Research in Veterinary Science 135 (2021) 335–342

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Research in Veterinary Science

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Common and specific mineral and metabolic features in dairy cows with
clinical metritis, hypocalcaemia or ketosis
Elda Dervishi a, *, Graham Plastow a, Brent Hoff b, 1, Marcos Colazo a
a
Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, AB, Canada
b
Animal Health Laboratory, University of Guelph, ON, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: The objectives were to evaluate differences in serum concentration of metabolites, macro minerals and hepatic
Dairy diseases enzymes at pre and postpartum time-points in dairy cows diagnosed with clinical metritis, hypocalcaemia or
Metabolites ketosis postpartum. A total of 144 Holstein cows from 11 commercial dairy herds in Alberta, (Western Canada)
Macro minerals
were enrolled in this study. Cows with clinical metritis had lower serum concentrations of glutamate dehydro­
Hepatic enzymes
genase (GLDH) at pre and postpartum and lower total Ca, albumin, urea, and cholesterol at postpartum when
compared to control cows. Cows with hypocalcaemia had greater serum concentrations of Na, Cl, and calculated
osmolarity (CalOsmo) at prepartum and lower concentration of total serum Ca, glucose, cholesterol, gamma-
glutamyl transpeptidase (GGT), GLDH, total protein and albumin at postpartum. Prepartum serum concentra­
tions of non-esterified fatty acids (NEFA), beta hydroxybutyrate (BHB), Cl, albumin/globulin ratio (A/G), Na, K
and sum of Na and K were greater in ketotic cows when compared with control cows. Cows with ketosis had also
greater postpartum serum concentrations of NEFA, BHB, GGT and aspartate transaminase (AST) when compared
with control cows. Prepartum serum Na and Cl concentrations and CalOsmo were greater in cows diagnosed with
hypocalcaemia or ketosis when compared with control cows. Furthermore, postpartum serum concentrations of
total Ca, cholesterol, albumin and GLDH were significantly affected by hypocalcaemia or clinical metritis and
concentrations of GGT by hypocalcaemia or ketosis. Finally, postpartum serum concentrations of haptoglobin
increased in all disease groups when compared with control cows. These results suggest common metabolic
features for clinical metritis, hypocalcaemia and ketosis in dairy cows in addition to the specific ones.

1. Introduction and 30.6% of cows were diagnosed with metritis, hypocalcaemia or

The so-called transition diseases such as metritis, hypocalcaemia and
ketosis negatively impact animal welfare and the profitability of dairy
farms due to high culling rates, costs of treatment, lost productivity and
impaired reproduction (Duffield et al., 2009; LeBlanc, 2010; Dubuc
et al., 2011). It has been reported that 30 to 50% of cattle experience a
metabolic or inflammatory disease soon after calving (LeBlanc, 2010).
Moreover, cows with one disease are at greater risk of developing other
diseases (DeGaris and Lean, 2008). Indeed, Macmillan et al. (2020a)
reported that 39.7% of cattle becoming sick during postpartum had
more than one disease. Addressing animal health, animal welfare and
reproduction are priorities identified for sustainable milk production in
Canada. In a recent study conducted by Macmillan et al. (2020a) in
several dairy farms in Alberta, Canada, it was reported that 21.4, 41.0
ketosis respectively. Therefore, predicting these transition diseases
before their occurrence will help dairy farmers to make nutritional ad­
justments and/or take better management decisions in order to reduce
their negative impact. However, until now the attempts to discover
specific predictive biomarkers of transition diseases have remained at an
experimental stage.
Clinical metritis is an inflammatory disease, which occurs during the
first 21 days after parturition, characterized by an enlarged uterus and
watery red-brown fluid discharge without fever (Sheldon et al., 2006).
Hypocalcaemia and ketosis are both considered metabolic diseases that
develop during peripartum or early postpartum. During early lactation
cows undergo a state of negative energy balance (NEB) due to lower feed
intake and increased energy demand for milk production (Huzzey et al.,
2007). To cope with NEB, body fat mobilisation takes place, leading to

* Corresponding author at: 4/10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
E-mail address: dervishi@ualberta.ca (E. Dervishi).
1
Deceased.

https://doi.org/10.1016/j.rvsc.2020.10.012
Received 24 April 2020; Received in revised form 16 September 2020; Accepted 14 October 2020
Available online 17 October 2020
0034-5288/© 2020 Elsevier Ltd. All rights reserved.
E. Dervishi et al.
an increase of non-esterified fatty acids (NEFA) and beta hydrox­
ybutyrate (BHB) in blood. Currently, Ca and BHB blood concentrations
remain the gold standard for diagnosis of hypocalcaemia and ketosis
respectively. In addition, hypocalcaemia and ketosis diseases have their
clinical and subclinical states. In this regard, cows with blood concen­
tration of Ca ≤ 2.1 mmol/L are considered subclinical cases of hypo­
calcaemia, meanwhile cows with Ca concentrations < 1.5 mmol/L are
considered as clinical cases (Reinhardt et al., 2011). Subclinical ketosis
is characterized by BHB concentrations between 1.1 and 2.9 mmol/L
and clinical ketosis by BHB concentration ≥ 3 mmol/L (Oetzel, 2007;
Macmillan et al., 2017). Currently, efforts are focused in early diagnosis
to reduce the negative impact of these diseases in lactating dairy cows.
There is now a wealth of evidence, which indicates that cows with
metritis, hypocalcaemia or ketosis experience alteration in some of the
innate immunity reactants and metabolites related to carbohydrate
metabolism weeks before the occurrence of disease (Dervishi et al.,
2016; Zhang et al., 2016; Zhang et al., 2018). In this regard, Zhang et al.
(2018) reported that serum concentrations of tumor necrosis factor α
(TNFα), lactate and NEFA, 8 weeks before parturition and a combination
of TNFα, serum amyloid alpha (SAA) and BHB at 4 weeks before
parturition are useful predictive biomarkers for hypocalcaemia. In
another study, serum concentrations of interleukin (IL)-6, lactate and
SAA at 8 weeks before parturition and IL-6, BHB and lactate at 4 weeks
before parturition were proposed to have very good discrimination
power between ketotic and control cows (Zhang et al., 2016). Moreover,
Dervishi et al. (2016) suggested that prepartum SAA can be used to
screen cows for the potential occurrence of postpartum uterine in­
fections. Another acute phase protein that has been suggested as an early
indicator of metritis and inflammation is haptoglobin (Huzzey et al.,
2009, 2011). A wide range of serum cut off values from ≥ 0.2 g/L to ≥
1.4 g/L have been proposed for haptoglobin, depending on the type of
intervention choice (Huzzey et al., 2009). However, SAA and hapto­
globin are shown to be increased during mastitis or hypocalcaemia as
well (Grönlund et al., 2005; Zhang et al., 2018). Data from the literature
would indicate that many metabolic features are not specific to one
disease, suggesting common metabolic response to infections and
metabolic diseases in dairy cows. In the current study, we hypothesized
that clinical metritis, hypocalcaemia and ketosis share common meta­
bolic features among them in addition to their specific response.
Therefore, the objectives of this study were to evaluate differences in the
serum concentration of metabolites, macro minerals and three hepatic
enzymes at pre and postpartum periods in cows with clinical metritis,
hypocalcaemia or ketosis postpartum.
2. Materials and methods
2.1. Animals, diet and blood collection
This was a retrospective study conducted from April to November
2015 on 11 commercial dairy farms in Alberta, Canada. Current and
historical weather conditions for the province of Alberta can be found at
https://acis.alberta.ca/acis/. Samples and data were collected from
1,096 cattle. Herd-selection criteria included herd size (≥ 100 lactating
cows), free stall facility for fresh cows, quality of available records,
subscription to the milk recording service, CanWest DHI (dairy herd
improvement) or use of DairyComp 305 (Valley Agricultural Software,
Tulare, CA, USA), and an established relationship with the local veter­
inary clinic to ensure consistency and integrity of the data used.
Reproductive management and outcomes and 305-d mature equivalent
(ME) milk yield by herd have been described in Macmillan et al.
(2020b). Herds enrolled in this study were monitored to identify cows
that suffered from retained fetal membranes, ketosis, clinical metritis,
mastitis, hypocalcaemia, fatty liver, or displaced abomasum (DA). At
each farm, only cows without any signs of health disorders during the
dry period were enrolled. Ten herds used a milking frequency of twice
daily and the remaining herd milked cows thrice daily. All farms fed a
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Research in Veterinary Science 135 (2021) 335–342
totally mixed ration, once daily, to meet or exceed the dietary re­
quirements for a lactating cow weighing approximately 680 kg and
producing 45 kg of 3.5% fat-corrected milk (NRC, 2001). All procedures
were conducted in accordance with guidelines of the Canadian Council
on Animal Care (CCAC, 2009).
In the present study, cows were observed for clinical metritis,
hypocalcaemia or ketosis from calving to 60 days in milk. In the study
conducted by Macmillan et al. (2020a), which involves the same dairy
herds as here, cattle were categorized based on the type of disease (e.g.
inflammatory, metabolic or having both). However, in the present study
we retrospectively selected cows that only exhibited one transition
disease (i.e. clinical metritis, hypocalcaemia or ketosis) and both clinical
and subclinical cases were considered for hypocalcaemia and ketosis.
Cattle that experienced multiple diseases were excluded. The disease
diagnoses were conducted by four herd veterinarians. Clinical metritis
was defined as described by Sheldon et al. (2006). A cow was considered
to have clinical hypocalcaemia if she was unable to rise within 72 h
following calving and responded to intravenous calcium treatment or
remained prostrated with cold extremities and decreased rectal tem­
perature (<38◦ C). Subclinical hypocalcaemia was characterized as a
postpartum circulating concentration of Ca ≤ 2.1 mmol/L (Martinez
et al., 2012). Clinical ketosis was defined as depressed appetite and
lethargy with evidence of elevated blood ketones (postpartum circu­
lating concentration of BHB ≥ 3.0 mmol/L). Finally, cows without signs
of sickness with postpartum circulating concentration of BHB ≥ 1.2 and
< 3.0 mmol/L, were considered as having subclinical ketosis (Macmillan
et al., 2017). Every disease case was matched with a control case from
the same herd (based on parity, body condition score (BCS) season and
month of calving)). Criteria for selection of control cases included cows
with no clinical signs and/or records of any of the seven diseases. The
cows were attributed to each group after 60 days in milk. Each disease
group was compared with its own control group, however in some cases,
a control case was used to match more than one disease case. The BCS
were assessed by two well-trained technicians at 6.0 ± 4.2 d prepartum
using a 5-point scale with 0.25 increments (1 = thin and 5 = fat) as
previously described (Edmonson et al., 1989).
Blood samples were collected from all 144 cows at 6.0 ± 4.2 d pre­
partum and at 9.0 ± 3.6 d postpartum (median ± SD) by members of the
research team during bi-weekly farm visits. Blood was collected from the
coccygeal vessels into vacuum tubes containing no preservative (Vacu­
tainer; Becton Dickinson and Co., Franklin Lakes, NJ, USA). Serum
sample tubes were rested at room temperature for 3 to 4 h, and then
centrifugated (3,000 x ɡ for 20 min; Rotanta 460 R, Hettich Zentrifugan,
Tuttlingen, Germany). Serum was collected and samples frozen, and
stored at − 20◦ C until submission to the Animal Health Laboratory (AHL;
University of Guelph, Guelph, ON, Canada) for determination of con­
centrations of all analytes.
2.2. Determination of serum analytes profile
The serum concentrations of total Ca, P, Mg, Na, K, Cl, urea, glucose,
NEFA, cholesterol, albumin, globulin, total protein, gamma-glutamyl
transpeptidase (GGT), aspartate transaminase (AST), glutamate dehy­
drogenase (GLDH), BHB and haptoglobin were determined as previously
described by Gobikrushanth et al. (2019) and Macmillan et al. (2020a).
NaK was calculated as the sum of Na and K. Osmolarity (CalOsmo) was
calculated using the formula: 1.86*(Na + K) + (Urea+Glucose). Albu­
min/globulin (A/G), Ca/P, and Na/K, NaK/Cl ratios were calculated and
included in the statistical analysis.
2.3. Statistical analyses
Statistical analyses were performed using RStudio (Version 1.2.5).
Before statistical analysis each of the variables were checked for their
normality. Variables that were not normally distributed were log
transformed. To investigate differences between diseased cows vs.
E. Dervishi et al.
control cows each metabolic indicator was analysed with a Linear
Model, before and after parturition with the full model being:
y = μ + Farm + Lactation + BCS + Season + SDay + Group + Lact*Group + e
Where: y is the response variable, μ represents the population mean,
farm, lactation (1–4 or more), BCS, season (spring, summer and fall),
sampling day (0–5 d; 6–10; 11–15 before/after parturition), group
(control vs. sick) and group and its interaction with lactation number,
were included as fixed effects, and e refers to the common error term.
Results are shown as Least Square Mean (±SEM) after Tukey correction
was applied for multiple comparisons.
Before the cluster analysis each metabolic indicator, hepatic enzyme
and mineral concentration was adjusted for the fixed effects except for
health status and analysed together using One-way Analysis of Variance
(ANOVA). Significant differences were considered if P ≤ 0.05 and P >
0.05 and ≤ 0.10 it was considered as a tendency. Cluster analysis was
performed using MetaboAnalyst software (Xia et al., 2015). For hierar­
chical cluster analysis, we considered two parameters namely similarity
measure and the clustering algorithm. For distance measurements we
used the Euclidian and Ward algorithm for clustering. The results of
cluster analysis are shown as a heatmap (Fig. 1a and 1b).
3. Results
The results of univariate analysis are presented separately for each
disease group, which was compared to its control group. Cows diag­
nosed with clinical metritis were first (64.3%), second (28.6%) and third
lactation (7.1%). Among cows with hypocalcaemia, 38.0, 27.5, and
34.5% were second, third and ≥ four lactation and cows diagnosed with
ketosis 20.0, 26.6, 30.0 and 20.0% were first, second, third and ≥ four
lactation.
3.1. The effect of diseases on pre and postpartum serum concentrations of
nutritional and metabolic indicators
In total, we analysed samples from 144 cows out of 1,096 available
cows: 29 metritic, 29 hypocalcemic (clinical n = 15 and subclinical n =
14), 30 ketotic (clinical n = 15 and subclinical n = 15) and 56 control.
Cows that experienced multiple diseases were excluded from the present
study. Supplementary Table 1 summarizes the total number of animals
per group, lactation number, season and day of sampling at prepartum
and postpartum period. At postpartum period the number of cows with
hypocalcaemia was fewer due to culling or deaths.
Table 1 shows the significant prepartum metabolic indicators change
by postpartum diseases. Cows with clinical metritis had lower serum
concentration of GLDH prepartum when compared to control group (P
< 0.05). Cows that developed hypocalcaemia had greater serum con­
centrations of Na, Cl, CalOsmo and NaK prepartum when compared with
control cows (P < 0.05). The prepartum serum concentrations of nine
indicators were significantly affected in ketotic cows. Non-esterified
fatty acids, BHB, Cl, Na, K, A/G ratio, and NaK were significantly
greater (P < 0.05), meanwhile globulin and Ca/P ratio were lower (P <
0.05) in ketotic cows when compared with control cows.
At postpartum, cows with clinical metritis continued to have lower
serum concentration of GLDH, and also had lower total Ca, albumin,
urea and cholesterol when compared to control group (P < 0.05). In
addition, postpartum haptoglobin concentration in cows with clinical
metritis was greater when compared to control cows (P < 0.05; Table 2).
The number of analytes that were affected by hypocalcaemia increased
postpartum. Cows with hypocalcaemia had greater serum haptoglobin
concentrations and lower concentrations of total Ca, glucose, choles­
terol, GGT, GLDH, albumin and total protein (P < 0.05; Table 2) when
compared to control group. At postpartum, cows with ketosis had
greater serum concentrations of NEFA, BHB, haptoglobin, GGT and AST
(P < 0.05), meanwhile Mg was lower (P < 0.05) compared to control
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cows (Table 2).
3.2. Effect of season, lactation and sampling day
Season, farm, lactation and sampling day were all factors associated
with changes in the concentration of nutritional and metabolic in­
dicators. Prepartum serum concentrations of GDHL were affected by the
interaction of disease and lactation. In this regard, third lactation cows
with clinical metritis had lower concentrations of GDLH compared to
their control counterparts (0.59 ± 0.16 vs. 1.2 ± 0.16 U/L; P = 0.05).
Interestingly, when analysing hypocalcaemia vs. control, season
affected the prepartum serum concentrations of Na and NaK (Table 1).
Prepartum serum concentrations of Na and NaK were greater in summer
when compared to fall (Table 3; P < 0.05). When analysing ketosis vs.
control, the prepartum serum concentration of BHB was greater in
spring when compared to summer (Table 3; P < 0.05) and tended to be
greater in spring when compared to fall (Table 3; P = 0.06). Moreover,
the serum concentration of NEFA was affected by day of sampling.
Pairwise comparison showed that cows whose sample was collected
between 0 and 5 d before parturition, had greater NEFA concentrations
when compared to the cows whose samples were collected 6–10 days
and 11–15 days, before parturition (Table 3; P < 0.05).
At postpartum, the studied metabolic indicators were mainly
affected by lactation number and the interaction between lactation
number and disease (Table 2). For example, when comparing clinical
metritis vs. control group, second lactation cows had greater serum al­
bumin concentrations when compared to that inin first lactation cows
(Table 4; P < 0.05), meanwhile serum cholesterol concentration was
significantly lower in third lactation cows when compared to first and
second lactation cows (Table 4; P < 0.05). In addition, postpartum
serum cholesterol concentration was also significantly affected by the
sampling day. Samples collected 0–5 days after parturition had lower
concentrations than samples collected at 11–15 days after parturition
(1.9 ± 0.19 vs. 2.5 ± 0.20 mmol/L; P < 0.05).
When analysing hypocalcaemia vs. control group, the interaction
between lactation number and disease significantly affected serum
GLDH and total protein content. Cows in their second lactation diag­
nosed with hypocalcaemia had lower serum concentration of total
protein content when compared with second lactation control cows
(58.0 ± 2.95 vs. 71.8 ± 3.07 g/L; P < 0.05).
Postpartum serum concentration of BHB was significantly affected
by lactation and the interaction between lactation and disease in the
ketosis vs. control group analysis. First lactation cows had lower BHB
concentration when compared with cows in their ≥ fourth lactation
(Table 4; P < 0.05). Ketotic cows in their ≥ fourth lactation had greater
BHB when compared to counterpart control cows (2.7 ± 0.28 vs. 0.58 ±
0.34 mmol/L; P < 0.05). In addition, ketotic cows in their second
lactation had lower BHB concentration when compared with ketotic
cows in their ≥ fourth lactation (1.4 ± 0.24 vs. 2.7 ± 0.28 mmol/L; P <
0.05). Moreover, third lactation cows diagnosed with ketosis had lower
serum concentration of GGT when compared with ketotic cows in their
≥ fourth lactation (19.5 ± 3.08 vs. 37.8 ± 4.47 U/L). Finally sampling
day affected the concentration of haptoglobin. Samples collected at 0–5
days had greater concentrations when compared to samples collected at
6–10 days and at 11–15 days postpartum (Table 4; P < 0.05).
3.3. Results of cluster analysis
After adjusting for all the fixed effects, the three diseases and their
control groups were compared together. Results showed that prepartum
serum concentrations of Na (P < 0.0001), NaK (P < 0.0001), CalOsmo
(P < 0.0001) and Cl (P < 0.0001) were significantly greater in cows with
ketosis or cows with hypocalcaemia when compared with control and
metritic cows. Greater prepartum NEFA concentrations were observed
only in cows with ketosis (P < 0.001) when compared to control, clinical
metritis, or hypocalcaemia groups. The results of cluster analysis of two
E. Dervishi et al. Research in Veterinary Science 135 (2021) 335–342

Fig. 1. Hierarchical clustering analysis of the adjusted

features measured in the serum at a) prepartum period
and b) postpartum period in cows classified as control,
clinical metritis, hypocalcaemia or ketosis. Each cell is
colored based on the feature concentration. Dark brown
represents high concentration, blue indicates low con­
centration, and gray indicates the intermediate level.
(For interpretation of the references with different col­
ours in this figure legend, the reader is referred to the
web version of this article.)

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Table 1
The differences in least square mean (±SEM) in circulating serum concentrations of prepartum analytes by postpartum diseases.
Feature Group Factors, P-value

Clinical Metritis (n = 29) Control (n = 27) Group Lactation Season DS GxL

GLDH (U/L) 0.87 ± 0.04 1.07 ± 0.04 0.008 0.002 0.79 0.56 0.04
Hypocalcaemia (n = 29) Control (n = 24)
Na (mmol/L) 143 ± 1.31 136 ± 1.16 <0.0001 0.12 0.03 0.17 0.48
Cl (mmol/L) 99.7 ± 1.01 95.1 ± 0.90 <0.0001 0.12 0.22 0.23 0.56
CalOsmo 282 ± 2.60 268 ± 2.31 <0.0001 0.15 0.02 0.05 0.53
NaK 148 ± 1.39 140 ± 1.24 <0.0001 0.16 0.02 0.20 0.47
Ketosis (n = 30) Control (n = 25)
Na (mmol/L) 145 ± 1.68 137 ± 2.01 <0.0001 0.51 0.009 0.25 0.91
K (mmol/L) 4.80 ± 0.11 4.52 ± 0.13 0.03 0.06 0.57 0.64 0.64
Cl (mmol/L) 100.9 ± 1.21 96.8 ± 1.44 0.002 0.22 0.02 0.12 0.83
CalOsmo 287.0 ± 3.01 271 ± 3.66 <0.0001 0.49 0.01 0.25 0.98
NaK 150 ± 1.75 141 ± 2.08 0.0001 0.49 0.01 0.25 0.89
NaK/Cl 1.48 ± 0.008 1.46 ± 0.01 0.02 0.61 0.88 0.58 0.97
Ca/P 1.14 ± 0.05 1.29 ± 0.06 0.04 0.07 0.70 0.46 0.38
BHB (mmol/L) 0.51 ± 0.04 0.37 ± 0.05 0.01 0.99 0.009 0.35 0.55
NEFA (mmol/L) 0.30 ± 0.08 0.016 ± 0.09 0.005 0.08 0.008 0.001 0.81
Globulin (g/L) 32.3 ± 1.62 37.1 ± 1.93 0.01 0.19 0.98 0.12 0.25
AG ratio 1.18 ± 0.06 1.01 ± 0.07 0.01 0.27 0.82 0.78 0.57

DS, day of sampling; G x L, interaction between group and lactation number.

CalOsmo = calculated osmolality; GLDH = glutamate dehydrogenase; BHB = beta hydroxybutyrate; NEFA = non-esterified fatty acid.

Table 2
The differences in least square mean (±SEM) in circulating serum concentrations of postpartum analytes by postpartum diseases.
Feature Group Factors, P-value

Clinical Metritis (n = 29) Control (n = 27) Group Lactation Season DS GxL

Ca (mmol/L) 2.26 ± 0.03 2.33 ± 0.03 0.009 0.03 0.10 0.59 0.28
Urea (mmol/L) 3.40 ± 0.26 3.98 ± 0.26 0.02 0.03 0.53 0.49 0.29
Cholesterol (mmol/L) 2.05 ± 0.17 2.29 ± 0.17 0.04 0.02 0.27 0.001 0.73
GLDH log10 (U/L) 1.18 ± 0.11 1.48 ± 0.11 0.008 0.04 0.28 0.60 0.80
Albumin (g/L) 31.8 ± 0.84 33.7 ± 0.87 0.008 0.01 0.04 0.64 0.52
Haptoglobin (g/L) 0.82 ± 0.18 0.22 ± 0.19 0.02 0.51 0.98 0.72 0.18
Hypocalcaemia (n = 21) Control (n = 24)
Ca (mmol/L) 2.01 ± 0.08 2.39 ± 0.06 <0.0001 0.08 0.67 0.15 0.79
Glucose (mmol/L) 2.31 ± 0.20 2.75 ± 0.16 0.02 0.43 0.61 0.15 0.55
Cholesterol (mmol/L) 2.02 ± 0.28 2.62 ± 0.21 0.005 0.06 0.24 0.04 0.23
GGT (U/L) 15.2 ± 2.37 19.2 ± 1.83 0.01 0.83 0.55 0.18 0.37
GLDH log10 (U/L) 1.11 ± 0.07 1.30 ± 0.05 0.001 0.36 0.77 0.29 0.05
Albumin (g/L) 32.4 ± 1.58 36.7 ± 1.22 0.0007 0.16 0.18 0.84 0.19
Haptoglobin (g/L) 0.46 ± 0.13 0.20 ± 0.10 0.02 0.02 0.74 0.91 0.89
Total protein (g/L) 67.2 ± 2.90 73.1 ± 2.24 0.001 0.02 0.05 0.04 0.007
Ketosis (n = 30) Control (n = 26)
Mg (mmol/L) 0.85 ± 0.02 0.92 ± 0.02 0.01 0.48 0.59 0.32 0.53
NEFA (mmol/L) 0.91 ± 0.09 0.36 ± 0.09 <0.0001 0.08 0.06 0.23 0.76
BHB (mmol/L) 1.6 ± 0.17 0.5 ± 0.18 <0.0001 0.0007 0.07 0.14 0.01
GGT (U/L) 22.7 ± 2.33 14.9 ± 2.47 0.02 0.13 0.94 0.06 0.0007
AST (U/L) 97.1 ± 8.88 75.4 ± 9.42 0.03 0.02 0.27 0.60 0.06
Haptoglobin (g/L) 0.45 ± 0.10 0.22 ± 0.10 0.004 0.17 0.63 0.004 0.28

DS, day of sampling; G x L, interaction between group and lactation number; GGT = gamma-glutamyl transpeptidase; AST = aspartate aminotransferase; GLDH =
glutamate dehydrogenase; NEFA = non-esterified fatty acid; BHB = beta hydroxybutyrate.

time points are shown in Fig. 1a and 1b. Fig. 1a displays the differences
between control and the three disease groups. A cluster of indicators
such as Cl, CalOsmo, Na and NaK were increased in ketosis and hypo­
calcaemia groups when compared to control demonstrating that these
indicators are not specific to one disease.
At postpartum, the serum concentration of haptoglobin increased in
all three diseases (P < 0.0001; Fig. 1b) when compared with control.
However, we only observed greater NEFA (P < 0.0001) and BHB (P <
0.0001) concentrations in cows with ketosis, and lower concentrations
of Ca (P < 0.0001), cholesterol (P < 0.0001), total protein (P < 0.001),
and albumin (P < 0.0001) in cows with hypocalcaemia when compared
to all other groups.
4. Discussion
In the present study, we hypothesized that clinical metritis,
hypocalcaemia and ketosis share common metabolic features in addition
to their specific ones. To test our hypothesis, we evaluated differences in
the serum concentration of metabolites, minerals, and hepatic enzymes
at pre and postpartum in cows that developed clinical metritis, hypo­
calcaemia or ketosis postpartum. Results from this study indicate that
prepartum serum concentrations of Na, Cl, NaK (Na and K) and CalOsmo
were significantly greater in cows diagnosed with ketosis or hypo­
calcaemia when compared to control cows. Sodium, K and Cl are very
important minerals responsible for maintaining acid/base balance in the
body in addition to regulation of excitability of cells and cell volume. It
has been hypothesized that metabolic alkalosis is the underlying cause
of hypocalcaemia in dairy cattle. In support of that, there is evidence
that the ratio of cations Na+, K+ and anions Cl− and SO4− , plays a sig­
nificant role in the incidence of hypocalcaemia (Ender et al., 1971) and
by feeding dry cows anionic salts, the incidence of hypocalcaemia de­
creases (Block, 1984). Recently, excess of K in the diet has been found to

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Table 3
Least square mean (±SEM) in serum concentrations of prepartum analytes affected by lactation, season or day of sampling in each comparison between control and
disease group.
Feature Lactation Season DS

First Second Third ≥Fourth Spring Summer Fall 0–5 6–10 11–15

Clinical Metritis vs. Control

GLDH (U/L) 10.95 ± 14.29 ± 9.17 ± 11.6 ± 2.25 9.6 ± 2.31 13.2 ± 1.79 11.1 ± 1.77 12.5 ± 1.80 10.8 ± 2.60
1.51a 1.53b 3.60ab

Hypocalcaemia vs. Control

Na (mmol/L) 140 ± 1.08 142 ± 1.51 139 ± 1.34 139 ± 143 ± 1.23a 140 ± 1.04b 142 ± 1.21 141 ± 1.24 138 ± 1.53
1.61ab
CalOsmo 276 ± 2.12 280 ± 2.97 275 ± 2.64 274 ± 3.17a 282 ± 2.42b 276 ± 281 ± 279 ± 272 ± 3.01b
2.04ab 2.39a 2.43ab
NaK 144 ± 1.14 147 ± 1.60 144 ± 1.42 143 ± 1.7ab 147 ± 1.3a 144 ± 1.1b 147 ± 1.28 146 ± 1.31 142 ± 1.62

Ketosis vs. Control

K (mmol/L) 4.25 ± 0.20a 4.91 ± 0.12b 4.86 ± 4.63 ± 4.76 ± 0.15 4.64 ± 0.11 4.58 ± 0.16 4.60 ± 0.09 4.60 ± 0.22 4.78 ± 0.12
0.16ab 0.20ab
BHB (mmol/L) 0.46 ± 0.08 0.47 ± 0.05 0.43 ± 0.06 0.39 ± 0.07 0.55 ± 0.06a 0.40 ± 0.37 ± 0.48 ± 0.04 0.46 ± 0.05 0.38 ± 0.08
0.04b 0.06ab
y
NEFA (mmol/ 0.35 ± 0.14 ×
0.11 ± 0.08 0.17 ± 0.14 ± 0.10 0.22 ± 0.23 ± 0.11 ± 0.10b 0.43 ± 0.16 ± 0.12 ±
L) 0.11xy 0.10ab 0.08a 0.06a 0.08b 0.05b

GLDH = glutamate dehydrogenase; NaK = sum of Na and K; BHB = beta hydroxybutyrate; NEFA = non-esterified fatty acid; DS = day of sampling.
a,b,c Least square mean with different superscripts within the same category and row differed (P ≤ 0.05).
x,y, Least square mean with different superscripts within the same category and row tended to differ (P > 0.05 and ≤0.10).

Table 4
Least square mean (±SEM) in serum concentrations of postpartum analytes affected by lactation, season or day of sampling in each comparison between control and
disease group.
Clinical Metritis vs. Control

Feature Lactation Season DS

First Second Third ≥Fourth Spring Summer Fall 0–5 6–10 11–15

Ca (mmol/L) 2.30 ± 0.02 2.34 ± 0.02 2.24 ± 0.06 2.33 ± 2.24 ± 2.31 ± 2.26 ± 2.31 ± 0.03 2.31 ±
0.05ab 0.03a 0.03b 0.03 0.03
Urea (mmol/L) 3.45 ± 3.94 ± 0.19y 3.68 ± 3.86 ± 0.40 3.64 ± 0.25 3.57 ± 3.87 ± 3.47 ± 0.30 3.74 ±
0.21× 0.49xy 0.25 0.29 0.24
a
Cholesterol (mmol/ 2.47 ± 2.56 ± 0.13 1.48 ± 2.45 ± 0.26 2.05 ± 0.16 2.01 ± 1.93 ± 2.09 ± 2.49 ±
L) 0.14a 0.32b 0.16 0.19a 0.15ab 0.20b
Albumin (g/L) 32.1 ± 33.9 ± 0.64b 32.2 ± 32.7 ± 1.31 33.1 ± 0.83 32.3 ± 33.2 ± 32.4 ± 0.77 32.6 ±
0.69a 1.61ab 0.82 0.96 1.00

Hypocalcaemia vs. Control

Haptoglobin (g/L) 0.47 ± 0.46 ± 0.05 ± 0.19 ± 0.16 0.34 ± 0.17 0.45 ± 0.08 0.27 ± 0.14 0.43 ± 0.12 0.27 ±
0.10× 0.14×y 0.17y 0.14

Ketosis vs. Control

BHB (mmol/L) 0.61 ± 1.0 ± 0.02ab 1.1 ± 0.02ab 1.5 ± 0.03b 1.0 ± 0.02 1.2 ± 0.02 1.0 ± 0.02 1.1 ± 0.03 0.88 ± 0.02 1.3 ± 0.02
0.03a
Haptoglobin (g/L) 0.32 ± 0.59 ± 0.09a 0.10 ± 0.30 ± 0.41 ± 0.12 0.22 ± 0.09 0.36 ± 0.76 ± 0.10 ± 0.13 ±
0.16ab 0.13b 0.17ab 0.13 0.16a 0.10b 0.10b

GLDH = glutamate dehydrogenase; NaK = sum of Na and K; BHB = beta hydroxybutyrate; NEFA = non-esterified fatty acid; gamma-glutamyl transpeptidase = GGT;
aspartate transaminase = AST; DS = day of sampling.
a,b,c Least square mean with different superscripts within the same category and row differed (P ≤ 0.05).
x,y, Least square mean with different superscripts within the same category and row tended to differ (P > 0.05 and ≤0.10).

be involved in the pathogenesis of hypocalcaemia (Goff et al., 2014).
Therefore, it has been suggested that negative dietary cationic anionic
difference diets decrease blood pH, causing a slight metabolic acidifi­
cation and increasing tissues responsiveness to parathyroid hormone
(PTH), which in turn stimulates Ca mobilisation from bones and in­
creases intestinal Ca absorption (Goff, 2006; Horst et al., 1997; Goff,
2008). However, the role of Na, K and Cl in the incidence and devel­
opment of ketosis have not been studied extensively. Zhang et al. (2011)
did not find significant changes in serum concentrations of Na and K in
cows with subclinical ketosis and concluded that Na and K are not
involved in its pathogenesis. More recently, Zhang et al. (2017) reported
multiple alterations of mineral elements in the serum and urine of dairy
cows weeks before the diagnosis of ketosis, but again serum concen­
trations of Na and K did not differ between ketotic and control cows.
Conversely, in the current study, the prepartum concentrations of both
Na and K and their sum NaK were greater in cows subsequently found to
develop hypocalcaemia or ketosis when compared to control cows, thus
supporting the concept of prepartum metabolic alkalosis as one of the
underlying mechanisms for both diseases. Therefore, the findings sup­
port our hypothesis that these diseases share common prepartum
metabolic features, which deserve further investigation.
Prepartum serum concentrations of GLDH were lower only in cows
that developed clinical metritis. Cows with clinical metritis continued to
have lower serum concentration of GLDH postpartum, in addition to
lower total serum Ca, albumin, urea, cholesterol. Similarly, Dervishi
et al. (2018) reported that cows diagnosed with metritis had lower
postpartum concentrations of urea in urine compared to control cows.
Interestingly, in the present study the serum concentration of the liver

340
E. Dervishi et al.
enzyme GLDH that is required for urea synthesis, was lower pre and
postpartum in cows developing clinical metritis. Hence, a lower con­
centration of GLDH could have led to a lower urea concentration after
parturition in cows diagnosed with clinical metritis. Furthermore,
postpartum serum concentrations of total Ca, cholesterol, albumin and
GLDH were lower in both hypocalcaemic and metritic cows. Currently,
total serum Ca concentration along with clinical signs are used to di­
agnose hypocalcaemia. It is generally accepted that a blood concentra­
tion of total Ca ≤ 2.1 mmol/L is indicative of subclinical hypocalcaemia,
meanwhile a blood concentration of Ca below 1.5 mmol/L is indicative
of clinical hypocalcaemia. In the present study, the mean total serum Ca
concentration in cows with hypocalcaemia was 2.01 mmol/L, which is
in the range of subclinical hypocalcaemia. The maintenance of Ca ho­
meostasis in the body is a complex process that is influenced by several
components, one of them being circulating albumin (Peacock, 2010).
Extracellular Ca is transported mainly by albumin, and a small amount
by globulin (Peacock, 2010), therefore it can be argued that a lower
serum albumin concentration could contribute to a lower serum Ca
concentration. It should be pointed out that postpartum perturbation of
Ca and albumin concentrations were not exclusive to cows diagnosed
with hypocalcaemia. Cows diagnosed with clinical metritis also had
lower concentrations of Ca and albumin when compared to their control
group, although the average concentration of postpartum total Ca in
cows with clinical metritis fall within the normal range (2.26 ± 0.03
mmol/L). Our results, in cows with clinical metritis, are in agreement
with those from Pinedo et al. (2017), who reported that cows diagnosed
with puerperal metritis had lower postpartum Ca concentration
compared to control. In addition, both hypocalcaemic and metritic cows
had lower GLDH concentration when compared to their control group.
Decreased GLDH has been reported as an indicator of impaired liver
function (Du et al., 2017), therefore it is reasonable to speculate that
cows with hypocalcaemia or clinical metritis experience impaired liver
function in addition to Ca metabolism perturbation.
In the current study, postpartum haptoglobin concentration
increased in cows diagnosed with clinical metritis, hypocalcaemia or
ketosis compared to control cows, however, prepartum haptoglobin did
not differ among groups, therefore limiting its use as a prepartum
screening marker to predict the incidence of transition diseases. Pre and
postpartum serum concentrations of haptoglobin have been previously
associated with uterine postpartum diseases (Dubuc et al., 2010; Huzzey
et al., 2015). Haptoglobin increases markedly during active inflamma­
tory disease and has been reported as a useful indicator of acute
response, with a significant increase within one to five days after the
occurrence of inflammation. Huzzey et al. (2009) proposed a wide range
of serum haptoglobin cut off values (≥ 0.2 g/L to ≥ 1.4 g/L) depending
on the type of intervention choice. Authors reported that a cut-point of ≥
0.4 g/L might identify animals that warrant daily monitoring of body
temperatures to detect the first signs of illness meanwhile a cut-point of
1 g/L may help in the early diagnosis of postpartum metritis in dairy
cows. In agreement, our findings suggest that an increase in serum
haptoglobin concentration is a good indicator of an inflammatory pro­
cess, but it is not associated with a particular disease. Results from the
current study show that postpartum serum BHB and NEFA concentra­
tions were greater only in cows with ketosis, which is in agreement with
a previous report by Ospina et al. (2010). Blood concentrations of BHB
between 1.2 and 2.9 mmol/L are used as reference for subclinical ketosis
and concentrations ≥3.0 mmol/L for clinical ketosis (Oetzel, 2007). In
the present study the average serum concentration of BHB in ketotic
cows was 1.6 ± 0.17 mmol/L, which is higher than the commonly
accepted cut off value to diagnose subclinical ketosis. We did observe
significant differences in the postpartum serum concentration of BHB
when the ketosis group was not only compared with its control group
but also with the metritic or hypocalcaemic group, supporting the sug­
gestion that the predictive value of BHB is higher during the postpartum
period (Overton et al., 2017). Prepartum NEFA concentrations were
greater in cows with ketosis, but the serum concentration of NEFA was
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Research in Veterinary Science 135 (2021) 335–342
affected by sampling day. In agreement with the results reported by
Oetzel (2004), cows whose sample was collected between 0 and 5 days
before parturition, had greater NEFA concentrations.
Other environmental factors such as season and number of lactations
significantly affected the concentration of indicators studied before and
after parturition. In this regard, we observed that prepartum serum
concentration of Na was greater in summer when compared to fall in the
analysis conducted between hypocalcaemic and control cows. In addi­
tion, BHB was greater in spring when compared to summer in the
analysis between ketotic and control cows. It is possible that changes in
the serum concentration of indicators studied might be the result of
changes in the diet. There is evidence that season has a significant effect
on circulating metabolic indicators in dairy cows (Yokus and Cakir,
2006; Cozzi et al., 2011). It is commonly accepted that traits such as milk
production or milk fat content are affected by parity and stage of
lactation, and environmental factors such as season and temperature
(Yang et al., 2013), therefore it can be argued that environmental factors
could probably impact physiological indicators as well. We believe this
is an important piece of information for the dairy industry as nutritional
adjustments might be required for dairy cattle during certain periods to
avoid negative effects on health, productive and reproductive perfor­
mance. Another important implication of our results is that environ­
mental factors need to be taken into consideration for the appropriate
interpretation of serum metabolic indicators in dairy cattle.
In conclusion, results from the present study support our hypothesis
that hypocalcaemia and ketosis share common prepartum metabolic
features, before disease occurrence. Hypocalcaemia and clinical metritis
share common postpartum metabolic features. The increase of pre­
partum serum concentrations of Na and K might be one of the under­
lying mechanisms of hypocalcaemia and ketosis, which deserve further
investigation. This is also a critical piece of information, which might
help dairy producers to adjust the nutritional management in a short
term and design nutritional strategies in order to mitigate the negative
impact of these diseases on production and reproduction. Another
implication of our study is that other factors such as metabolic com­
monalities between diseases and environmental factors should be taken
into account when investigating specific biomarkers of disease in dairy
cows and for appropriate interpretation of the metabolic indicators.
Acknowledgements
This project was financially supported by Growing Forward 2 (a
federal-provincial-territorial initiative), Alberta Agriculture and
Forestry, (2018F009R), NSERC, Canada Research Chairs Program,
Genome Canada, and Alberta Innovates. The authors thank the partici­
pating dairy producers, and veterinarians for their cooperation. Authors
also thank Dr. Irene Lopez Helguera (University of Lleida, Spain), Dr.
Mohanathas Gobikrushanth (University of Saskatchewan, Canada) and
Mr. Amir Behrouzi (University of Alberta, Canada) for assistance during
the study.
References
Block, E., 1984. Manipulating dietary anions and cations for prepartum dairy cows to
reduce incidence of milk fever. J. Dairy Sci. 67, 2939–2948.
Canadian Council on Animal Care, 2009. Canadian Council on Animal Care CCAC
Guidelines on: The Care and Use of Farm Animals in Research, Teaching and Testing.
CCAC, Ottawa, ON, Canada.
Cozzi, G., Ravarotto, L., Gottardo, F., Stefani, A.L., Contiero, B., Moro, L., Brscic, M.,
Dalvit, P., 2011. Short communication: reference values for blood parameters in
Holstein dairy cows: effects of parity, stage of lactation, and season of production.
J. Dairy Sci. 94, 3895–3901.
DeGaris, P.J., Lean, I.J., 2008. Milk fever in dairy cows: a review of pathophysiology and
control principles. Vet. J. 176, 58–69.
Dervishi, E., Zhang, G., Hailemariam, D., Goldansaz, S.A., Deng, Q., Dunn, S.M.,
Ametaj, B.N., 2016. Alterations in innate immunity reactants and carbohydrate and
lipid metabolism precede occurrence of metritis in transition dairy cows. Res. Vet.
Sci. 104, 30–39.
E. Dervishi et al.
Dervishi, E., Zhang, G., Hailemariam, D., Mandal, R., Wishart, D.S., Ametaj, B.N., 2018.
Urine metabolic fingerprinting can be used to predict the risk of metritis and
highlight the pathobiology of the disease in dairy cows. Metabolomics 14, 83.
Du, X., Chen, L., Huang, D., Peng, Z., Zhao, C., Zhang, Y., Zhu, Y., Wang, Z., Li, X.,
Lui, G., 2017. Elevate apoptosis in the liver of dairy cows with ketosis. Cell. Physiol.
Biochem. 43, 568–578.
Dubuc, J., Duffield, T.F., Leslie, K.E., Walton, J.S., Leblanc, S.J., 2010. Risk factors for
postpartum uterine diseases in dairy cows. J. Dairy Sci. 93, 5764–5771.
Dubuc, J., Duffield, T.F., Leslie, K.E., Walton, J.S., Leblanc, S.J., 2011. Effects of
postpartum uterine diseases on milk production and culling in dairy cows. J. Dairy
Sci. 94, 1339–1346.
Duffield, T.F., Lissemore, K.D, McBride, B.W., and Leslie, K.E., 2009. Impact of
hyperketonemia in early lactation dairy cows on health and production. J. Dairy Sci.
92, 571–580.
Edmonson, A.J., Lean, I.J., Weaver, L.D., Farver, T., Webster, G., 1989. A body condition
scoring chart for Holstein dairy cows. J. Dairy Sci. 72, 68–78.
Ender, F., Dishington, I.W., Helgebostad, A., 1971. Calcium balance studies in dairy cows
under experimental induction and prevention of hypocalcaemic paresis puerperalis.
The solution of the aetiology and the prevention of milk fever by dietary means. Z. Z.
Tierphysiol. Tierernahr. Futtermittelkd 28, 233–256.
Gobikrushanth, M., Macmillan, K., Behrouzi, A., Hoff, B., Colazo, M.G., 2019. The factors
associated with postpartum body condition score change and its relationship with
serum analytes, milk production and reproductive performance in dairy cows. Livest.
Sci. 228, 151–160.
Goff, J.P., 2006. Major advances in our understanding of nutritional influences on bovine
health. J. Dairy Sci. 89, 1292–1301.
Goff, J.P., 2008. The monitoring, prevention, and treatment of milk fever and subclinical
hypocalcemia in dairy cows. Vet. J. 176, 50–57.
Goff, J.P., Liesegang, A., Horst, R.L., 2014. Diet-induced pseudohypoparathyroidism: a
hypocalcemia and milk fever risk factor. J. Dairy Sci. 97, 1520–1528.
Grönlund, U., Hallén Sandgren, C., Persson Waller, K., 2005. Haptoglobin and serum
amyloid a in milk from dairy cows with chronic sub-clinical mastitis. Vet. Res. 36,
191–198.
Horst, R.L., Goff, J.P., Reinhardt, T.A., Buxton, D.R., 1997. Strategies for preventing milk
fever in dairy cattle. J. Dairy Sci. 80, 1269–1280.
Huzzey, J.M., Veira, D.M., Weary, D.M., von Keyserlingk, M.A.G., 2007. Prepartum
behaviour and dry matter intake identify dairy cows at risk for metritis. J. Dairy Sci.
90, 3220–3233.
Huzzey, J.M., Duffield, T.F, LeBlanc, S.J, Veira, D.M, Weary, D.M., and von Keyserlingk
M.A.G., 2009. Haptoglobin as an early indicator of metritis. J. Dairy Sci. 92,
621–625.
Huzzey, J.M., Nydam, D.V., Grant, R.J., Overton, T.R., 2011. Associations of prepartum
plasma cortisol, haptoglobin, fecal cortisol metabolites, and nonesterified fatty acids
with postpartum health status in Holstein dairy cows. J. Dairy Sci. 94, 5878–5889.
Huzzey, J.M., Mann, S., Nydam, D.V., Grant, R.J., Overton, T.R., 2015. Associations of
peripartum markers of stress and inflammation with milk yield and reproductive
performance in Holstein dairy cows. Prev. Vet. Med. 120, 291–297.
LeBlanc, S.J., 2010. Monitoring metabolic health of dairy cattle in the transition period.
J Reprod Dev. 56, S29–S35.
Macmillan, K., López Helguera, I., Behrouzi, A., Gobikrushanth, M., Hoff, B., Colazo, M.
G., 2017. Accuracy of a cow-side test for the diagnosis of hyperketonemia and
hypoglycemia in lactating dairy cows. Res. Vet. Sci. 115, 327–331.
Macmillan, K., Gobikrushanth, M., Behrouzi, A., López Helguera, I., Cook, N., Hoff, B.,
Colazo, M.G., 2020a. The association of circulating pre partum metabolites,
minerals, cytokines and hormones with postpartum health status in dairy cattle. Res.
Vet. Sci. 130, 126–132.
342
Research in Veterinary Science 135 (2021) 335–342
Macmillan, K., Gobikrushanth, M., López Helguera, I., Behrouzi, A., Colazo, M.G., 2020b.
Relationships between early postpartum nutritional and metabolic profiles and
subsequent reproductive performance of lactating dairy cows. Theriogenology. 151,
52–57.
Martinez, N., Risco, C.A., Lima, F.S., Bisinotto, R.S., Greco, L.F., Ribeiro, E.S.,
Manusell, F., Galvão, K., Santos, J.E., 2012. Evaluation of peripartal calcium status,
energetic profile, and neutrophil function in dairy cows at low or high risk of
developing uterine disease. J.Dairy Sci. 95, 7158–7172.
National Research Council, 2001. National Research Council Nutrient Requirements of
Dairy Cattle, 7th revised ed.
Oetzel, G.R., 2004. Monitoring and testing dairy herds for metabolic disease. Vet. Clin.
North Am. Food Anim. Pract. 20, 651–674.
Oetzel, G.R., 2007. Herd-level ketosis–diagnosis and risk factors. In: American
Association of Bovine Practitioners. 40th Annual Conference. Vancouver, BC,
Canada.
Ospina, P.A., Nydam, D.V., Stokol, T., Overton, T.R., 2010. Associations of elevated
nonesterified fatty acids and beta-hydroxybutyrate concentrations with early
lactation reproductive performance and milk production in transition dairy cattle in
the northeastern United States. J. Dairy Sci. 93, 1596–1603.
Overton, T.R., McArt, J.A.A., Nydam, D.V., 2017. A 100-year review: metabolic health
indicators and management of dairy cattle. J. Dairy Sci. 100, 10398–10417.
Peacock, M., 2010. Calcium metabolism in health and disease. Clin. J. Am. Soc. Nephrol.
5, S23–S30.
Pinedo, P, Velez, J., Solano, G., Rodriguez, N., Naves, J., Schuenemann, G.M, and Risco,
C., 2017. Effect of oral calcium administration on the cure and reproductive
performance of Holstein cows diagnosed with puerperal metritis. J. Dairy Sci. 100,
2917–2927.
RStudio Team, 2019. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA.
http://www.rstudio.com/.
Reinhardt, T.A., Lippolis, J.D., McCluskey, B.J., Goff, J.P., Horst, R.L., 2011. Prevalence
of subclinical hypocalcemia in dairy herds. Vet. J. 188, 122–124.
Sheldon, M.I., Lewis, G.S., LeBlanc, S., Gilbert, R.O., 2006. Defining postpartum uterine
disease in cattle. Theriogenology. 65, 1516–1530.
Xia, J., Sinelnikov, I.V., Han, B., Wishart, D.S., 2015. MetaboAnalyst 3.0––making
metabolomics more meaningful. Nucleic Acids Res. 43, W251–W257.
Yang, L., Yang, Q., Yi, M., Pang, Z.H., Xiong, B.H., 2013. Effects of seasonal change and
parity on raw milk composition and related indices in Chinese Holstein cows in
northern China. J. Dairy Sci. 96, 6863–6869.
Yokus, B., Cakir, U.D., 2006. Seasonal and physiological variations in serum chemistry
and mineral concentrations in cattle. Biol. Trace Elem. Res. 109, 255–266.
Zhang, Z., Li, X., Wang Guo, C., Gao, L., Zhang, Y., Li, P., Wang, Z., Li, Y., Liu, G., 2011.
Concentrations of sodium, potassium, magnesium, and iron in the serum of dairy
cows with subclinical ketosis. Biol. Trace Elem. Res. 144, 525–528.
Zhang, G., Hailemariam, G., Dervishi, E., Goldansaz, S.A., Deng, Q., Dunn, S.M.,
Ametaj, B.N., 2016. Dairy cows affected by ketosis show alterations in innate
immunity and lipid and carbohydrate metabolism during the dry off period and
postpartum. Res. Vet. Sci. 107, 246–256.
Zhang, G., Dervishi, E., Dunn, S.M., Mandal, R., Liu, P., Han, B., Wishart, D.S., Ametaj, B.
N., 2017. Metabotyping reveals distinct metabolic alterations in ketotic cows and
identifies early predictive serum biomarkers for the risk of disease. Metabolomics.
13, 43.
Zhang, G., Dervishi, E., Ametaj, B.N., 2018. Milk fever in dairy cows is preceded by
activation of innate immunity and alterations in carbohydrate metabolism prior to
disease occurrence. Res. Vet. Sci. 117, 167–177.