2001).
Due
to
different
HA
structures,
the
content
of
functional
groups
and
various
qualities
(colloidal,
spectral,
electrochemical
and
ion
exchange)
their
considerable
adsorption
capacity
is
assumed
(Klocking
1994;
Alvarez
Puebla
et
al.
2005).
Due
to
the
colloidal
characteristics
and
the
ability
to
form
chelates,
HA
and
their
salts
can
significantly
modify
the
toxic
effects
of
a
number
of
xenobiotics
and
undesirable
substances
that
enter
the
digestive
tract
together
with
feeds
and
water
(Livens
1991).
They
have
strong
affinity
for
mutagens
(Cozzi
et
al.
1993),
pesticides
(Negre
et
al.
2001;
Li
et
al.
2003),
monoaromatic
and
polycyclic
aromatic
compounds
(Nanny
and
Maza
2001;
Kollist
Siigur
et
al.
2001),
heavy
metals
(Livens
1991;
Madronova
et
al.
2001;
Hammock
et
al.
2003;
Herzig
et
al.
2007;
Zral'
et
al.
2008),
aflatoxin
Bi
(Van
Rensburg
et
al.
2006)
and
microorganisms
(Fein
et
al.
1999).
Positive
effects
of
oxihumolite
resulting
in
a
decreased
ammonia
emission
in
the
inside
environment
of
broiler
and
pig
housing
were
noted
by
Such'
et
al.
(1999)
and
Herzig
et
al.
(2001).
Address
for
correspondence:
Phone
+420
533
331
612
MVDr.
Zdenak
Zraly,
CSc.
Fax
+420
541
211
229
Veterinary
Research
Institute
E-mail:
zsaly@vri.cz
Hudcova
70,
621
00
Brno,
Czech
Republic
http://www.vfu.cz/acta-vet/actavet.htm
74
Furthermore,
Ji
et
al.
(2006)
found
reduced
ammonia
emission
from
the
environment
of
market
pigs
after
supplementation
of
the
diet
with
humic
compounds.
In
the
past,
humic
substances
were
studied
above
all
owing
to
their
positive
effect
on
the
animal
organism.
After
feeding
sodium
humate
to
calves,
Griban
(1988)
found
an
increased
body
weight
gain
and
decreased
mortality;
after
feeding
the
same
product
to
dairy
cows,
decreased
occurrence
of
mastitis
and
milk
somatic
cell
counts
were
detected.
Antiflogistic,
antitoxic,
antibacterial
and
antiviral
effects
were
documented
for
humic
acids;
these
can
be
used
for
therapy
and
prevention
(Lenk
and
Benda
1989;
Klocking
1994).
Mechanism
of
adaptation
and
bioregulation
properties
of
HA
and
their
salts
consist,
besides
others,
in
their
direct
effect
on
the
immune
responses,
detoxicating
activity
of
liver
and
sulphhydryl-disulphide
balance
in
metabolism
of
proteins
and
saccharides
(Lind
and
Glynn
1999;
Santos
et
al.
2004).
Due
to
the
fact
that
no
toxic,
allergic,
mutagenic
and
teratogenic
effects
of
the
HA
were
found,
HA
and
its
sodium
salt
(HNa)
were
allowed
for
oral
treatment
of
all
food
animals
(EMEA
1999).
They
have
been
applied
in
horses,
ruminants,
pigs
and
poultry
for
treatment
of
diarrhoea,
dyspepsia
and
poisoning.
Despite
the
above
mentioned
positive
adsorption
effects,
their
possible
adverse
effect
on
the
levels
of
essential
nutrients
-
above
all
trace
elements,
which
play
an
important
role
in
a
series
of
physiological
functions
-
have
been
discussed.
The
aim
of
the
present
experiment
was
to
test
the
effect
of
sodium
humate
feeding
on
the
content
of
trace
elements
in
the
organs
of
weaned
piglets,
their
growth
efficiency
and
biochemical
indicators.
Materials and
Methods
Twenty
hybrid
Pietrain
x
(Large
White
x
Landrace
x
Duroc)
pigs
in
equal
numbers
of
barrows
and
gilts
at
the
age
of
28
days
were
used
in
the
study.
The
experiment
was
performed
under
good
hygienic
conditions
of
accredited
animal
facilities
in
the
Veterinary
Research
Institute.
The
animals
were
allocated
to
two
groups
based
on
individual
live
body
weight
and
sex.
The
average
live
body
weight
of
control
(C)
and
experimental
(HNa)
piglets
was
7.95
±
0.87
kg
(V%
=
10.9)
and
8.55
±
0.90
kg
(V%
=
10.5),
respectively.
In
the
course
of
the
experiment
(21
days)
pigs
were
housed
in
pens,
fed
ad
libitum
the
diet
for
early
weaned
piglets
twice
a
day
at
7:00
and
16:00
h.
They
had
free
access
to
feed
and
water
(supplied
through
nipple
drinkers).
The
control
group
diet
contained
85.7%
of
dry
matter
with
the
following
nutrients:
204.2
g
crude
protein
(N
x
6.25),
14.3
g
lysine,
4.9
g
methionine,
9.2
g
threonine,
35.0
g
lipids,
31.9
g
fibre,
52.6
g
ashes,
9.2
g
Ca,
4.9
g
P,
2.2
g
Na/kg
and
the
content
of
metabolizable
energy
(MEp)
was
13.2
MJ/kg.
All
the
experimental
animals
were
fed
the
same
diet
in
which
1%
of
the
wheat
content
was
replaced
by
HNa
(JV
24)
provided
by
Research
Institute
of
Inorganic
Chemistry
(Usti
nad
Labem,
Czech
Republic).
The
chemical
analysis
(mass
%)
revelaed
77.80%
dry
matter,
27.21%
ash
in
dry
matter,
72.79%
humic
substances
(HS)
in
dry
matter.
The
concentrations
of
trace
elements
in
dry
matter
(X-ray
fluorescent
analysis
RFA)
were
as
follows:
22
mg
Cu,
22
mg
Zn,
<
1
mg
Se,
25
mg
Mn
and
21
mg
Co/
kg.
Supplementation
of
the
diet
for
experimental
animals
with
1%
inclusion
of
HNa
did
not
significantly
alter
the
content
of
essential
nutrients.
Feed
consumption
was
recorded
in
each
group.
Throughout
the
experiment
the
health
state
was
monitored.
Samples
taken
from
the
dead
piglet
were
examined
using
bacteriological
methods
according
to
A
le
xa
et
al.
(1997).
At
the
end
of
the
trial,
the
weight
of
the
animals
was
taken
and
blood
samples
were
drawn
from
v.
cava
cranialis
for
biochemical
analysis.
The
live
body
weight
gains
(BWG)
of
each
animal
and
average
weight
of
respective
groups
were
the
differences
between
the
live
body
weight
detected
at
the
beginning
and
the
end
of
the
experiment.
Feed
conversion
rate
(FCR)
was
calculated
from
the
efficiency
of
pigs
in
converting
feed
mass
into
increased
body
mass.
After
slaughter,
liver,
kidney
and
muscle
tissue
samples
(m.
longissimus
lumborum
et
thoracis)
were
collected
for
detection
of
trace
element
content.
The
content
of
dry
matter,
crude
protein
(N
x
6.25),
lipids,
crude
fibre
and
ash
in
the
diet
were
analyzed
by
methods
AOAC
(2001).
Total
protein,
albumin,
glucose,
triacylglycerols,
cholesterol,
alkaline
phosphatase
(ALP),
aspartate
and
alanine
aminotransferases
(AST,
ALT),
calcium,
phosphorus
and
magnesium
blood
serum
concentrations
were
detected
spectrophotometrically
using
Bio-La-Tests
(PLIVA
Lachema
Brno,
Ltd.,
Czech
Republic).
The
Cu,
Zn,
Mn,
and
Co
concentrations
in
the
feed
mixture,
organs
and
tissues
were
detected
using
the
validated
method
F-AAS
in
a
Perkin
Elmer
Model
2100
spectrometer
with
the
following
detection
limits
(mg/kg):
0.03,
0.02,
0.07
and
0.04,
respectively,
and
wave
lengths
(nm):
324.8,
217.0,
279.5
and
240.7,
respectively.
The
amount
of
samples
taken
into
analysis
was
5-10
g;
mineralization
occurred
via
decomposition
in
dry
air
(450
±
20
°C).
The
Se
concentration
was
assessed
by
a
hydride
generation
method
(HG-AAS)
using
AAnalyst-300
+
Fias
400
75
(Perkin
Elmer)
spectrometer
at
the
sensitivity
of
0.005
mg/kg;
the
wave
length
was
196.0
nm.
The
amount
of
0.5-
1
g
was
taken
into
analysis,
mineralization
occurred
via
reduction
of
Se->Se
4-
'
performed
by
addition
of
HC1.
The
reliability
of
the analysis
was
checked
using
the
certified
reference
material
Milk
Powder
(BCR-151®,
in
the
case
of
Co,
Bovine
Liver-1577®,
Belgium).
Each
sample
was
analyzed
in
duplicates.
The
results
were
processed
by
statistical
and
graphic
software
STAT
Plus
(VRI,
Brno,
Czech
Republic).
Data
were
analysed
using
basic
statistical
characteristics;
the
mean
differences
were
tested
by
t-test.
Results
and
Discussion
Experimental
diet
with
the
inclusion
of
HNa
contained
the
following
trace
elements:
166.0
mg
Cu,
203.1
mg
Zn,
104.5
mg
Mn,
0.378
mg
Se
and
0.61
mg
Co/kg.
Control
diet
contained
the
same
amounts
of
trace
elements.
The
content
of
the
investigated
trace
elements
was
in
accordance
with
the
recommended
supply
for
weaned
piglets
(g
ime6ek
et
al.
2000);
the
content
of
iron
and
cobalt
were
at
the
upper
and
lower
range
of
recommended
amounts,
respectively.
The
trace
elements
found
in
blood,
liver,
kidney
and
muscles
of
the
control
animals
reflected
the
current
level
of
dietary
mineral
supply
to
the
piglets.
The
between-group
differences
in
copper,
zinc
and
cobalt
content
in
all
investigated
tissues
were
non-significant.
In
experimental
vs.
control
group,
significantly
lower
content
of
manganese
in
liver
(2.04
±
0.07
vs.
2.33
±
0.13
mg/kg,
P
<
0.01)
and
kidney
(0.82
±
0.03
vs.
0.87
±
0.03
mg/kg,
P
<
0.05)
was
found
(Table
1).
Significant
reduction
of
selenium
content
in
the
investigated
tissues
and
organs
of
experimental
animals
was
confirmed
by
blood
(P
<
0.05)
and
muscle
(P
<
0.01)
analyses.
Table
1.
Trace
elements
in
blood
serum
and
organs
of
piglets
after
feeding
sodium
humate
(mg/kg)
Indicator
Blood
serum
Liver
Kidneys
Muscle
tissue
C
FINa
C
FINa
C
FINa
C
HNa
Cu
x
1.53
1.55
23.10 20.50
4.96
4.91
0.96
1.01
SD
0.11
0.16
7.69
5.85
0.46
0.65
0.13
0.08
Zn
x
1.94
1.88
69.60
63.70
16.70 16.50
12.30
12.30
SD
0.13
0.13
12.80
21.00
1.00
1.45
0.79
0.71
Se
x
0.124a
0.117
1
0.310
0.240 0.710 0.660
0.078'
0.045
8
SD
0.005
0.004
0.030
0.070 0.060 0.060
0.012
0.006
Mn
x
<
0.07
<
0.07
2.33'
2.04
8
0.87a
0.82b
0.16
0.14
SD
0.13
0.09
0.03 0.03
0.04
0.04
Co
x
0.05
0.05
<
0.04
<
0.04
<
0.04
<
0.04
<
0.04
<
0.04
SD
0.01
0.01
A
'
B
significant
differences
at
P
<
0.01;
a
'
b
significant
differences
at
P
<
0.05
Considering
the
use
of
adsorbents
and
investigating
new
feed
supplements
for
farm
animals,
the
authors
discussed
the
hypothesis
of
potential
adsorption
of
biologically
important
substances,
such
as
trace
elements,
followed
by
a
decrease
in
their
biological
availability
for
animal
organisms.
The
obtained
results
confirmed
that
feeding
HNa
to
animals
had
no
significant
adverse
effect
on
the
copper,
zinc
and
cobalt
content
in
the
investigated
organs and
tissues;
this
finding
was
in
agreement
with
Doyle
and
Spaulding
(1978),
Kirchgessner
et
al.
(1994)
and
Jorhem
et
al.
(1996).
On
the
other
hand,
elevated
concentrations
of
trace
elements
in
pig
tissues
were
reported
by
Lop
ez
-Alonso
et
al.
(2007).
The
content
of
copper
in
muscular
tissues
detected
in
the
present
study
was
higher
in
comparison
with
data
presented
by
Lombardi-Boccia
et
al.
(2005).
The
detected
trace
element
levels
in
blood
serum
reflected
the
current
level
of
dietary
mineral
supply
to
the
animals
and
were
consistent
with
the
finding
of
Stowe
et
al.
(1992),
Kim
and
Mahan
(2001)
and
others.
The
highest
content
of
trace
elements,
except
selenium,
was
detected
in
the
liver
which
is
a
depot
organ
of
a
higher
76
diagnostic
value
than
muscular
tissue
for
the
assessment
of
dietary
mineral
supply
to
the
animals.
Kidneys,
where
highest
concentrations
of
selenium
were
detected
in
the
present
study,
are
the
most
important
organ
involved
in
selenium
disposition
(L
op
ez
-
Alons
o
et
al.
2007).
Owing
to
sodium
humate
feeding,
the
levels
of
manganese
and
above
all
selenium
(which
is
physiologically
important)
in
blood
serum
and
muscular
tissue
was
significantly
decreased.
The
presented
results
showed
that
the
effect
of
humic
substances
from
a
diet
on
different
trace
elements
was
unequal.
Potential
antagonistic
interactions,
such
as
between
iron
and
manganese
or
iron,
copper
and
zinc
etc
should
also
be
taken
into
consideration
(Creech
et
al.
2004).
A
high
proportion
of
humic
acids
in
water
was
associated
with
increased
prevalence
of
thyroid
dysfunctions
in
human
population
(Huang
et
al.
1994).
The
availability
of
selenium
was
impaired
due
to
its
inhibition
by
humic
substances
present
in
drinking
water
(Wang
et
al.
1992).
Humic
substances
can
form
complexes
between
humic
acid
and
heavy
metal
ions
and
also
between
humic
acid
and
metal
ions:
Zn,
Cu,
Mn,
Au,
Fe,
Al,
and
Se
and
thus
affect
their
availability
to
the
animal
organism
(Livens
1991).
From
this
aspect,
physiological
effects
of
complexes
prepared
from
humic
acids
and
targeted
biogenic
elements
could
be
tested.
The
average
body
weight
gain
detected
over
the
whole
experimental
period
in
control
and
experimental
animals
was
5.41
±
0.76
kg
and
6.36
±
0.95
kg,
respectively
(Table
2).
It
is
in
accordance
with
the
HNa
effect
on
the
average
daily
body
weight
gain,
which
was
0.303
±
0.04
vs.
0.258
±
0.03
(P
<
0.05)
in
experimental
and
control
group.
The
average
daily
feed
intake
was
higher
in
the
experimental
(0.48
kg)
compared
to
control
group
(0.44
kg).
A
favourable
effect
of
HNa
treatment
on
the
feed
conversion
was
noted;
it
was
improved
by
6.4%
in
the
experimental
group
(1.60
vs.
1.71
kg/kg
body
weight
gain).
Table
2.
Indicators
of
growth
efficiency
in
piglets
fed
a
diet
with
the
inclusion
of
1%
I-INa
(x
±
SD)
Indicator
C
I-INa
Index
(%)
Number
of
piglets
10/9*
10
Average
live
body
weight
DO
(kg)
7.95
±
0.87
8.55
±
0.90
107.5
Average
live
body
weight
D21
(kg)
13.36
±
1.06
14.91
±
1.70
111.6
Average
live
body
weight
gain
(kg/day)
0.258
±
0.03'
0.303
±
0.04
1
'
111.8
Feed
conversion
(kg/kg)
1.71
1.60
93.6
*one
piglet
dead
b
significant
differences
(P
<
0.05)
A
positive
effect
of
humic
substances
in
diet
or
water
for
poultry
on
the
growth
descriptors
was
observed
by
Bailey
et
al.
(1996),
Islam
et
al.
(2005),
Demeterova
and
Mari
scakova
(2006).
Our
results
are
in
accordance
with
the
study
of
S
lavik
(1999).
Verifying
the
effects
of
0.6%
inclusion
of
humic
acids
in
the
feed
for
piglets,
they
found
a
decreased
feed
conversion
by
1.27
to
5.26%
and
increased
daily
body
weight
gain
by
2.19
to
9.42%.
On
the
other
hand,
S
chuhmacher
and
Gropp
(2000)
found
a
non-significant
effect
of
HS
treatment
of
weaned
piglets.
The
positive
effect
of
humic
substances
can
be
explained
by
enhancement
of
the
metabolic
activity
of
cell
membranes
by
acceleration
of
oxidative
processes
that
due
to
increased
nutrient
uptake
stimulate
the
vital
functions
(Islam
et
al.
2005).
One
piglet
with
clinical
symptoms
of
acute
gastroenteritis
died
during
the
experiment.
Infection
with
enterotoxigenic
E.
coli
infection
was
diagnosed
in
the
piglet.
Except
for
this
case,
no
clinical
signs
of
a
disease
were
observed
in
the
piglets.
It
was
confirmed
by
biochemical
test
results
presented
in
Table
3.
Significantly
increased
glucose
and
triacylglycerol
levels
(P
<
0.01)
as
well
as
Ca
metabolism
(P
<
0.05)
were
detected
in
experimental
animals.
77
Table
3.
Selected
biochemical
indicators
in
the
blood
serum
of
piglets
(x
±
SD;
n
=
9)
Indicator
C
HNa
Total
protein
g/l
51.80
±
5.60
52.40
±
7.30
Albumin
g/l
27.20
±
2.95
27.30
±
3.80
Glucose
nuno1/1
4.67
±
0.53'
5.60
±
0.56B
Triacylglycerols
nuno1/1
0.44
±
0.10"
0.62
±
0.13's
Cholesterol
nuno1/1
1.63
±
0.30
1.61
±
0.27
ALT
µkat/1
1.10
±
0.33
0.90
±
0.22
AST
µkat/1
0.89
±
0.31
0.81
±
0.25
ALP
µkat/1
4.33
±
1.14
4.88
±
0.63
Calcium
nuno1/1
2.53
±
0.16a
2.70
±
0.13
1
'
Phosphorus
nuno1/1
2.86
±
0.22
3.07
±
0.31
Magnesium
nuno1/1
1.02
±
0.10
0.98
±
0.10
A
'
B
significant
differences
at
P
<
0.01;
a
'
a
significant
differences
at
P
<
0.05
As
indicated
by
clinical
and
biochemical
tests,
no
adverse
effects
of
sodium
humate
feeding
on
health
of
experimental
animals
were
noted.
Significantly
increased
glucose,
triacylglycerol,
and
calcium
gave
evidence
of
increased
energy
and
mineral
metabolism
(Kreutz
and
Schlikekewey
1992).
Other
indicators
of
internal
environment
of
the
animals
were
within
the
physiological
range
(T1u6hof
2001).
The
results
of
the
present
study
show
that
feeding
sodium
humate
to
animals
can
cause
a
certain
loss
of
manganese
and
selenium
from
the
body.
If
humic
substances
are
used
in
pig
feeding,
attention
should
be
paid
to
dietary
supplementation
of
these
trace
elements
to
the
animals.
Vliv
huminovych
hitek
na
obsah
stopovfch
prvkii
v
orginech
a
uiltkovost
odstavenSrch
selat
Cilem
studie
bylo
ovefit
vliv
21
denniho
podivani
krmne
smesi
suplementovane
1
%
humatu
sodneho
(HNa)
na
obsah
stopovSrch
prvku
(med',
zinek,
selen,
mangan
a
kobalt)
ye
tkanich
odstavenSrch
selat,
ulitkovost
a
biochemicke
ukazatele.
Po
aplikaci
1
%
HNa
byl
zjigten
priikazne
niIgi
obsah
manganu
v
jatrech
(P
<
0,01)
a
ledvinach
(P
<
0,05)
vil6i
kontrolnim,
neogetfenSan
zvifatilm.
Signifikantne
niIgi
koncentrace
selenu
byly
prokazany
v
krevnim
seru
(P
<
0,05)
a
svalovine
(P
<
0,01)
pokusnSrch
zvifat.
U
pokusnSrch
selat
byl
zaznamenan
prfikazne
vyggi
prumernST
denni
pfirfistek
live
hmotnosti
ve
srovnani
s
kon-
trolnimi
(0,303
vs.
0,258
kg,
P
<
0,05)
a
lepgi
konverze
krmiva
o
6,4
%
(1,60
vs.
1,71
kg).
U
pokusnSrch
selat
byly
v
krevnim
seru
zjigteny
vyggi
koncentrace
glukozy,
triacylglyce-
rolu
(P
<
0,01)
a
vapniku
(P
<
0,05)
ve
srovnani
s
kontrolnimi
selaty.
VSTsledky
doloIily,
le
humat
sodnST,
i
pi
-
es
pozitivni
vliv,
sniIil
koncentrace
fyziologicky
vSrznamneho
manganu
a
selenu
ye
tkanich.
Acknowledgement
This
work
was
supported
by
grant
MZE-0002716202
and
MSMT
project
Admire
Vet.
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Acta
Vet
Brno
77:
439-445