Table 1 Correlation coefficients, intercept and slope by Least Square Regression analysis between blood pH 7.40 corrected ionised calcium (ICA7.4 mmol/L) concentrations analysed by EDAN and GEM and other blood parameters in 125 Holstein cows
Parameter
|
Device for ICA7.4 analysis
|
r
|
r2
|
Intercept+Slope (y)
|
p
|
TC (mmol/L)
|
EDAN
|
0.515
|
0.265
|
y = 0.700+0.18x
|
0.000
|
GEM
|
0.566
|
0.320
|
y = 0.623+1.555x
|
0.000
|
Lactate* (mmol/L)
|
EDAN
|
0.059
|
0.003
|
y = 0.810+0.238x
|
0.516
|
GEM
|
0.270
|
0.073
|
y = -0.417+1.097x
|
0.002
|
pH*1
|
EDAN
|
0.094
|
0.009
|
y = 7.471-0.022x
|
0.299
|
GEM
|
0.034
|
0.010
|
y = 7.448+0.008x
|
0.709
|
TP (g/L)
|
EDAN
|
0.117
|
0.014
|
y = 6.245+0.559x
|
0.194
|
GEM
|
0.107
|
0.011
|
y = 6.353+0.484x
|
0.237
|
Alb (g/L)
|
EDAN
|
0.133
|
0.018
|
y = 2.869+0.279x
|
0.139
|
GEM
|
0.138
|
0.019
|
y = 2.888+0.275x
|
0.124
|
GL** (g/L)
|
EDAN
|
0.059
|
0.003
|
y = 3.376+0.280x
|
0.515
|
GEM
|
0.046
|
0.002
|
y = 3.465+0.209x
|
0.609
|
TC: serum total calcium. Alb: serum albumin. GL: serum globulin. TP: serum total protein. *: Lactate and pH values refer to each blood gas device in which they were analysed. **: GL was calculated by deduction of Alb from TP. 1: Correlation between blood pH and real value of ionised calcium concentration. Serum TC, TP and Alb concentrations were analysed by BS120
Table 2 Average concentrations (mean and standard errors, x±se) of serum and whole blood gas parameters analysed by EDAN and GEM blood gas analysers and BS120 automated wet-biochemistry analyser in 125 Holstein between calving to postpartum day 3 (group 1) and postpartum day 4 to day 27 (group 2)
Parameter
|
Device
|
All animals (n=125)
|
Group 1 (n=87)
|
Group 2 (n=38)
|
p (1)
|
TC (mmol/L)
|
BS120
|
2.25±0.49
|
2.14±0.48
|
2.49±0.43
|
0.000
|
ICA7.4 (mmol/L)
|
EDAN
|
1.09±0.17
|
1.06±0.17
|
1.19±0.14
|
0.000
|
GEM
|
1.04±0.18*
|
1.01±0.18*
|
1.13±0.14*
|
0.000
|
ICA (mmol/L)
|
EDAN
|
1.08±0.17
|
1.04±0.17
|
1.17±0.14
|
0.000
|
GEM
|
1.02±0.17*
|
0.98±0.18*
|
1.10±0.13*
|
0.000
|
pH
|
EDAN
|
7.45±0.04
|
7.45±0.04
|
7.44±0.04
|
0.713
|
GEM
|
7.46±0.04*
|
7.46±0.04*
|
7.45±0.03*
|
0.452
|
Lactate (mmol/L)
|
EDAN
|
1.07±0.69
|
1.21±0.73
|
0.76±0.42
|
0.001
|
GEM
|
0.73±0.72*
|
0.83±0.82*
|
0.50±0.28*
|
0.001
|
CI (mmol/L)
|
EDAN
|
102.66±3.86
|
103.69±3.79
|
100.29±2.91
|
0.000
|
TP (g/L)
|
BS120
|
68.58±8.09
|
67.80±8.59
|
70.38±6.59
|
0.102
|
Alb (g/L)
|
BS120
|
31.75±3.54
|
31.33±2.99
|
32.72±4.46
|
0.084
|
GL (g/L)
|
BS120
|
36.83±8.06
|
36.47±8.70
|
37.66±6.39
|
0.452
|
TC/TP ratio (%)
|
BS120
|
0.13±0.03
|
0.13±0.03
|
0.14±0.03
|
0.011
|
TC/Alb ratio (%)
|
BS120
|
0.29±0.07
|
0.27±0.06
|
0.31±0.07
|
0.010
|
TC/GL ratio (%)
|
BS120
|
0.25±0.07
|
0.25±0.07
|
0.27±0.07
|
0.061
|
Alb/TP ratio (%)
|
BS120
|
46.77±6.26
|
46.81±6.35
|
46.67±6.13
|
0.913
|
GL/TP ratio (%)
|
BS120
|
53.23±6.26
|
53.19±6.35
|
53.33±6.13
|
0.913
|
ICA7.4/TC ratio (%)
|
EDAN
|
50.28±9.21
|
50.82±8.86
|
49.03±9.98
|
0.321
|
GEM
|
47.56±8.13*
|
47.98±7.82*
|
46.60±8.83*
|
0.386
|
*: p<0.01 compared to EDAN value in the same column for the same parameter. p (1): significance between group 1 and group 2. ICA7.4: blood pH 7.40 corrected ionised calcium concentration, CI: chloride, TC: serum total calcium, TP: serum total protein, GL: serum globulin, Alb: serum albumin concentrations. **: TC values in mmol/L were converted to mg/dl by dividing with 0.02495 to calculate the respective ratio; TP, Alb and GL values in g/L were converted to mg/dl to calculate the ratio to serum TC
Table 3 Correlation coefficients, intercept and slope between parity (min: 1, max: 7, average: 2.68±1.46) and blood parameters in 125 lactating Holstein
Parameters
|
Units
|
r
|
r2
|
Intercept+Slope (y)
|
p
|
ICA7.4 by GEM
|
mmol/L
|
-0.175
|
0.031
|
y=1.100-0.021x
|
0.051
|
ICA7.4 by EDAN
|
mmol/L
|
-0.233
|
0.054
|
y=1.169-0.027x
|
0.009
|
ICA by GEM
|
mmol/L
|
-0.178
|
0.032
|
y=1.076-0.021x
|
0.047
|
ICA by EDAN
|
mmol/L
|
-0.224
|
0.050
|
y=1.146-0.026x
|
0.012
|
Serum TC by BS120
|
mmol/L
|
-0.162
|
0.026
|
y= 2.391-0.055x
|
0.070
|
Serum TP by BS120
|
g/L
|
0.208
|
0.043
|
y=65.485+1.156x
|
0.020
|
Serum GL by BS120
|
g/L
|
0.219
|
0.048
|
y=33.585+1.212x
|
0.017
|
Serum Alb by BS120
|
g/L
|
-0.023
|
0.001
|
y=31.900-0.056x
|
0.797
|
Ratio of ICA7.4 /TC by GEM
|
%
|
-0.016
|
0.000
|
y=47.800-0.089x
|
0.859
|
Ratio of ICA7.4 /TC by EDAN
|
%
|
-0.021
|
0.000
|
y=50.628-0.131x
|
0.818
|
Ratio of TC/GL by BS120
|
%
|
-0.261
|
0.068
|
y=0.289-0.013x
|
0.003
|
Ratio of TC/TP by BS120
|
%
|
-0.243
|
0.059
|
y=0.146-0.005x
|
0.006
|
Ratio of TC/Alb by BS120
|
%
|
-0.136
|
0.018
|
y=0.302-0.006x
|
0.132
|
CI by EDAN
|
mmol/L
|
0.145
|
0.021
|
y=101.625+0.385x
|
0.107
|
Blood pH by GEM
|
pH
|
-0.035
|
0.001
|
y=7.458-0.001x
|
0.700
|
Blood pH by EDAN
|
pH
|
-0.057
|
0.003
|
y=7.450-0.002x
|
0.531
|
Lactate by GEM
|
mmol/L
|
-0.046
|
0.002
|
y=0.789-0.023x
|
0.609
|
Lactate by EDAN
|
mmol/L
|
-0.037
|
0.001
|
y=1.118-0.018x
|
0.680
|
TC: serum total calcium, ICA7.4: blood pH 7.40 corrected ionised calcium (ICA), TP: serum total protein, GL: serum globulin, Alb: serum albumin, CI: chloride concentration. BS120: an automated serum wet-biochemistry analyser
Table 4 Cut-points and respective test characteristics of EDAN and GEM to detect hypocalcemia (number of cases and prevalence) in coccygeal blood based on Receiver Operating Characteristics (ROC) analysis using different cut-points of serum total calcium concentration of <2.15 mmol/L (subclinical hypocalcemia, SCH) and <1.50 mmol/L (severe SCH) analysed by BS120 as reference (automatic wet-biochemistry analyser)
ROC analysis (n=125)
|
Detection of hypocalcemia
|
All cows (n=125)
|
Group 1
(n=87)
|
Group 2 (n=38)
|
Cut-point of Ca (mmol/L) for devices
|
Spec (%)
|
Sens (%)
|
AUC (%95)
|
j
|
p
|
n
|
%
|
n
|
%
|
n
|
%
|
BS120
|
<2.15
|
100
|
100
|
1.00
|
1.000
|
0.000
|
54
|
43.2
|
46
|
52.9
|
8
|
21.1
|
EDAN
|
1.05
|
81.7
|
57.4
|
0.73
|
0.391
|
0.000
|
44
|
35.2
|
38
|
43.7
|
6
|
15.8
|
GEM
|
1.02
|
90.3
|
57.4
|
0.76
|
0.377
|
0.000
|
45
|
36.0
|
37
|
42.5
|
8
|
21.1
|
BS120
|
<1.50
|
100
|
100
|
1.00
|
1.000
|
0.000
|
7
|
5.6
|
7
|
8.0
|
0
|
0
|
EDAN
|
0.86
|
95.8
|
100
|
0.98
|
0.958
|
0.000
|
12
|
9.6
|
12
|
13.8
|
0
|
0
|
GEM
|
0.78
|
96.6
|
100
|
0.99
|
0.966
|
0.000
|
11
|
8.8
|
11
|
12.6
|
0
|
0
|
Spec: specification, Sens: sensitivity, AUC: area under the curve, j: Youden-Index. Group 1 (cows between calving to postpartum day 3), group 2 (cows between postpartum day 4 to 27)
Table 5 Average concentrations (mean and standard errors, x±se) of serum total calcium (TC), total protein (TP), albumin (Alb) and globulin (GL) concentrations analysed by BS120 (wet-biochemistry analyser), whole blood pH 7.40 adjusted ionised calcium (ICA7.4), lactate and chloride (CI) concentrations and their ratio analysed by EDAN and GEM in primiparous (PRP, n=35) and multiparous (MUL, n=90) Holstein between calving and postpartum day 3 (group 1) and postpartum day 4 to 27 (group 2)
Parameter
|
Parity
|
All animals (n=125)
|
Group 1 (n=87)
|
Group 2 (n=38)
|
p(1)
|
TC (mmol/L)
|
PRP
|
2.41±0.49
|
2.28±0.46
|
2.70±0.46
|
0.017
|
MUL
|
2.18±0.48*
|
2.09±0.48+
|
2.40±0.40*
|
0.002
|
EDAN ICA7.4 (mmol/L)
|
PRP
|
1.15±0.11
|
1.14±0.09
|
1.17±0.15
|
0.463
|
MUL
|
1.08±0.18*
|
1.03±0.18**
|
1.19±0.13
|
0.000
|
GEM ICA7.4 (mmol/L)
|
PRP
|
1.08±0.12
|
1.07±0.12
|
1.08±0.14
|
0.944
|
MUL
|
1.03±0.19+
|
0.98±0.19**
|
1.15±0.13
|
0.000
|
TP (g/L)
|
PRP
|
66.60±8.15
|
65.50±8.67
|
69.01±6.62
|
0.242
|
MUL
|
69.35±7.98+
|
68.68±8.46
|
70.93±6.62
|
0.221
|
Alb (g/L)
|
PRP
|
31.87±3.77
|
31.16±2.67
|
33.42±5.30
|
0.100
|
MUL
|
31.70±3.47
|
31.39±3.13
|
32.43±4.15
|
0.249
|
GL (g/L)
|
PRP
|
34.73±7.58
|
34.33±8.25
|
35.58±6.15
|
0.658
|
MUL
|
37.65±8.13+
|
37.29±8.79
|
38.50±6.41
|
0.519
|
GEM lactate (mmol/L)
|
PRP
|
0.73±0.55
|
0.81±0.62
|
0.55±0.31
|
0.209
|
MUL
|
0.73±0.78
|
0.83±0.90
|
0.48±0.28
|
0.006
|
EDAN lactate (mmol/L)
|
PRP
|
1.08±0.47
|
1.15±0.51
|
0.93±0.34
|
0.199
|
MUL
|
1.07±0.76
|
1.23±0.81
|
0.68±0.43
|
0.357
|
EDAN Cl (mmol/L)
|
PRP
|
102.09±3.36
|
102.71±3.58
|
100.72±2.45
|
0.107
|
MUL
|
102.88±4.04
|
104.06±3.82
|
100.11±3.11
|
0.000
|
EDAN ICA7.4 /TC ratio (%)
|
PRP
|
49.38±10.90
|
51.48±10.19
|
44.79±11.48
|
0.092
|
MUL
|
50.63±8.51
|
50.57±8.38
|
50.76±8.96
|
0.922
|
GEM ICA7.4 /TC ratio (%)
|
PRP
|
46.58±10.44
|
48.85±9.64
|
41.61±10.84
|
0.056
|
MUL
|
47.94±7.07
|
47.65±7.07
|
48.63±7.14+
|
0.547
|
TC/TP ratio (%)
|
PRP
|
0.15±0.03
|
0.14±0.03
|
0.16±0.03
|
0.101
|
MUL
|
0.13±0.03**
|
0.12±0.03*
|
0.14±0.02*
|
0.047
|
TC/Alb ratio (%)
|
PRP
|
0.31±0.06
|
0.29±0.05
|
0.33±0.08
|
0.121
|
MUL
|
0.28±0.06*
|
0.27±0.06
|
0.30±0.07
|
0.022
|
TC/GL ratio (%)
|
PRP
|
0.29±0.08
|
0.28±0.07
|
0.31±0.09
|
0.165
|
MUL
|
0.24±0.07**
|
0.24±0.07*
|
0.26±0.05+
|
0.191
|
PRP cows: group 1 consists n=24, group 2 consists n=11. MUL cows: group 1 consists n=63, group 2 consists n=27. *: p<0.05 versus PRP in the same group, **:p<0.001 versus PRP in the same group, +: p<0.07 versus PRP in the same group, p(1): significance of the difference between group 1 and group 2
Table 6 Average concentrations (mean and standard errors, x±se) of serum total calcium (TC), total protein (TP), albumin (Alb) and globulin (GL) concentrations analysed by BS120 (wet-biochemistry analyser), whole blood pH 7.40 adjusted ionised calcium (ICA7.4), lactate and chloride (CI) concentrations and their ratio analysed by EDAN and GEM in Holstein with subclinical hypocalcemia (SCH) (TC<2.15 mmol/L) and without SCH (TC≥2.15 mmol/L)
Parameter
|
Cows with SCH (n=54)
|
Cows without SCH (n=71)
|
p(1)
|
TC (mmol/L)
|
1.82±0.27
|
2.57±0.36
|
0.000
|
EDAN ICA7.4 (mmol/L)
|
1.02±0.18
|
1.16±0.13
|
0.007
|
GEM ICA7.4 (mmol/L)
|
0.95±0.19*
|
1.11±0.14*
|
0.003
|
EDAN ICA7.4 /TC ratio (%)
|
56.10±8.07
|
45.85±7.41
|
0.000
|
GEM ICA7.4 /TC ratio (%)
|
52.14±6.99*
|
44.08±7.19*
|
0.000
|
TP (g/L)
|
66.38±8.19
|
70.26±7.66
|
0.008
|
Alb (g/L)
|
30.31±2.93
|
32.84±3.60
|
0.000
|
GL (g/L)
|
36.06±8.34
|
37.42±7.85
|
0.353
|
TC/TP ratio (%)
|
0.11±0.02
|
0.15±0.03
|
0.000
|
TC/GL ratio (%)
|
0.21±0,06
|
0.29±0.07
|
0.000
|
TC/Alb ratio (%)
|
0.24±0.04
|
0.32±0.06
|
0.000
|
EDAN Cl (mmol/L)
|
103.93±3.75
|
101.69±3.69
|
0.001
|
EDAN lactate (mmol/L)
|
1.04±0.49
|
1.10±0.81
|
0.184
|
GEM lactate (mmol/L)
|
0.65±0.46*
|
0.79±0.87*
|
0.107
|
SCH: subclinical hypocalcemia, TC: serum total calcium, ICA7.4: pH 7.4 adjusted ionised calcium, TP: serum total protein, Alb: serum albumin, GL: serum globulin, CI: chloride. *: p<0.01 indicates the difference between GEM and EDAN for the same parameter in the same column. p(1) : significance between cows with and without SCH
Table 7 Average concentrations (mean and standard errors, x±se) of serum total calcium (TC), total protein (TP), albumin (Alb) and globulin (GL) concentrations analysed by BS120 (wet-biochemistry analyser), whole blood pH 7.40 corrected ionised calcium (ICA7.4), lactate and chloride (CI) concentrations and their ratio analysed by EDAN and GEM in lactating Holstein with or without subclinical hypocalcemia (with SCH = serum TC<2.15 mmol/L, without TC≥2.15 mmol/L) in group 1 (calving to postpartum day 3) and group 2 (postpartum day 4 to 27)
Parameters
|
Group 1
|
p*
|
Group 2
|
p*
|
With SCH (n=46)
|
Without SCH (n=41)
|
With SCH (n=8)
|
Without SCH (n=30)
|
TC (mmol/L)
|
1.81±0.28
|
2.51±0.37
|
0.000
|
1.92±0.15
|
2.64±0.35
|
0.000
|
GEM ICA7.4 (mmol/L)
|
0.93±0.19
|
1.09±0.14
|
0.000
|
1.06±0.15
|
1.15±0.13
|
0.112
|
EDAN ICA7.4 (mmol/L)
|
0.99±0.17
|
1.13±0.13
|
0.000
|
1.15±0.18
|
1.20±0.13
|
0.391
|
EDAN ICA7.4 /TC ratio (%)
|
55.44±7.69
|
45.63±7.09
|
0.000
|
59.89±9.70
|
46.14±7.95
|
0.000
|
GEM ICA7.4 /TC ratio (%)
|
51.59±6.76
|
43.93±6.96
|
0.000
|
55.32±7.90
|
44.28±7.61
|
0.001
|
EDAN Cl (mmol/L)
|
104.65±3.21
|
102.61±4.12
|
0.011
|
99.75±4.13
|
100.43±2.57
|
0.563
|
EDAN lactate (mmol/L)
|
1.08±0.48
|
1.35±0.93
|
0.089
|
0.79±0.48
|
0.75±0.41
|
0.816
|
GEM lactate (mmol/L)
|
0.69±0.48
|
0.98±1.08
|
0.118
|
0.40±0.21
|
0.53±0,30
|
0.268
|
TP (g/L)
|
66.37±8.47
|
69.40±8.54
|
0.100
|
66.41±6.85
|
71.43±6.21
|
0.054
|
Alb (g/L)
|
30.56±2.79
|
32.19±3.02
|
0.010
|
28.91±3.52
|
33.73±4.16
|
0.005
|
GL (g/L)
|
35.81±8.58
|
37.21±8.88
|
0.456
|
37.50±7.09
|
37.70±6,32
|
0.939
|
TC/TP (%)
|
0.11±0.02
|
0.15±0.03
|
0.000
|
0.12±0.02
|
0.15±0.03
|
0.003
|
TC/GL (%)
|
0.21±0.06
|
0.28±0.07
|
0.000
|
0.21±0.05
|
0.29±0.07
|
0.005
|
TC/Alb (%)
|
0.24±0.04
|
0.32±0.06
|
0.000
|
0.27±0.03
|
0.32±0.07
|
0.007
|
p*: between cows with and without SCH in the respective group.