Ketone and NEFA testing as diagnostic

tools in assessing transition dairy cows

Stephen LeBlancOABP/OABA meeting

April 14, 2005

Monitoring Programs for Transition Cows

1. Monitor Current Transition Cow Program – HERD LEVEL– Track success and compliance with existing program– Early detection of problems

2. Monitor for Subclinical Disease - INDIVIDUAL LEVEL

– Early treatment to prevent clinical disease

• Helps to quantify problems and direct investigation

Options for Monitoring or Investigating

• Clinical disease incidence• Milk production• DMI

– Why doesn’t it get done??– Group average; distribution within

group– Target > 12 kg DMI average in close-up

(heifers & cows; 3 weeks before due)– Fresh group

• Metabolic tests

Daily Dry Matter Intake Around Calving

*

CALVING

Re-esterified triglyceride

Stored in liver

Exported in VLDL

Gluconeo-genesis

PropionateAA

GlycerolNEFA

Glucose

Fetus Mammary gland

Incompletely

oxidized ketones

Completely oxidized energy BHB

Acetoacetate

Acetone

Re-esterified triglyceride

Stored in liver

Exported in VLDL

Gluconeo-genesis

PropionateAA

NEFA

Glucose

Fetus Mammary gland

Incompletely

oxidized ketones

Completely oxidized energy BHB

Acetoacetate

Acetone

Unsuccessful response

to NEB – Ketosis and Fatty liver

Typical patterns of DMI and NEFA

Overton/Burhans, 2001

Associations with health and

performance

•Pre-partum NEFA associated with:– ~ 4X increased risk of LDA

(Cameron et al, 1998; LeBlanc et al, 2005)

– ~ 1.5X increased risk of RP(Dyk, 1995; LeBlanc et al, 2004)

– 2 – 3 X increased risk of subclinical ketosis

(Osborne, 2003; Gooijer et al, 2004)

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10

Weeks from Calving

Incidence of

subclinical

ketosis (%)

Median time to diagnosis of clinical ketosis = 11 DIM

Duffield, 2000

Incidence of Subclinical Ketosis

0

5

10

15

20

25

30

35

-4 -2 0 2 4 6 8 10

Weeks From Calving

% of Cows with

Subclinical Ketosis

Serum BHBMilk ketones

Prevalence of

Subclinical Ketosis

Duffield et al 1998

Oetzel, 2003

Clinical ketosis treatment rate is a poor estimate of

ketosis

0

20

40

60

80

8 15 14 11 12 2 1 17 13

Herd

% S

ub

clin

ical

Ket

osi

s

012345678910

% C

lin

ical

Ket

osi

s In

cid

ence

SCK 1400 BHBA Clinical Ketosis

(Duffield et al 1998)

Associations with health and

performance• BHB (subclinical ketosis) in early

lactation is associated with:– 4-8X increased risk of LDA

(Geishauser, 2000; LeBlanc et al, 2005)

– Decreased milk production(Duffield, 2000)

– Increased severity of mastitis(Suriyasathaporn et al, 2000)

– 50% decrease in pregnancy at first AI(Walsh et al, 2004)

Effect of subclinical ketosis in week 2 on CR at 1st AI

.1.2

.3.4

Pro

bability o

f P

regnancy

0 1000 2000 3000 4000Week 2 BHBA

(Walsh et al, 2004)

Cow-side tests for ketosis

(relative to serum BHB ≥1400 µmol/L)MilkKeto-Test• 100 µmol/L

– Sensitivity = 83%– Specificity = 82%

• 200 µmol/L– Sensitivity = 54%– Specificity = 94%

Oetzel, 2004

• Powder lacks sensitivity

UrineKetostix (read at 5

seconds)• “small” (15µmol/L)

– Sensitivity = 79%– Specificity = 96%

Carrier et al, 2004

• Acetest tablet lacks specificity

Subclinical Ketosis Monitoring Programs

(True Prevalence = 20%)Test PV + PV- Apparent

Prevalence

Keto-Test (100 umol/L) 62% 93% 23%

Keto-Test (200 umol/L) 80% 87% 11%

Ketocheck (Milk) 90% 86% 8%

Acetest(Urine) 38% 100% 53%

Sampling logistics

• In a herd with 50 to 1000 cows, if a prevalence of “positive” tests– e.g. NEFA ≥ 0.5 or BHB ≥ 1400

• And ≥ 10% is the threshold of interest• And you wish to be 75% confident of

detecting this level of problem, then…• 13 samples are required• Oetzel proposes using 12 samples for

NEFA and BHB blood testing for investigations

Metabolic Predictors of LDA

• 1184 animals in 20 herds• Weekly visit by technician• Same day, same time (AM)• Cows enrolled 4 - 10 d prior to

expected calving• Sampled weekly until the week after

calving (Total of 2 - 4 samples)

LeBlanc et al, 2005

Days from calving

-20 -15 -10 -5 0 5 10

Se

rum

NE

FA

(m

Eq

/L)

0.0

0.5

1.0

1.5

2.0

Cows without DA (n = 1078)Cows with DA (n = 53)

LeBlanc et al, 2005

Days from calving

-20 -15 -10 -5 0 5 10

Ser

um

h

ydro

xyb

uty

rate

( m

ol/

L)

0

500

1000

1500

2000

2500

3000

3500

Cows without DA (n = 1078)Cows with DA (n = 53)

LeBlanc et al, 2005

Prepartum DA model

• Among all variables measured in last week before calving:

OR 95% CI PNEFA 0.5 mEq/L 3.5 1.9 – 7.1

.0001

Sensitivity = 64% Specificity = 66%

LeBlanc et al, 2005

Simple Association of NEFA 4-10 d before

DUE with LDANEFA OR Se Sp LR

0.3 2.3 63 56 1.4

0.4 2.6 50 72 1.8

0.5 4.1 46 82 2.6

0.6 3.0 30 89 2.6

0.8 2.6 17 93 2.5

1.0 4.1 15 96 3.8LeBlanc et al, 2005

Simple Association of NEFA 4-10 d before

DUE with LDANEFA OR OR

*Se Se* Sp Sp* LR LR*

0.3 2.3 2.6 63 61 56 61 1.4 1.6

0.4 2.6 2.9 50 47 72 77 1.8 2.0

0.5 4.1 5.1 46 43 82 87 2.6 3.3

0.6 3.0 3.7 30 26 89 92 2.6 3.2

0.8 2.6 3.0 17 12 93 96 2.5 3.1

1.0 4.1 5.1 15 12 96 98 3.8 5.2

* Excluding cows within 2 days of actual calving

LeBlanc et al, 2005

Postpartum DA model

Variable OR 95% CI P

RP 1.7 1.1 – 2.7 .01

Metritis 4.8 2.0 – 11.2

.0003

BHB per 100 mol/L *

1.08 1.06 – 1.1

.0001

NEFA per 1.0 mEq/L *

2.4 1.4 – 4.3 .002

Season, parity, MF, twins all NSMinimum significant cut-points in the model:

BHB 1000 mol/L ; NEFA 0.6 mEq/L

LeBlanc et al, 2005

Postpartum Simple Associations with DA

Test Cutpoint

OR Se Sp LR

NEFA 0.6 4.8 86 43 1.5

0.8 3.9 68 64 1.9

1.0 4.8 56 79 2.6

BHB 1000 6.3 69 74 2.6

1200 8.0 63 82 3.5

1400 8.0 53 88 4.3

LeBlanc et al, 2005

Postpartum Simple Associations with DA

Test Cutpoint

OR Se Sp LR

Milk BHB

100 2.8 64 62 1.7

200 3.4 48 80 2.4

LeBlanc et al, 2005

Sample handling

• Serum (red top) or plasma (purple top)• Avoid hemolysis• Ideal: keep chilled, separate within a few

hours, ship chilled to arrive at lab in 1-2 days

• Serum can be frozen for at least 1 month• What you could get away with: delay of <

24 h to separate; serum at room temp for < 24 h or in fridge for < 3 days

(Stokol & Nydam, 2004)

Monitoring Energy Metabolism in Transition

Cows•Pre-Calving - NEFA

•Post-Calving - Ketones– Routine monitoring (milk or

urine)

Monitoring Energy Metabolism in Transition

Cows•Helps to direct

investigation– What is the problem?– Where/when is the problem?

•Rarely answers “WHY?”– Need to look further and test

hypotheses

Evaluation of a Rapid, On-Site Serum NEFA Test

• 10 Guelph-area farms• Prepartum blood sample • (-7 to –4 days)• Harvested serum and aliquoted• Measure NEFA concentrations:

– Animal Health Laboratory (Hitachi 911 analyzer)

– DVM NEFA

Gooijer et al, ICPD 2004

R2 = 0.80

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

AHL NEFA

DV

M N

EF

A

Correlation between testsPearson’s r = 0.89

Gooijer et al, ICPD 2004

DVMNEFA

AHL NEFA

0.5 <0.5

>0.4 133 7 140

0.4 25 185 210

158 192 350

Test Characteristics of DVM NEFA

(Gold Standard = AHL > 0.4 mEq/L)

Sensitivity = 84% Specificity = 96%

Gooijer et al, ICPD 2004

Maintaining Peripartum DMI

• Fresh feed daily• Adequate bunk space

(>60 cm)• > 100 ft2/cow of pack• < 100% stocking• Separate heifer

groups• Moderate BCS (3.5)• Adaptation to new

rations (3-4 weeks)• Adequate eNDF

• Minimize group/pen changes

• Heat abatement – THI > 72– T > 27 C

• Free choice water• 0.5 – 0.75% BW in

concentrates• 60:40

Forage:concentrate• Rumensin CRC