03/03/2016

1

Low Emission Animal Feed

Effect of feed additives on methane emission from dairy cows

02-03-2016 – Geronda Klop

Feed additives in the LEAF project

Two main aspects:

1. Interaction between feed additives

In vivo experiment

2. Adaptation to feed additives

Stepwise approach

Additives and mode of action

Nitrate (NO3) and docosahexaenoic acid (DHA)

- Different mode of action

NO3

- Alternative H2 sink in the rumen

- Persistently lowers CH4

- Shift towards higher acetate proportion in rumen fluid

DHA

- Affects microbial metabolism in the rumen

- Lower CH4 production in vitro

- Shift towards higher propionate proportion in rumen fluid

Why interested in testing interaction?

Same effect on CH4 at lower dose of additives

Alleviate potential adverse effects – e.g.:

● NO3 methemoglobinemia

● DHA reduced feed intake

4

Materials and Methods

2x2 factorial arrangement of treatments:

7 blocks of 4 cows

20 multiparous and 8 primiparous

(26 cows included in analyses – feeding error)

NO3 No Yes No Yes

DHA No No Yes Yes

Diets and feeding

Fed as total mixed ration (TMR)

NO3: 21 g/kg DM

DHA: 3.0 g/kg DM

On DM basis:

20% Grass silage

50% Corn silage

30% Concentrates (TRT)

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2

Materials and Methods

Before start of experiment ‘pre-adaptation’ for cows assigned to a NO3 diet

No DHA in the diet during pre-adaption

Blood samples to monitor MetHb levels

Day % control concentrate

% NO3

concentrate

1-6 75% 25%

7-13 50% 50%

14-20 25% 75%

21-.... 0% 100%

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Housing Tie stalls Climate resp.

chambers

Feeding ad libitum restricted

Materials and Methods

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Housing Tie stalls Climate resp.

chambers

Feeding ad libitum restricted

Measurements

- DMI - Milk production

Materials and Methods

Measurements/samples

- Gaseous exchange (CH4, CO2, O2) - DMI - Milk production - Milk composition (fat protein lactose) - E and N balance - Digestibility(marker)

Day 13 14 15 16 17

Housing Climate resp.

chambers

Feeding Restricted .

Materials and Methods

5.9 5.9 5.6 5.6

1.3 1.3 2.5 3.4

0.0

5.0

10.0

15.0

20.0

25.0

30.0

25% 50% 75% 100%

NO3 dose (% of treatment dose)

avg Hb (mmol/L) avg MetHb (%Hb)

Results: Hb and MetHb

Subclinical methemoglobinemia

363

263

369

298

0

50

100

150

200

250

300

350

400

CON NO3 DHA NO3+DHA

CH

4 g

/d

Results: CH4 in g/d Significance

NO3 ***

DHA *

N×D †

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3

22.0

16.9

22.4

18.2

0

10

20

30

40

CON NO3 DHA NO3+DHA

CH

4 (

g/

kg

DM

I)

Results: CH4 in g/kg DMI Significance

NO3 ***

DHA †

N×D NS

13.1

10.8

15.4

12.6

0

5

10

15

20

25

CON NO3 DHA NO3+DHA

CH

4 (

g/

kg

FP

CM

)

Results: CH4 in g/kg FPCM Significance

NO3 ***

DHA **

N×D NS

Results: Feed intake and milk yield

Protein g/kg

31.2 30.4 31.0 29.5 * NS NS

Item CON NO3 DHA NO3+DHA

Significance

NO3 DHA NO3×DHA

Milk kg/d

27.8 25.1 28.0 28.0 NS NS NS

FPCM kg/d

27.9 24.7 24.2 23.8 NS † NS

Fat g/kg

40.9 39.5 29.8 29.4 NS *** NS

DMI kg/d

16.5 15.7 16.5 16.4 * * †

Conclusions

CH4 emission lowered by dietary NO3, but not by DHA

In this trial both additives affected milk composition

DHA – reduced milk fat%

NO3 – reduced milk protein%

No apparent interaction effects on output parameters of interest (with restricted feeding)

For more details.... Feed additives in the LEAF project

Two main aspects:

1. Interaction between feed additives

In vivo experiment

2. Adaptation to feed additives

Stepwise approach

03/03/2016

4

Adaptation to feed additives: Background

Problem: Adaptation to feed additives with potential to reduce CH4

Alternating use of feed additives Solution?

Required:

● Two suitable additives (rumen adaptation)

● Info on duration of the effect of each additive on CH4

● Indication for mitigation potential and adaptive effects needed

Alternating use of feed additives

Three steps required for adequate testing of the concept:

1 •1st step: In vitro screening

2

•2nd step: In vitro/In vivo (adaptation) experiment

3

•3rd step: In vivo experiment using climate respiration chambers (CRC)

1st step: In vitro screening

Activated Charcoal (2.5% of DM)

Activated Charcoal (5% of DM)

Vitamin C (ascorbic acid) (5% of DM)

Coconut Oil (5% of DM)

Krabok Oil (5% of DM)

Lauric acid (C12:0) (5% of DM)

Myristic acid (C14:0) (5% of DM)

Agolin Ruminant® (0.01% of DM)

BES (positive control) (3% of DM)

Alternating use of feed additives

Three steps required for adequate testing of the concept:

1 •1st step: In vitro screening

2

•2nd step: In vitro/In vivo (adaptation) experiment

3

•3rd step: In vivo experiment using climate respiration chambers (CRC)

2nd step: In vitro / in vivo

9 fistulated cows

3 experimental diets

CON Control (no additive)

AR Agolin Ruminant®

~1 g/cow/d

C12:0 Lauric acid ~3% of total DM

In vitro measurements of adaptation in vivo

2nd step: In vitro / in vivo

Inoculum Substrate

Cow Diet

1 CON CON, AR, C12:0, -

2 AR CON, AR, C12:0, -

3 C12:0 CON, AR, C12:0, -

4 CON CON, AR, C12:0, -

5 AR CON, AR, C12:0, -

6 C12:0 CON, AR, C12:0, -

7 CON CON, AR, C12:0, -

8 AR CON, AR, C12:0, -

9 C12:0 CON, AR, C12:0, -

9 cows

3 exp. diets

3 substrates (similar to exp. diets) + 1 blank

Duplicate CH4

measurements

Gas production

measurements in triplicate

90 bottles

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2nd step: In vitro / in vivo Measurements

Cows - DMI intake - Milk production - Feed composition IVGPT - Total gas production - CH4

- VFA, pH, OMD (end of run)

wk1 wk2 wk3 wk4 wk5 wk6 wk7

End of Exp.

- Housing in freestall barn - Start adaptation to basal diet

TMR (DM basis) 40% corn silage 30% grass silage 30% control concentrate

Housing in tie stalls Adaptation to basal diet Feed restriction from d12 onwards Day 15: 1st IVGPT run (no additives)

- Start feeding additives - 5 IVGPT runs (48h): day 1,4,8,15 and 22

Preliminary results (in vivo)

Diet CON AR C12:0

DMI, kg/d 19.8 20.1 16.2

Milk production, kg/d 30.0 30.8 28.9

FPCM, kg/d 30.7 31.7 26.2

Fat, g/kg 41.8 42.6 32.5

Protein, g/kg 34.5 33.0 31.1

Lactose, g/kg 46.7 45.6 44.2

Preliminary results (in vitro)

150.0

200.0

250.0

300.0

350.0

0 2 4 6 8 10 12 14 16 18 20 22

gas p

ro

du

cti

on

ml/

g

OM

in

cu

bate

d (

48

h) CONdiet

ARdiet

C12diet

150.0

200.0

250.0

300.0

350.0

0 2 4 6 8 10 12 14 16 18 20 22

gas p

ro

du

cti

on

ml/

g

OM

in

cu

bate

d (

48

h)

Days relative to start of TRT

CONsub

ARsub

C12sub

no substrate x diet interaction

Preliminary results (in vitro)

0.0

10.0

20.0

30.0

40.0

50.0

0 2 4 6 8 10 12 14 16 18 20 22

CH

4 p

ro

du

cti

on

ml/

g

OM

in

cu

bate

d (

48

h) CONdiet

ARdiet

C12diet

0.0

10.0

20.0

30.0

40.0

50.0

0 2 4 6 8 10 12 14 16 18 20 22

CH

4 p

ro

du

cti

on

ml/

g

OM

in

cu

bate

d (

48

h)

Days relative to start of TRT

CONsub

ARsub

C12sub

no substrate x diet interaction

Preliminary results (VFA in rumen fluid)

Differences in VFA profile before incubation

Similar difference in fermentation fluid after incubation

Different microbial composition of rumen fluid?

Total VFA (mmol/L)

Diet CON AR C12:0

Day 0 88.0 89.8 89.4

Day 1 76.6 82.5 60.1

Day 4 86.3 90.2 67.5

Day 8 81.5 88.5 70.0

Day 15 82.0 91.8 74.2

Day 22 81.4 94.9 88.5

Acetate:Propionate

CON AR C12:0

4.54 4.09 4.19

4.17 4.23 2.93

3.88 4.07 2.72

4.34 3.98 2.56

4.41 4.05 2.15

4.19 4.18 2.41

Preliminary results (OMD)

Organic matter degraded (48h)

Diet Control AR C12:0

Sub CON AR C12:0 CON AR C12:0 CON AR C12:0

Day 0 844 845 760 843 823 782 835 845 786

Day 1 837 841 771 839 823 767 842 817 739

Day 4 837 843 783 836 824 774 775 808 731

Day 8 829 816 775 838 836 765 809 798 718

Day 15 825 831 741 831 834 779 791 792 746

Day 22 832 821 763 832 828 769 794 788 748

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Summary

C12:0 at inclusion level of 3% of DM reduced intake and milk- and milk fat yield

Indications for adaptive response to AR

Effects of donor diet on VFA profile in rumen and fermentation fluid

Effects of donor diet on OM digestibility

Alternating use of feed additives

Three steps required for adequate testing of the concept:

1 •1st step: In vitro screening

2

•2nd step: In vitro/In vivo (adaptation) experiment

3

•3rd step: In vivo experiment using climate respiration chambers (CRC)

3rd step: In vivo experiment

Feed additive treatment

CON Only Agolin Ruminant® (AR) ~1 g/cow/d

ALT Weekly rotation of Agolin Ruminant® (AR) ~1 g/cow/d and lauric acid (C12:0) ~2% of total DMI

Again basal diet composed of 30% grass silage, 40% corn silage and 30% concentrates (treatments) on a DM basis.

Fistulated cows (group 1)

Non-fistulated cows (group 2)

3rd step: In vivo experiment

Cow Block (pair)

1 1

2 1

3 2

4 2

5 3

6 3

7 4

8 4

TRT

CON

ALT

CON

ALT

CON

ALT

CON

ALT

Experimental setup

Alternate use repeated 5 times (10 weeks)

“Background” CH4 production was measured on a similar basal diet without TRT additives

Thereafter setup as follows (per block):

TRT CRC Tie stalls CRC Tie stalls CRC

Wk 1 2 3 4 5 6 7 8 9 10

CON AR AR AR AR AR AR AR AR AR AR

ALT AR C12 AR C12 AR C12 AR C12 AR C12

Materials and Methods

Housing CRC Tie stalls

Week 1 2 3 4

CON AR AR AR AR

ALT AR C12 AR C12

• Gaseous exchange (CO2, O2, CH4) • DMI • Milk production and composition • Digestibility (marker)

• DMI • Milk production

• Rumen fluid (pH, vfa) on days 1 and 2 after each diet switch

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Preliminary results

CH4

g/d

408 405 378 336 386 328 386 316

g/kg FPCM

14.5

17.2 13.2 12.1 13.6 12.9 14.1 13.1

Basal diet (no additives)

Week 1+2 Week 5+6 Week 7+8

CON ALT CON ALT CON ALT CON ALT

... To be continued ...

Thank you!