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