Subacute ruminal acidosis and the

global profile of ruminal and fecal

microbiota of dairy cows

J.L. Martinez, E. Sandri, Y. Couture, R. Gervais, J. Levesque,

D. Roy, and D. E. Rico*

Oesophagus

Rumen

Reticulum

Omasum

Abomasum

Caecum

Rectum

Small intestin

Inflammation

cascade

LPS

in blood

Permeability

Permeability

LPS

LPS

LBP*

*Lipopolysacharide

binding protein

Lourenço et al., 2010

Linoleic acid

Linolenic acid

Hartfoot and Hazlewood, 1997

*Group A B

A

A

B

A + B

Propionibacterium acnes

A + B

A B

Ruminal biohydrogenationof PUFA

Vlæminck et al. 2005Fievez et al. 2012

Odd chain FA

Branched chain FA

Milk OBCFA

anteiso

Amylolytic

bacteria

Amylolytic bacteria

iso

Cellulolytic

bacterua

Propionate, Valerate(odd chain VFA)

Val, Leu, Ile(branched chain AA)

Rumen

Mammary

gland

Credit: Rachel Gervais

COOH

COOH

COOH

COOH16:0

17:0

iso 17:0

anteiso 17:0

acetate C2

propionate C3

Volatile branched chain

Fatty acids

Credit: Rachel Gervais

Twelve ruminally cannulated cows (120 ± 52 DIM; 35.5 ± 8.9 kg of milk/d; mean ± SD)

1) SARA induction; (29% starch, 24% NDF, and

2.8% fatty acids)

2) Control / Recovery ; (20% starch, 31% NDF,

and 2.3% fatty acids)

Cow Pre-Exp. Period 1 Period 2 Period 3

1 Control Control Induction Recovery

2 Induction Recovery Control Induction

3 Control Induction Recovery Control

pH recorded every 5 min over a

24h period on d 0, 3, 7, 14, 21

• Milk fat (d 0, 3, 7, 14, 21) extracted and profile determined by gas

chromatography

• Genomic DNA from whole rumen digesta (liquid and solid fractions; d 7 and 21)

were extracted and subjected to Illumina sequencing of 16S rRNA (V3-V4

region).

• Default QIIME pipeline was used to identify operational taxonomic units

• Data analyzed using MicrobiomeAnalyst

5,0

5,2

5,4

5,6

5,8

6,0

6,2

6,4

0 7 14 21

Time, d

A

A

0

1

2

3

4

5

6

7

8

0 7 14 21

Time, d

A

R

SARA Recovery Control

A

Mean rumen pH Time below 5.6, h/d

Trt x time P < 0.001

Trt x time P < 0.001

Ecow, UK

CON vs. SARA (A = P < 0.05; a = P < 0.10)CON vs. Recovery (R = P < 0.05; r = P < 0.10)

2,5

3,0

3,5

4,0

4,5

5,0

0 7 14 21

%

Time, d

R

A

r

Trt x time P = 0.001

0,8

1,0

1,2

1,4

1,6

0 7 14 21

kg

/d

Time, d

a

rA

Trt x time P = 0.001

SARA Recovery Control

Milk fat concentration Milk fat yield

0,0

0,2

0,4

0,6

0,8

1,0

0 7 14 21Time, d

AR

Trt x time P = 0.001

trans-10/trans-11 ratio

SARA Recovery Control

% o

f m

ilk f

at

Positively associated with the trans-10/trans-11 ratio (r=0.70 to 0.83)

trans-10, cis-12 CLA not detected

0 0,2 0,4 0,6 0,8 1 1,2

c__Gammaproteobacteria

o__Aeromonadales

f__Succinivibrionaceae

p__Proteobacteria

g__5_7N15 2

f__Veillonellaceae

g__Acidaminococcus

g__

g__Shuttleworthia

ratio t10/t11

Ratio trans-10/trans-11 18:1

FecalRumen

p__Actinobacteriap__Bacteroidetesp__Elusimicrobiap__Fibrobacteresp__Firmicutesp__Proteobacteriap__Spirochaetesp__SR1p__TM7p__Verrucomicrobiap__WPS_2

Chao 1 diversity.

P value: < 0.0001

*

*

*

*

*

Shannon diversity. P value < 0.05Chao 1 diversity. P value = 0.13

-5 -3 -1 1 3 5

f__

p__SR1

f__Paraprevotellaceae

f__Christensenellaceae

p__TM7

c__TM7_3

o__CW040

f__F16

g__CF231

g__Selenomonas

p__Verrucomicrobia

c__Verruco_5

o__WCHB1_41

f__WCHB1_25

g__Mogibacterium

p__Actinobacteria

c__Actinobacteria

o__Bifidobacteriales

f__Bifidobacteriaceae

LDA score, Log 10

Rumen Feces

-6 -1 4

f__Ruminococcaceae

g__

g__Bacteroides

c__Erysipelotrichi

o__Erysipelotrichales

f__Erysipelotrichaceae

g__Roseburia

f__Lachnospiraceae

LDA score, Log 10

SARA Control

50

80

110

140

170

200

0 7 14 21

Time, d

Aa

Trt x time P < 0.001

No effects detected on plasma cytokines:IFNα, IFNγ, IL-13, IL-1α, IL-1 F5, IL-21, IP-10, MIG, MIP-1β, and TNFα

LBP, ng/mL

SARA Recovery Control

-0,4 -0,2 0 0,2 0,4 0,6 0,8 1

f__Paraprevotellaceae

p__Proteobacteria

f__Succinivibrionaceae

c__Gammaproteobacteria

o__Aeromonadales

g__Acidaminococcus

g__

LBP

LBP and rumen microbiota

-0,8 -0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1

g__ 2

f__Ruminococcaceae 2

s__ 2

c__Clostridia 2

o__Clostridiales 2

g__Acidaminococcus 2

p__Actinobacteria 2

f__Bifidobacteriaceae 2

c__Actinobacteria 2

o__Bifidobacteriales 2

g__Epulopiscium 2

g__Ruminococcus 2

f__S24_7 2

g__Bifidobacterium 2

s__pseudolongum 2

LBP

LBP and fecal microbiota

Possible

markers of

endotoxemia

Vlæminck et al. 2005Fievez et al. 2012

Odd chain FA

Branched chain FA

Milk OBCFA

anteiso

Amylolytic

bacteria

Amylolytic bacteria

iso

Cellulolytic

bacterua

Propionate, Valerate(odd chain VFA)

Val, Leu, Ile(branched chain AA)

Rumen

Mammary

gland

Credit: Rachel Gervais

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0 7 14 21

Time, d

AR

Trt x time: P < 0.001

C13:0

0,8

1,0

1,2

1,4

1,6

1,8

2,0

0 7 14 21

Time, d

AR

Trt x time: P < 0.001

C15:0

SARA Recovery Control

% o

f m

ilk f

at%

of

milk

fat

0 0,2 0,4 0,6 0,8 1

g__Mitsuokella

s__multacida

g__Shuttleworthia

f__Bacteroidaceae

g__5_7N15

g__Succiniclasticum

g__Acidaminococcus

p__Proteobacteria

c__Gammaproteobacteria

o__Aeromonadales

f__Succinivibrionaceae

s__

f__Veillonellaceae

g__

odd chain

Odd chain FA

0,30

0,35

0,40

0,45

0,50

0 7 14 21

Time, d

AR

r

AATrt x time: P < 0.001

C15:0 anteiso

C17:0 anteiso

0,3

0,34

0,38

0,42

0,46

0 7 14 21

Time, d

ARTrt x time: P = 0.02

SARA Recovery Control

% o

f m

ilk f

at%

of

milk

fat

0 0,2 0,4 0,6 0,8 1

s__F. succinogenes

p__Fibrobacteres

c__Fibrobacteria

o__Fibrobacterales

f__Fibrobacteraceae

g__Fibrobacter

p__SR1

anteiso

Anteiso FA

0,00

0,05

0,10

0,15

0,20

0 7 14 21

Time, d

A

R

Trt x time: P < 0.001

R

C14:0 iso

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0 7 14 21

Time, d

A

R

Trt x time: P < 0.001

C16:0 iso

SARA Recovery Control

% o

f m

ilk f

at%

of

milk

fat

0 0,2 0,4 0,6 0,8 1

p__Fibrobacteresc__Fibrobacteria

o__Fibrobacterales

f__Fibrobacteraceae

g__Fibrobacter

c__Clostridia

o__Clostridiales

p__Firmicutes

g__Selenomonas

s__ruminantium

g__Moryella

s__D168

g__Butyrivibrio

f__RF16f__Paraprevotellaceae

f__Mogibacteriaceae

s__succinogenes

g__YRC22

g__Pseudobutyrivibrio

f__Clostridiaceae

g__Clostridium

g__Ruminococcus

f__Ruminococcaceae

f__Christensenellaceae

f__

isoIso FA

• Induction of SARA resulted in a rapid and transient

reduction in milk fat synthesis, which was not strongly

associated with ruminal biohydrogenation .

• Milk odd and branched fatty acids closely followed

acidosis.

• Ruminal and fecal microbiota were affected by SARA

and may be used as predictors of observed responses.

Thanks!

CRSAD, Deschambault, QC, Canada

Daniel Rico, Ph.D.email: daniel.rico@crsad.qc.ca.

Questions?

25

29

33

37

41

45

0 7 14 21

kg

/d

Time, d

A

R

r

Trt x time P < 0.001

20

22

24

26

28

30

32

0 7 14 21

kg

/d

Time, d

Ar

A

A

Trt x time P < 0.001

SARA Recovery Control

Dry matter intake Milk yield

40

60

80

100

120

140

0 7 14 21

μEq

/L

Time, d

Aa

r

5

10

15

20

25

30

0 7 14 21

μg

/L

Time, d

A

R

r

0,03

0,05

0,07

0,09

0,11

0 7 14 21

mm

ol/

L

Time, d

A

SARA

Recovery

Control

Insulin

NEFA BHBA

All Trt x time P = 0.001

[PERMANOVA] R-squared: 0.11; p-value < 0.057

Adapted from Shingfield and Griinari, 2007

trans-10, cis-12 CLA, g/100 g of fat

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

0

-10

-20

-30

-40

-50

-60

Milk fat

change, %

Abomasal infusion of CLA t10c12(n =11); R2 = 0.86

High concentrates+ vegetable oil

Fish oil

Fish + vegetable oil

?

ALC

Oesophagus

Rumen

Reticulum

Omasum

Abomasum

Caecum

Rectum

Small

intestin

Milk fat?

Insulin

NEFA?

Inflammation

cascade

LPS in blood