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Approaches to improve efficiency
of N utilisation on dairy cow level
Mogens Vestergaard
Aarhus University, Denmark
Final REDNEX Conference, FIAP, Paris 30 August 2013
2
URINE N
37%
MILK N
28%
FAECAL N
33%
N INTAKE
503 g/day
Mills et al 2009
Why are dairy cows a concern?
3
0
50
100
150
200
250
300
350
400
0 200 400 600 800 1000
Nit
roge
n in
milk
, fae
ces
or
uri
ne
, g/d
ay
Nitrogen intake, g/day
Urine N Faecal N Milk N
Mills et al., 2009.
Meta Analysis of N Balance in Dairy Cows
Milk N/N Intake vs. N Intake
Mills et al., 2009
Approaches to improve efficiency
of N utilisation on dairy cow level
Research on N digestion and N metabolism:
Rumen (WP2)
Amino acid and nutrient metabolism (WP3)
N recycling (WP4)
WP2 Maximisation of absorption of feed
and microbial protein in low-N diets
Michel Doreau (WP leader),
Pierre Nozière, Diego Morgavi
Jamie Newbold, Jon Moorby
Alejandro Belanche
Sergio Calsamiglia, Alfred Ferret
Andreas Foskolos
Peter Lebzien
Martina Aschemann
rumen small intestine
1 Innovative ways to decrease
ruminal protein degradation
Ways to optimise
microbial protein
synthesis
2 Role of the microbial
ecosystem
especially protozoa
3
NH3
energy
minerals
Microbial
Protein
Feed Crude
Protein Degraded
Protein
Undegraded Protein
Microbial Protein
1. Innovative ways to decrease ruminal protein
degradation
A first innovative approach was the inclusion of essential oils in silages.
Essential oils in ryegrass silage are efficient in reducing silage protein
degradation but the dose required and thus the cost may be too high for
practical use
State-of-the-art : the most efficient additives for decreasing ruminal
protein degradation are various essential oils, but their effect in vivo
remains unclear
A second innovative approach was the inclusion of polyclonal antibodies
against proteolytic or deaminating bacteria. Several attempts failed to
decrease protein degradation
Capsicum oil and PTSO (propyl-propyl thiosulphate, derived from garlic oil
processing) were promising additives. We observed a trend to an incrase in
the ratio between N in milk and N intake with both additives (see next figure)
• Total N balance (g/day)
N intake
CTR 539
CAP 561
PTSO 505
N milk
CTR 160
CAP 171
PTSO 175
Urine N
CTR 216
CAP 219
PTSO 211
Fecal N
CTR 203
CAP 211
PTSO 188
MNE (%)
CTR 30.3
CAP 30.8
PTSO 34.3
1. N Balance study with two essential oils
(Capsicum oil and PTSO)
2. Ways to optimise microbial protein synthesis
Decreasing crude protein level to 11-12% of dietary DM in dairy cows
resulted in a moderate decrease in milk production in 1st experiment, but not
in 2nd experiment. In both experiments, urinary N was strongly decreased
Niacin supplementation to low-N diets did not change microbial protein flow
and efficiency of synthesis but increased protozoa population; the use of
fermentable protein by microbes may be changed (see next Table)
The source of dietary carbohydrates, starch or fibre, had a minor effect on
ruminal protein metabolism; a trend to a higher microbial protein flow and
efficiency of synthesis was observed with starch (see next Table)
Decreasing crude protein level to 11-12% of dietary DM in dairy cows had a
minor effect on the efficiency of microbial protein synthesis: no change in 1st
experiment, and only a trend to a decrease in 2nd experiment
In vitro, microbial protein yield did not differ between ryegrass and red clover,
but microbes were more efficient with ryegrass for capturing N and in
efficiency of N utilization in the rumen. Comparison of varieties (ryegrass
varying in sugar content, red clover varying in polyphenol oxidase) needs
further research
Normal N (14% CP) Low N (11% CP)
Starch Fibre Starch Fibre
Microbial N efficiency
g N / kg OM fermented
28 24 26 21 ns
OM digestibility, % 70 68 66 66 N**
Fanchone et al., 2013
Urea (15.6% CP)
Low (12% CP)
Microbial N efficiency
g N / kg OM fermented
32 30 ns
OM digestibility, % 72a 69b 71ab *
Aschemann et al., 2012
Low +Niacin (12% CP)
27
3. Characterisation and role of the microbial
ecosystem, especially protozoa
The role of different protozoa in bacterial breakdown has been specified.
Entodinium and Epidinium are especially active, whereas Holotrichs have a
minor predatory activity. Therefore, lowering numbers of Entodinum and
Epidinium species in the rumen may be a strategy for improving microbial
synthesis (see next Figure)
The identification of key bacteria involved in protein metabolism by using
DNA-Stable Isotope Probing was faced with strong methodological issues
and results were inconsistent
In normal- or low-N diets, defaunation (or faunation with an Holotrich
species) did not change rumen ammonia in sheep suggesting a better use
of N by rumen microbes, which results in a lower urinary N
Cows are able to adapt themselves to fibrous diets by increasing the
complexity of the rumen microbial community and the concentrations of
protozoa, anaerobic fungi, and methanogens. On the contrary, rumen
protozoa, fungi, methanogens and certain bacterial species are sensitive to
N shortage which can explain the observed decrease in OM digestibility
(Figure)
High Protein Low Protein
FIB STA FIB STA
Concentration (per g DM)
Bacteria (mg) 2.93 2.95 2.59 2.53
Protozoa (mg) 0.71 0.47 0.54 0.43
Anaerobic fungi (µg) 1.70a 1.40b 1.58ab 0.66c
Archaea (107 copies) 4.87 3.94 3.60 2.80
Bacterial diversity 147bc 148b 152a 138c
Fungal diversity 34a 32a 33a 27b
The relative abundance of the 6
major protozoal groups in rumen
of cattle and bacterial breakdown
attributed to each of these
protozoa groups.
(Belanche et al., 2012. J. Anim.
Sci)
Effect of the level of protein and type of carbohydrate on the rumen concentration of
certain microbial groups and their biodiversity. (Belanche et al., 2012. J. Nutr.)
3. Characterisation and role of the microbial
ecosystem, especially protozoa
The role of different protozoa in bacterial breakdown has been specified.
Entodinium and Epidinium are especially active, whereas Holotrichs have a
minor predatory activity. Therefore, lowering numbers of Entodiniomorphids
in the rumen may be a strategy for improving microbial synthesis (Figure)
The identification of key bacteria involved in protein metabolism by using
DNA-Stable Isotope Probing was faced with large challenges and results
were inconsistent
In normal- or low-N diets, defaunation (or faunation with an Holotrich
species) did not change rumen ammonia in sheep suggesting a better use
of N by rumen microbes, which results in a lower urinary N
Cows are able to adapt themselves to fibrous diets by increasing the
complexity of the rumen microbial community and the concentrations of
protozoa, anaerobic fungi, and methanogens. On the contrary, rumen
protozoa, fungi, methanogens and certain bacterial species are sensitive to
N shortage which can explain the observed decrease in OM digestibility
(Figure)
Take-home messages from WP2
• Improved knowledge of the relation between rumen
microbes (especially protozoa) and N ruminal
metabolism
• Lowering dietary N below present recommendations
decreases OM and fibre digestibility
• There is no adaptation to N underfeeding and
microbial protein synthesis is not more efficient
• No real innovative way to decrease ruminal protein
degradation was detected
Factors affecting the conversion of absorbed AA into milk protein – Understanding the determinants of the efficiency of dietary Nitrogen Utilisation
Reducing Nitrogen Excretion WP3
Improving the nitrogen economy of the dairy cow
Chris K. Reynolds, Sophie Lemosquet, Isabelle Ortiques et al.
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
100 125 150 175 200 225 250 275 300 325
Mil
k N
/Ap
pa
ren
tly d
iges
ted
N
Apparently digested N, g/d
EFFICIENCY OF N CONVERSION
STARCH
FIBRE
Feeding trial – INRA Theix
11% improvement in N milk / N intake with high starch diets JDS submitted
=> Metabolism trial
AVAILABLE MP, g PDIE/d
MIL
K P
RO
TEIN
, g
/d
18001600140012001000800600
900
800
700
600
500
400
ENERGY
FIBER
STARCH
Y = 225.4*** + 0.360X
Y = 173.1*** + 0.363X
EFFICIENCY OF MP UTILIZATION
improved with starch
N = 48; 3 Rednex experiments WP2.2.2 + WP3.4
Cantalapiedra-Hijar et al., 2012 (3R)
Main results so far
EFFECTS OF STARCH
Indications of higher microbial
protein synthesis (purines in urine)
JDS submitted
Increased whole-body metabolic
use of Leu (IRL) in favor of protein
synthesis
EAAP 2012
19
• Determine the effects of metabolisable protein (MP) supply fed both above and below metabolic requirement on post ruminal nutrient absorption and metabolism
• Determine the effects of different forage types (maize vs. grass silage) on post ruminal nutrient absorption and metabolism
Objectives – Univ. Reading
20 Protein (Linear), P < 0.001; Forage, P < 0.13
Nitrogen Intake
Barratt et al., 2013.
21
Efficiency of N Dietary Use
Barratt et al., 2013.
22 Forage, P < 0.06; Protein (Linear), P < 0.001
Arterial urea concentration
Barratt et al., 2013.
23
Efficiency of N Dietary Use
Milk
Barratt et al., 2013.
24
Conclusions Univ. Reading
Effects of increased protein intake
• Linear increase in DMI, milk yield, milk N, rumen ammonia and
PDV ammonia flux and arterial urea concentration
• Decrease in N efficiency with increasing dietary protein
Effects of forage
• Higher N intake on grass silage based diets
• No forage effect on DMI or milk yield
• Increased rumen ammonia, PDV ammonia flux and arterial urea
on grass silage based diets
• No forage effect on N efficiency (milk N/N intake)
• No forage × protein interactions seen
Clear positive relationship between N intake and rumen ammonia,
arterial urea, and milk urea concentration, all of which are
negatively related to N utilization efficiency
Barratt et al., 2013.
Research Questions - INRA Rennes
1. Does balancing the EAA profile increase milk protein yield and metabolisable protein (MP) efficiency both at low and high MP supply?
5 experiments: • 3 balancing the whole EAA profile through duodenum
infusions (4 to 6 cows)
Are mammary uptakes of AA modified? • 2 experiments with Lys, Met and Leu balanced through diet
(16 to 32 cows):
2. Among the 9 EAA to balance are Val, Ile and Arg important?
Milk protein and efficiency increased when balancing AA profile at low and high MP supply
Milk protein yield, g/d
1300
800
900
1000
1100
1200
700
80 90 100 110 120 PDI, g/kg DM
Exp 1
Exp 2
Exp 3
Exp 4
AA+
AA-
(CP from 13.5% to 19% of DM)
Rumen Intestin
AA
AA
AA
AA
Mammary net uptake of only the limiting EAA increased: a higher waste of nitrogen when increasing both intestinal
supplies of EAA and NEAA through increased MP
LPHP + 72 g/d of N
AA - AA+ = 0 g/d of N
LPHP Intestine EAA: + 28 g/d of N NEAA: + 24 g/d of N
AA-AA+ Intestine EAA: +28 g/d of N NEAA: - 28 g/d of N
Mammary Uptake = Output EAA only:
+ 10 g/d of N
Mammary Uptake = Output
EAA only: + 12 g/d of N
N efficiency 0.360.34
N efficiency 0.330.35
Take home messages WP3 1. Dietary starch increases efficiency of dietary N utilization through
effects on digestion and metabolism
2. Forage type has less of an effect when total rations are balanced for
major components (starch, NDF, etc.)
3. Clear relationships between N intake and NPN metabolism and thus
milk urea N is negatively correlated to N efficiency (this is relative to
WP6).
4. Improving the balance of EAA provided to the mammary gland
increases milk protein production across a range of metabolizable
protein supplies.
5. Further work is needed on BCAA requirements
6. Masses of new knowledge of the metabolism of amino acids and
other nutrients in lactating dairy cows fed diets below and above
requirements for MP.
Innovative and practical management approaches to reduce nitrogen
excretion by ruminants
WP4
New feeding strategies
improving N recycling while
reducing N inputs
Betina Amdisen Røjen,
Niels Bastian Kristensen &
Mogens Vestergaard
Dept. of Animal Science, AU-Foulum, Aarhus University,
Denmark
Increased N utilization by:
1. Reduced dietary N concentration
2. Increased blood to gut transport of urea-N
N-efficient dairy cow
3. Role of urea transporter proteins in
urea transport across ruminal epithelia
Feed protein
Salivary urea-N
Blood urea-N
Feed protein
Salivary urea-N
Blood urea-N
Kidney
protein degradation Hindgut
1. Optimize urea recycling
to the GI tract while reducing
N inputs
5. Competition between kidney and
gut for urea with increased salt and
water intake
4. Increased hindgut fermentation on
urea recycling and N utilization
2. Ability of blood urea to sustain rumen
ammonia from recycling with ‘infrequent’
N supply
Arterial urea-N concentration, mmol/L
2 4 6 8 10 12 14
Ru
min
al e
xtr
acti
on
of
art
eri
al u
rea-N
, %
0
5
10
15
20
25
30
35
r = -0.732; P <0.01
The total amount of urea transferred from blood to gut
does not increase with decreasing N level
The permeability of the gut epithelia for
urea is up-regulated with reductions in N
intake thus adapting to dietary conditions,
but it is not up-regulated enough when N
status of the cow get insufficient to sustain
optimal microbial protein synthesis
Arterial urea-N concentration, mmol/L
0 2 4 6 8 10 12 14
Ne
t p
ort
al u
rea
-N f
lux
, m
mo
l/h
-1000
-800
-600
-400
-200
0
Arterial urea-N concentration, mmol/L
0 2 4 6 8 10 12 14
Net
po
rtal u
rea-N
flu
x, m
mo
l/h
-1000
-800
-600
-400
-200
0
r = 0.185; P = 0.10
Arterial urea-N concentration, mmol/L
2 4 6 8 10 12 14
Ru
min
al e
xtr
ac
tio
n o
f a
rte
ria
l u
rea
-N, %
0
5
10
15
20
25
30
Low nitrogen diet
High nitrogen diet
1. A Low-N diet leads to increased ruminal tissue
permeability to urea
2. But immediate return of N in ammonia apparently not
equilibrated with the rumen ammonia pool
Time relative to urea infusion, min
-150 -100 -50 0 50 100 150Ru
min
al
vein
- a
rteri
al
am
mo
nia
, m
mo
l/L
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Low N
High N
1. 2.
Time relative to feeding, h
-0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5Ru
min
al
am
mo
nia
co
ncen
trati
on
, m
mo
l/L
0
2
4
6
8
10
12
14
16
Time relative to feeding, h
-0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5
Art
eri
al
blo
od
ure
a-N
, m
mo
l/L
0
2
4
6
8
10
12
Use of infrequent N supply (i.e., 6-h urea infusion, ▲) leads to sustained
increase in arterial urea concentrations 9-16 h after end of infusion
But there was no increase in urea recycling to the gut
So, the cow was unable to make use of blood urea via urea recycling to
sustain rumen ammonia concentrations during periods of the day where
rumen N supply was at a minimum
▲ 6-h inf urea
■ 24-h inf urea
● Water inf
Control Oligo
Ne
t p
ort
al fl
ux
, m
mo
l/h
-300
-200
-100
0
100
200
300
400
500
ammonia urea
Increased carbohydrate supply to the hindgut
induced the predicted reduction in blood urea,
but mainly through changes in ammonia fluxes -
not by increased urea recycling to the hindgut
Take home message - WP4:
Urea recycling is less efficient than hypothesized and difficult
to manipulate to increase N efficiency in dairy cows
This points to increased precision in dairy cattle nutrition as
the most feasible short-term strategy to improve N
efficiency
Silage
Additive
TM
R /
PM
R
COW1
COW2
COW3
COW4
Production
More use of
information we
already have
available
Better accuracy
and precision
Need for new tools to monitor
physiological status, nutritional
sufficiency, and nutrient utilization
Feces Urine
Silage
Premix
Feces Urine
Feces Urine
Feces Urine
Accuracy and precision obtained
through biological monitoring system
Milk
Milk
Milk
Milk
Conclusions
Ways to reduce N excretion
• Reduce N intake
• Adjust EAA composition in rations at a lower N intake
• Use starch-rich vs. fibre-rich rations
• Use of certain essential oils in the forage
• Reduce protozoa, especially large species
• Feed by-pass CHO to increase hindgut fermentation
• …
• Use precision feeding of protein …. and relevant
management tools (see also later talks)
Thank you for your attention
Questions ?
Culled organic dairy cows finishing their ‘service’ as
suckler cows for two bull calves on semi-natural pastures
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