attapol tiantong*, shuen-ei chen and chai-ju chang ... · attapol tiantong*, shuen-ei chen and...
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Attapol Tiantong*, Shuen-Ei Chen and Chai-Ju Chang
Department of Animal Science
National Chung Hsing University, Taiwan ROC
Enterococcus faecium SF68® (SF68®) is considered one of the best
characterized probiotic active ingredient worldwide.
The strain has been isolated in Sweden from a healthy newborn baby in
the sixties and has been demonstrated to have clinical benefits for
humans (Wunderlich et al., 1989).
Furthermore, this probiotic strain is also effective in modifying the
immune response of piglets, young dogs, mice and cats (Bakhshi et al., 2006;
Benyacoub et al., 2005; Bybee et al., 2011; Scharek et al., 2005; Simpson et al., 2009).
Manufacturer: Cerbios-Pharma S.A. Switzerland 2
Cernivet® LBC ME10 is registered in EU for the use in:
• Calves up to 6 months,
• Chickens for fattening,
• Sows (2 weeks before farrowing and during lactation),
• Piglets up to 4 months and
• Pigs for fattening.
(http://www.cerbios.ch/I/biological-product/cernivet®-lbc-me10-and-cernivet®-lbc-me20-plus)3
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The application of probiotics to ruminant animals is relatively rare due to
the presence of pre-gastric fermentative rumen.
Nevertheless, a novel route of probiotic application via teat canal directly
into the mammary lumen of dairy animals has been developed for the
prevention and treatment of mastitis (Ryan et al., 1998, 1999; Crispie et al., 2008;
Klostermann et al., 2008; Beecher et al., Frola et al., 2012).
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In our recent studies, a preparation of SF68 was used to substitute for the
commercial antibiotic dry cow formula in Holstein cows and was found
with extra benefits of enhancing the local innate immune function (Peng
et al. 2013) and accelerating the involution process (Tiantong et al. 2014)
of mammary gland during the acute phase of drying-off.
Journal of Dairy Research (2013), 80; 349-359.
Animal Science Journal (2014), DOI: 10.1111/asj.12243
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In the current study, the effects of applying probiotics SF68 as a dry
cow formula on the final stage of pregnancy and the subsequent
parturition and lactation of Holstein cows.
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METERIALS AND METHODSCow selection and management
Lactating, second parity Holstein cows at 24 - 56 months of age were raised at the
University Farm of National Chung Hsing University (Taichung, Taiwan).
Milk stasis was practiced when cows were reaching the final two months of pregnancy.
Milk yield was less than 5 kg/day.
The experimental procedures have been approved by the committee of Care and Use of
Agriculture Animals of the university.
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Drying-off
30 days
60 days
D 1 D 3 D 7 D 30 Parturition
SF68 infusion
Colostrum sampling
Newborn Calf
sampling n=3
n=2
Experimental design
METERIALS AND METHODS
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HEAD
Front right quarter
Rear left quarter
Rear right quarter
Front left quarter
2.5-ml of ultrasonicated
SF68 (2 × 108 cfu/ml)
2.5-ml of commercial
dry-cow therapy
METERIALS AND METHODS
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5-ml of commercial dry
cow therapy
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Cow analysis:
Plasma hormone level
Plasma total protein content and composition
Plasma gelatinase level
Blood leukocyte number and function
Colostrum analysis:
Total protein content and composition
Gelatinase level
METERIALS AND METHODS
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Neonatal calves analysis:
Plasma total protein content and composition
Plasma gelatinase level
Blood leukocyte number and function
Figure 1 Typical images of SDS-PAGE and gelatin zymography for the evaluation for cow plasma.
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0
5
10
15
20
1 3 7 30 PartumPlas
ma
prog
este
rone
leve
l(n
g/m
l)
Days in experiment
0
20
40
60
80
100
1 3 7 30 Partum
Plas
ma
estr
adio
l lev
el(p
g/m
l)
Days in experiment
0
20
40
60
80
100
1 3 7 30 Partum
Plas
ma
cort
isol
leve
l(p
g/m
l)
Days in experiment
*
Figure 2 Summarized results of two individual cows showing the levels of
plasma hormones (solid lines) and the means (dotted lines). * , Mean value
is significantly different form the corresponding mean of day 1. P < 0.05.
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SF68
SF68
SF68
0
20
40
60
80
100
1 3 7 30 Partum
Plas
ma
tota
l pro
tein
co
nten
t (g
/L)
Days in experiment
0
10
20
30
40
1 3 7 30 Partum
Plas
ma
gam
ma-
glob
ulin
leve
l(a
rbitr
ary
unit)
Days in experiment
0
10
20
30
40
1 3 7 30 Partum
Plas
ma
albu
min
leve
l(a
rbitr
ary
unit)
Days in experiment
Figure 3 Summarized results of two individual cows showing the levels of
plasma total protein content and compositions of gamma globulin and albumin
(solid lines) and the means (dotted lines).
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SF68SF68
SF68
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Figure 4 Summarized result of
two individual cows showing the
blood total leukocyte count and
function, and plasma MMP-2
level (solid lines) and the means
(dotted lines). * ,Mean value is
significantly different form the
corresponding mean of day 1. P <
0.05.
14
0
2
4
6
8
10
12
1 3 7 30 Partum
Blo
od le
ukoc
yte
coun
t(×
106
cell/
ml)
Days in experiment
*
0
4
8
12
16
20
1 3 7 30 Partum
Phag
ocyt
osis
rat
e (%
)
Days in experiment
*
*
*
0
500
1000
1500
2000
2500
1 3 7 30 Partum
CL
inde
x
Days in experiment
0
5
10
15
20
1 3 7 30 Partum
Plas
ma
MM
P-2
leve
l(a
rbitr
ary
unit)
Days in experiment
*
** * *
SF68
SF68
SF68
SF68
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Figure 5 Representative SDS-PAGE images of
initial colostrum of two individual cows.
Cow 361 control udder
Cow 361 SF68 udder
Cow 381 control udder
Cow 381 SF68 udder
0
10
20
30
40
50
gamma-Globulin Albumin Casein β-Lactoglobulin α-Lactalbumin
Col
ostr
um p
rote
in c
ompo
sitio
n(a
rbitr
ary
unit)
Figure 6 Summarized result of cow showing the colostral total protein content, compositions of major colostral proteins for
the control and SF68-infused udders of the two individual cows.16
0
20
40
60
80
100
Cow 361 Cow 381
Col
ostr
al to
tal p
rote
in
cont
ent
(g/L
)
SF68 SF68C C
Cow 1 control udder
Cow 1 SF68 udder
Cow 2 control udderCow 2 SF68 udder
0
50
100
150
200
MMP-2 MMP-9
Col
ostr
um M
MP
(arb
itrar
y un
it)
Figure 7 Summarized result of cow showing the colostral levels of MMP-2 and MMP-9 for the control and
SF68-infused udders of the two individual cows. 17
0
5
10
15
20
Cow 361 Cow 381
Col
ostr
al M
MP-
2 le
vel
(arb
itrar
y un
it)
0
5
10
15
20
Cow 361 Cow 381
Col
ostr
al M
MP-
9 le
vel
(arb
itrar
y un
it)
SF68
SF68
C
C
SF68
SF68
C
C
0
5
10
15
20
25
Cow 361 Cow 381
Plas
ma
MM
P-2
leve
l of n
eona
tal
calf
(arb
itrar
y un
it)
18
0
2
4
6
8
10
12
Cow 361 Cow 381
Blo
od le
ukoc
yte
coun
t of
neon
atal
cal
f (x
106
cell/
ml)
0
4
8
12
16
20
Cow 361 Cow 381
Phag
ocyt
osis
rat
e of
neo
nata
l ca
lf (%
)
0
500
1000
1500
2000
2500
Cow 361 Cow 381
CL
inde
x of
neo
nata
l cal
f
0
20
40
60
80
100
Plas
ma
tota
l pro
tein
con
tent
of
neon
atal
cal
f (g
/L)
0
20
40
60
80
100
Plas
ma
gam
ma-
glob
ulin
con
tent
of
neo
nata
l cal
f (a
rbitr
ary
unit)
0
20
40
60
80
100
Plas
ma
albu
min
con
tent
of
neon
atal
cal
f (a
rbitr
ary
unit)
Figure 8 Summarized result of
two individual neonatal calves
showing the blood total leukocyte
count and function, plasma total
protein content and composition
of gamma-globulin and albumin,
and plasma MMP-2 level.
The most prominent change observed in the current study in association with intramammary
SF68 infusion is the elevation of MMP-2 level, both systemic and local mammary gland.
The presence of MMP in animal tissues and fluids has been used as supplementary information
for diagnosing diseases monitoring organ remodeling, including horse arthritis (Jouglin et al.,
2000), cow mastitis (Rabot et al., 2002) and different stages of mammary gland involution (Yu et al.,
2012).
The higher MMP-2 level in plasma and colostrum is very likely related to some subclinical
proinflammation in the body and mammary gland.
No change in most of the conventional physiological parameters of experimental cows during
the nearly two-month period following SF68 infusion.
Regarded clinically normal of final stage of pregnancy, the parturition, the colostrum quality
and the neonatal calves of Holstein cows after intramammary SF68 infusion.
Higher levels of the novel proinflammatory index MMP-2 in cow plasma and initial colostrum
associated with SF68 infusion might imply subclinical systemic and mammary gland
inflammation.
Lower casein composition in the initial colostrum might reveal suboptimal mammary
synthetic capacity at partum, and, therefore, warrants more investigations.
TravelingMinistry of Science and Technology, Taiwan, ROC (MOST-103-2922-I-005-074)
SHIUH GER TRADING CO., LTD. Taiwan, ROC
Dr. Chen Shuen-Ei, Department of Animal Science, National Chung Hsing University
AdviserChai Ju Chang, Ph.D. (Texas A&M University, USA)
Professor,
Department of Animal Science,
National Chung Hsing University
Email : [email protected]
Thanks for your attention!
ACKNOWLEDGEMEMTS