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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
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