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© 2014 Cargill, Incorporated. All rights reserved.GGAA 2016 CONFIDENTIAL. This document contains trade secret information. Disclosure, use or reproduction outside Cargill or inside
Cargill, to or by those employees who do not have a need to know is prohibited except as authorized by Cargill in writing.
© 2013 Cargill, Incorporated. All rights reserved.
The Effect of Dietary Nitrate on
Enteric Methane Emissions and
Methaemoglobin in Ruminants:
a Meta-Analysis
www.cargill.com/feed
Jolien Veneman, John Newbold,
Hink Perdok, Jamie Newbold
February 2016
Introduction
NITRATE
• Nitrate acts as alternative hydrogen sink when being reduced to
ammonia (Ungerfeld and Kohn, 2006)
NO3- + H2 → NO2
- + H2O
NO2- + 3∙H2 + 2∙H+ → NH4
+ + 2∙H2O
• Both reactions s have a greater negative ΔG value than methane
formation, through the reduction of CO2, therefore nitrate reduction
would be favoured over methanogenesis as a hydrogen sink
• If nitrate is completely reduced to ammonia then 100 g nitrate could
‘compete’ with 25.8 g of methane (stoichiometric potential)
• When nitrite forms, and is not quickly reduced to ammonia, it can be
absorbed across the rumen wall to bind to Heamoglobin, forming
Metheamoglobin (MetHb). This can lead to reduced performance or in
extreme situations death at high levels of MetHb
2
Introduction
META- ANALYSIS
• Several newly published studies on nitrate supplementation and methane emissions
• Not included in Lee and Beauchemin (Dec., 2014) 11 comparisions.
Aim
• Study the effect of nitrate supplementation on methane emissions (g/kg DMI) Calculate efficacy of nitrate
• The effect of nitrate on MetHb%
• Which moderators influence this effect
3
Materials and Methods;Data selection
STUDIES
• 18 papers
Total comparisons between a control and nitrate treatment
• 27 for Methane yield, 21 for MetHb%
Inclusion criteria;
• Within each experiment, control and nitrate diets contained equal concentrations of crude protein.
• Methane yield was expressed as methane emissions per kg dry matter intake (DMI) and MetHb as % of total haemoglobin.
4
Materials and methods; analysisMETAFOR PACKAGE IN R (VIECHTBOUWER, 2010)
• Mean difference (MD) (Treatment – Control) can be used when units (and range) are the same
• Using Random effects model to test overall effect of nitrate;
• θi = µ + ui
• Mixed effects model to test effect of moderators;
• θi = β0 + β1 x1p + ……. + βp xip + ui,
• Each moderator was first tested individually and included if P<0.10 to construct final model
• Animal type (beef cattle, dairy cattle or sheep; class)
• Dose (g NO3/kg DM; continuous)
• Feeding management ( or ; class)
• Treatment duration (weeks; continuous)
• Treatment type (coated or plain Ca Nitrate; class)
5
Results METHB (N=21)
Overall significant increase in
MetHb% by 1.2% units
(±0.35SE; P<0.001)
Significant effect for feeding
management
Restricted fed (n=11)
• MetHb +2.4% (±0.47SE;
P<0.001)
• Max values increased at dose
>18 g NO3/kg DM
Ad libitum fed (n=10)
• MetHb +0.4% (±0.31SE;
P=0.17)
• Max MetHb level did not
exceeded 7.2%
6
0
5
10
15
20
25
30
0 5 10 15 20 25 30
Me
tHb
% M
ea
n a
nd
Ma
x
Nitrate dose (g/kg DM)
Restricted/fixed fed;
Treatment mean and maximum
0
5
10
15
20
25
30
0 5 10 15 20 25 30
Me
tHb
% M
ea
n a
nd
Ma
x
Nitrate dose (g/kg DM)
Ad libitum fed;
Treatment mean and maximum
Animal type (beef cattle, dairy
cattle or sheep)
Feeding management ( or
)
Results
METHANE (G/KG DMI; N=27)
Overall significant reduction in methane yield (-3.6 g/kg DMI; ±0.33SE;
P<0.001)
Significant effect of dose
• nitrate decreases methane by 0.21g (±0.035SE; P<0.001) per g nitrate
7
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management
( or )
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 5 10 15 20 25 30
Eff
ec
t s
ize
(T
rt -
Co
ntr
ol)
Nitrate dose (g/kg DM)
Treatment MD with nitrate dose
Results METHANE (G/KG DMI; N=27)
20.8 gram per 100 gram instead of 25.8 gram = 80.6% efficacy
No apparent decrease in methane decreasing efficacy with increasing
nitrate dose
8
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management
( or )
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30
eff
ica
cy (
rea
l re
du
cti
on
/sto
ich
iom
etr
ic p
ote
nti
al)
nitrate dose (g/kg DM)
Efficacy per comparison
with increasing dose of nitrate
© 2014 Cargill, Incorporated. All rights reserved.GGAA 2016-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
0 5 10 15 20 25 30
Eff
ect
siz
e M
D (
Trt
-C
on
tro
l)
Duration of study (weeks)
Results
DMI (N=39)
Small overall negative
effect -0.12kg
(SE±0.06:P<0.003)
• No significant effect of
dose, animal type,
coating or feeding
management.
• Significant effect of exp.
Duration with decrease
in DMI with longer
experiments
9
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
0 5 10 15 20 25 30
Eff
ect
siz
e M
D (
Trt
-C
on
tro
l)
Nitrate dose (g/kg DM)
Treatment Mean Difference
Treatment MD, effect of exp. duration
Results
PH (N=11)
• Overall increase in pH with
0.06 points (SE±0.003;
P=0.003)
• Significant effect of animal
(P=0.0148) with sheep
and beef cattle showing a
significant increase, but
not in dairy cattle.
10
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0 5 10 15 20 25 30
Ch
an
ge i
n p
H (
Trt
-C
on
tro
l)
Nitrate dose (g/kg DM)
pHTreatment MD with dose
-0.05
0.00
0.05
0.10
0.15
0.20
1
pHbeef cattle
dairy cattle
sheep
Results
TOTAL VFA
(n=11)
• Overall no effect of
nitrate on VFA
• Tendency (P=0.093) for
effect of animal type with
only sheep showing a
significant increase in
tVFA
11
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-50
-40
-30
-20
-10
0
10
20
30
0 5 10 15 20 25 30
Eff
ect
siz
e (
Trt
-C
on
tro
l)
Nitrate dose (g/kg DM)
tVFA
-4
-2
0
2
4
6
8
10
12
1
tVFAbeef cattle
dairy cattle
sheep
Treatment MD
Treatment MD
Remove ‘only’ before sheep
© 2014 Cargill, Incorporated. All rights reserved.GGAA 2016
Results
ACETATE %
(n=15)
• Overall nitrate increases
Acetate by 2.46%
(P<0.001)
• Effect of nitrate or coated
nitrate, with coated nitrate
not increasing Ac%
• Effect of feeding
management, with
restricted feeding
increasing Ac% more
12
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-2
0
2
4
6
8
10
0 5 10 15 20 25 30
Ch
an
ge i
n a
ceta
te %
(T
rt -
Co
ntr
ol)
Nitrate dose (g/kg feed DM)
Ac%Treatment
-2
-1
0
1
2
3
4
5
coated nitrate nitrate (not coated)
MD in Ac%
0
1
2
3
4
5
6
ad libitum restricted
MD in Ac%
Type of feeding managementType of nitrate
Results
PROPIONATE %
(n=15)
• Overall nitrate decreases
propionate wby 2.17% s
(P<0.001)
• Effect of type of nitrate,
with coated nitrate not
decreasing Pr%
13
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-12
-10
-8
-6
-4
-2
0
2
4
0 5 10 15 20 25 30
Ch
an
ge i
n p
rop
ion
ate
% (
Trt
-C
on
tro
l)
Nitrate dose (g/kg DM)
Pr%Treatment
-4
-3
-2
-1
0
1
2
coated nitrate nitrate (not coated)
MD in Pr%Type of nitrate
Results
N-BUTYRATE %
(n=15)
• Overall no effect of
nitrate on Butyrate %
(P=0.94)
• No other moderators had
an effect (e.g. dose,
duration)
14
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or )
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
0 5 10 15 20 25 30
Ch
an
ge i
n b
uty
rate
% (
Trt
-C
on
tro
l)
Nitrate dose (g/kg DM)
Bu%
Treatment
Results
AMMONIA
(n=14)
• Overall nitrate showed a
tendency to decrease
ammonia by 1.1 mmol/L
(P=0.08)
• Significant effect of
experiment duration
(P<0.001) with ammonia
decreasing with
increasing duration
15
Animal type (beef cattle,
dairy cattle or sheep)
Feeding management (
or ) -10
-8
-6
-4
-2
0
2
4
6
0 2 4 6 8 10 12 14
ch
an
ge i
n a
mm
in
mM
(T
rt -
Co
ntr
ol)
Treatment duration (weeks)
Ammonia, mM
-8
-6
-4
-2
0
2
4
6
0 5 10 15 20 25 30
ch
an
ge i
n a
mm
in
mM
(T
rt -
Co
ntr
ol)
Nitrate dose (g/kg DM)
Ammonia, mM
Treatment effect with nitrate dose
Treatment effect with duration of experiment
ConclusionMETA-ANALYSIS
MetHb%
• When nitrate-fed ruminants are limit-fed (e.g. to 95% of ad libitum intake) mean MetHb is increased significantly by 2.4% compared to the control.
• MetHb% is not different from the control when cattle or sheep are fed ad libitum
• Risk of increased MetHb% is higher in limit fed animals
Methane Yield
• Nitrate reduced methane yield in a linear dose response manner ( 80.6% efficacy)
16
© 2014 Cargill, Incorporated. All rights reserved.GGAA 2016
Acknowledgements
• ANIMAL CHANGE
• IBERS Aberystwyth University
• Cargill Animal Nutrition
17
Data (appendix)INCLUDED IN THE META-ANALYSIS
18
REF-ID YEARCOMPA
-RISONCOUNTRY ANIMAL
FIXED or
AD LIBTREATMENT
DURATION
IN WEEKS
MAX*
DOSEn CH4 DMI RUMEN METHB
deRaphélisSoissan 01 2014 1 Australia Sheep Fixed Nitrate 10 20 7 x x x x
El-Zaiat 01 2014 1 Brazil Sheep Ad lib Coated nitrate 13.1 27 6 x x x x
Guyader 01 2015 1 France Dairy Fixed Nitrate 5 23 4 x x x x
Guyader 02 2015 1 France Dairy Fixed Nitrate 5 23 4 x x x x
Hulshof 01 2012 1 Brazil Beef Ad lib Nitrate 3.3 22 14 x x x
Lee 01 2015 3 Canada Beef Ad lib Coated nitrate 4 23 8 x x
Lee 02 2015 3 Canada Beef Ad lib Coated nitrate 4 23 8 x x x
Li 01 2012 1 Australia Sheep Both Nitrate 7.7 19 5 x x x x
Li 02 2013 1 Australia Sheep Both Nitrate 9.3 19 5 x x x
Newbold 01 2014 5 USA Beef Fixed Nitrate 6.1 28 6 x x x
Newbold 01 2014 5 Brazil Beef Ad lib Nitrate 15.7 24 5 x
Nolan 01 2010 1 Australia Sheep Fixed Nitrate 3 25 4 x x x x
Troy 01 2015 4 UK Beef Ad lib Nitrate 24 22 6 x x x
vanZijderveld 01 2010 1 Netherlands Sheep Fixed Nitrate 4 26 5 x x x
vanZijderveld 04 2011 1 Netherlands Dairy Fixed Nitrate 12.7 21 10 x x x
Velazco 01 2013 1 Australia Beef Ad lib Nitrate 6 19 11 x x
Veneman 01 2015 1 New Zealand Dairy Both Nitrate 4.6 20 6 x x x x
Veneman 01 2015 1 UK Dairy Ad lib Nitrate 5 20 6 x x x x
Olijhoek 01 2014 3 Denmark Dairy Ad lib Nitrate 4 21 4 x x x x
* formulated dose of nitrate in g/kg feed DM
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