The Influence of Subclinical Hypocalcemia on Production and Reproduction Parameters in Israeli Dairy Herds

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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 16
INTRODUCTION
Parturient paresis is a metabolic disorder occurring close
to parturition especially in high producing dairy cows. Te
disease is characterized by a rapid decline in blood calcium
(Ca) concentrations. Nearly all mature cows experience some
degree of hypocalcemia during the frst day after calving as
the intestine and bone adapt to the Ca demands of lacta-
tion (1). In some cows, the mammary drain of Ca causes
extracelluar and blood Ca concentrations to decline to levels
that disrupt neuromuscular function, resulting in the clinical
syndrome of “Milk Fever”. Tis Ca decline lasts in some cases
for several days postpartum (2).
Post-parturient hypocalcemia is divided into clinical
and subclincal forms (3, 4). Te literature indicates that the
clinical form is associated with an increase in post-parturient
diseases (5, 6, 7, 8). In large parts of the United States and
some European countries it is assumed that the subclinical
form is also related to post parturient diseases (5, 9, 10, 11)
and has a negative impact on proftability. Terefore, many
dairies use expensive feed additives to reduce the incidence
of this form (12, 13). Fatty acid metabolism might difer
between cows with subclinical hypoclacemia and their nor-
mocalcemic counterparts (14) however, there is conficting
evidence regarding the impact of subclinical blood calcium
levels on milk production, reproduction parameters and
post-parturient disorders (10, 14, 15). Most of the research
Te Inf luence of Subclinical Hypocalcemia on Production and
Reproduction Parameters in Israeli Dairy Herds
Gild, C.,
1
* Alpert, N.
2
and van Straten, M.
3
1
HaChaklait, Kfar Tavor Hashkedim, Israel.
2
HaChaklait, Kibbutz Degania, Israel.
3
HaChaklait, Moshav Klahim, Israel.
*
Corresponding Author: HaChaklait, Kfar Tavor Hashkedim 50, P.O. Box 484, 15241 Israel. Email: gild@hachaklait.co.il
ABSTRACT
A large percentage of mature dairy cows experience some degree of hypocalcemia during the frst days
post-calving. In some cases calcium concentrations decline to levels that disrupt neuromuscular function,
resulting in the clinical syndrome known as parturient paresis or milk fever. Post-parturient hypocalcemia
is divided into clinical and subclinical forms. It has been established that cows sufering from clinical milk
fever are susceptible to a variety of secondary conditions, however to the best of the authors’ knowledge there
has been no evaluation of the impact of the subclinical form on production and reproductive parameters.
Te objective of this study was to investigate the association between subclinical hypocalcemia and post-
parturient disorders, production and reproductive parameters in Israeli dairy herds. Blood results for corrected
calcium concentrations were analyzed from 634 mature cows from 5 farms. Te subclinical hypocalcemic
cows produced 3.2, 2.7 and 1.9 kg more milk in the frst three milk recordings than the normocalcemic cows.
Subclinical hypocalcmic cows did not show an increased risk for post-parturient diseases nor compromised
reproduction parameters in comparison to normocalcemic cows. It was concluded that there was no negative
impact of subclinical hypocalcemia on production and reproductive parameters in Israeli dairy cows after
parturition.
Keywords: Bovine; Milk Fever; Calcium; Hypocalcemia; Milk Production.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 17 Subclinical Hypocalcemia in Dairy Cows
published targets methods to improve calcium homeostasis
through manipulations of dietary cationic anionic difer-
ence or through calcium binders (16, 17, 18, 19) however to
the best knowledge of the authors’ the direct impact of the
subclinical form on production and reproductive parameters
has not been adequately investigated. Te objective of this
study to investigate whether the subclinical hypocalcemia
state has an infuence on post-parturient diseases, reproduc-
tive parameters and milk production parameters in Israeli
dairy herds.
MATERIALS AND METHODS
Animals and study design
Te study was comprised of two separate entities: Te frst
study, the preliminary study was conducted on a 60 cow dairy
farm in the northern part of Israel. Cows were housed in large
covered loose housing systems and fed dry cow total mixed
ration (TMR) pre-calving and a standard milking TMR
post-calving both manufactured by Givaat Yoav Feeding
Center located in Moshav Givaat Yoav, Israel. Blood was
drawn from the coccygeal vein of 11 mature Holstein cows
at a 4 hour interval from the beginning of frst stage of la-
bor and up to 12 hours postpartum. Blood was drawn once
more at 24 hours postpartum. Samples were immediately
centrifuged and serum was harvested and frozen at -20°C
for laboratory analysis at the Kimron Veterinary Institute,
Beit Dagan. According to these results a post-calving calcium
curve was prepared to determine the time of nadir levels of
serum calcium.
Te second study, the main study was conducted on 4
commercial Israeli dairy herds of 200-450 milking cows each
between June 2006 and July 2007. Blood was drawn from
633 mature Holstein cows between 8-20 hours postpartum
when serum Ca levels were expected to be the lowest based
on the results of the frst preliminary study. Samples were
immediately centrifuged and serum was harvested and frozen
at -20°C for further laboratory analysis.
All cows were either housed in free stalls or large covered
loose housing systems. All herds were fed dry cow TMR pre-
calving and a standard milking TMR post-calving. All farms
received their feed from diferent feed manufactures (Table 1).
In all herds cows were milked 3 times daily identifed
by ear tags and freeze marking in computer controlled
milking parlors. Te annual milk production was 10,000-
12,500 kg/cow. Te herds were within the practice area of
the “HaChaklait”, a mutual society for veterinary medicine
which provides a complete herd-health service. Visits are
conducted by a veterinarian to the farms during the study
period at least twice weekly.
All cows were examined after calving by trained veteri-
narians who diagnosed, treated and recorded all the peripar-
turient disease conditions. Cases of retained fetal membranes
(RFM) were defined as the presence of placental tissues 24
hours or more after calving as observed by trained farm em-
ployees or the attending veterinarian. Animals with observed
or suspected RFM were submitted for veterinary examination
on the next routine veterinarian visit (1-4 days postpartum).
Animals without a history or diagnosis of RFM were submit-
ted for examination between 6 and 9 days postpartum. At this
examination, body condition scoring (BCS) of all animals
was recorded and cows were comprehensively examined by
intravaginal palpation after thoroughly cleaning the perineal
area. Te diagnosis of clinical endometritis (CEM) was based
on the combined characteristics of vaginal discharge obtained
by manual examination of the vagina. Afected cows with
CEM had a watery or purulent, fetid vaginal discharge as
previously described (20).
All cows were examined for ketosis by placing a drop of
urine obtained with a sterile disposable plastic catheter on
a reagent strip (Ketostix, Bayer, Germany). Te color reac-
tion was compared to the standardized color chart after 15
seconds. Cows with urine aceto-acetate concentrations above
15 mg/dl were recorded as ketotic (3). Cows with lower than
expected milk production and poor appetite were examined
for displacement of the abomasum (LDA) by auscultation
and percussion. BCS was further recorded approximately
40-60 days after calving and before the dry-of period. All
animals not observed in estrus by the end of the voluntary
waiting period at approximately 60 days postpartum were
recorded and submitted for examination.
Table 1: Farm name and feed manufacture
Farm Feed manufacturer
Kibbutz Geshur
Givaat Yoav Feeding Center, Moshav Givaat
Yoav, Israel
Kibbutz Beit Zera
Amabar Feeding Center, Moshav Kefar
Yehezkel, Israel
Kibbutz Givaat
Haim Meuhad
Amatz Feeding Center, Moshav Amatz, Israel
Kibbutz Afkim Kibbutz Afkim, Israel
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 18
Clinical, reproductive, production and management
data were computer recorded by the herd manager and the
attending veterinarians. Cows not observed in estrus were
recorded for further reproduction calculation as cows not
showing heat. Once a month, each cow’s milk was sampled
and analyzed for fat, protein, lactose and somatic cell count
by the Central Laboratory for Milk Recording at the national
service for udder health and milk quality located in Caesarea
industrial park, Israel.
Reproductive management was solely based on artifcial
insemination performed by trained technicians employed by
“Sion” Israeli Company for artifcial insemination and breed-
ing, Migdal Ha’emek, Israel. In all herds, cows were mainly
inseminated on observed estrus or computerized pedometry
system. Conception rates and cumulative pregnancy were
based on pregnancy diagnosis performed by rectal palpation
of the uterus and its contents 40-50 days post-insemination.
Blood analysis
Total serum calcium levels were determined using Arsenazo
III method and calcium levels were corrected for serum al-
bumin which was determined using the Bromocresol Green
method. Correction was done using the following equation (3).
Corrected Calcium (mg/dl) = Measured Calcium (mg/dl) –
Albumin (g/dl) + 3.5
Statistical analysis
All data editing and analysis were performed using SAS
version 9.0 (21). Results were considered to be of statistical
signifcance if the relevant P-value was < 0.05.
In general, data analysis followed a 3 step approach: (1).
Descriptive statistics which included calculation of the mean,
standard deviation and histogram for continuous variables,
and frequency tables for other variables (2). Bivariate analysis
in which associations between a dependent variable and an
independent variable were assessed using the chi-square
test for categorical variables and t-tests when one of the
variables was on a continuous scale. For time to event data,
i.e. days from calving to conception, survivor functions were
compared using the Kaplan-Meier method and log-rank test
(3). Multivariable analysis in which associations between the
dependent variable and two or more independent variables
were assessed. Multivariable analysis was only performed
if, for the same dependent variable, two or more signifcant
associations were found in the bivariate analysis. In our case,
this only occurred in the analysis of milk production data.
For the latter, average test-day milk (kg) was estimated from
monthly test-day data using a linear model with a marginal
efect to account for repeated measurements from the same
cow. A maximum of 10 test-days was allowed per cow.
Lactation number was grouped into four categories, ie.
second, third, fourth and ffth, or greater lactation. Summer
months were considered June to September, inclusive.
Subclinical hypocalcemia was defned in a cow with a
corrected serum Ca level of < 7.5 mg/dl and without clini-
cal hypocalcemia 12-24 h postpartum. Somatic cell counts
(cells/mL) were grouped in 4 categories: ≤ 100,000; 101,000-
200,000; 201,000-400,000 and > 400,000. Farms were mod-
eled as a fxed efect and the correlation matrix used for R
was autoregressive. Te model we used was:
Y = farm (4 index variables) + summer (2 index variables)
+ lactation (4 index variables) + MIM (10 index variables) +
HCOR (2 index variables) + MIM + HCOR*MIM + SCCL
(4 index variables) + CEM (2 index variables) + e.
Y was test-day fat percentage, summer represents test
days occurring in the summer months, lactation was lacta-
tion group, MIM was month in milk, HCOR was sub-
clinical hypocalcemia, SCCL was somatic cell count level,
and “e” a complex error term representing the within-cow
correlation of test-day fat percentage and the residual error.
Signifcance of the fxed efects was determined using the
F-test (21).
RESULTS
Preliminary study
Corrected serum calcium levels obtained from all 11 cows
were combined into an average level for each 4 hour inter-
val. Tese average levels were plotted by time from calving.
Calcium levels decreased between calving and reached a nadir
at 8 hours-calving and stayed low until 20 hours post-calving.
Although not statistically signifcant, based on these results,
8-20 hours post-calving was chosen as the period of the nadir
of serum calcium levels for the main study. (Figure 1).
Main study
Data sets included measurements from 634 cows. 247, 186,
103 and 97 cows from second, third, fourth, ffth or greater
lactations, respectively. Tere was missing data on calving dis-
ease incidence for one cow and 16 cows sufered from clinical
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 19 Subclinical Hypocalcemia in Dairy Cows
milk fever. Terefore a total of 617 cows were included in the
fnal data set.
Of all cows 18.9% sufered from subclinical hypocalcae-
mia. Milk fever incidence was 7.63% and 1.36% for the sub-
clinical hypocalcemic and normocalcemic cows respectively.
Te probability for development of clinical milk fever was
found to be statistically higher in the subclincial hypocalce-
mic group as opposed to the normocalcemic group of cows.
(P < 0.0007) (Table 2).
Te probability for a cow developing subclinical hypocal-
cemia increased with lactation number (P < 0.0001) (Table 3).
Table 2: Milk Fever by Subclinical hypocalcaemia
Subclinical
Hypocalcemia
Milk Fever
Total
No Yes
No 508 7 515
% 98.64 1.36
Yes 109 9 118
% 92.37 7.63
Total 617 16 633
% 97.47 2.53 100
Table 3: Subclinical hypocalcaemia by lactation
Lactation
Number
Subclinical Hypocalcemia
Total
No Yes
2 235 12 247
% 95.14 4.86
3 164 22 186
% 88.17 11.83
4 63 40 103
% 61.17 38.83
>=5 53 44 97
% 54.64 45.36
Total 515 118 633
% 81.36 18.64 100
Calving diseases
Of the normocalcemic cows, 12% versus 11% of subclinical
hypocalcemic cows sufered from retained placenta. Tere was
no statistical diference between normocalcemic and subclini-
cal hypocalcemic cows (P < 0.897). Of the normocalcemic
27.6% cows versus 19.3% of subclinical normocalcemic
cows sufered from metritis however there was no statistical
diference in the probability of a subclinical hypocalcemic
cow sufering from metritis (P < 0.087). 21.3% of normo-
calcemic cows versus 25.7% of subclinical normocalcemic
cows sufered from ketosis however there was no statistical
diference in the probability of a subclinical hypocalcemic
cow sufering from ketosis (P < 0.376). Only 2 cows from
the normocalcemic group sufered from an LDA. 5.7% of
normocalcemic cows versus 3.7% of subclinical hypocalcemic
cows had stillbirths. Tere was no statistical diference in
the probability of a subclinical normocalcemic cow to have
stillbirths (P < 0.533).
Although subclinical milk fever (defned as corrected
serum calcium below 7.5 mg/dl) was associated with milk
fever incidence no association was found between serum
calcium and calving diseases.
Reproduction
Tirty-two cows received a “do not breed” code or were culled
before frst insemination and therefore 585 cows were in-
cluded in the reproduction analysis study.
Tere was no statistical diference (P < 0.755) between
groups for the frst artifcial insemination (AI) conception
rate. 29.7% vs. 27.6% for the normocalcemic and subclini-
cal hypocalcemic cows respectively. Tere was no statistical
diference (P < 0.453) between groups for cows not showing
heat. 33.7% vs. 38.1% for the normocalcemic and subclini-
cal hypocalcemic cows respectively. Tere was no statistical
diference (P < 0.958) in the cumulative conception until 180
days in milk between both groups (Figure 2).
No associations were found between subclinical milk
fever (defned as corrected calcium below 7.5 mg/dl) and
reproduction parameters.
Milk production
Subclinical hypocalcemic cows produced signifcantly more
milk when compared with normoclacemic cows. 3.17, 2.71
and 1.90 kg more milk was produced on the frst, second and
third test days, respectively (Table 4).
Figure 1: Corrected serum calcium (mg/dl) by time from
calving (hours)
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 20
Table 4: Milk production by calcium group.
Test day Subclinical
Hypocalcemia
Estimate
Kg
Standard
error
P-value
1
st
test day No -3.1673 1.1607 0.0064
1
st
test day Yes 0
2
nd
test day No -2.7079 1.1508 0.0187
2
nd
test day Yes 0
3
rd
test day No -1.8951 1.1368 0.0956
3
rd
test day Yes 0
Subclinical hypocalcemic cows produced more milk in
the frst 6 milk test days (Figure 3).
DISCUSSION AND CONCLUSIONS
Many physiological pathways are dependent on blood ionized
calcium levels. Decreased ionized calcium levels have been
found to be associated with increased fat mobilization around
calving (14) and these decreased levels could presumably
infuence the gastrointestinal track motility (22) leading to
decreased feed intake and as a result an increased prevalence
of metabolic disorders and other post-parturient diseases. It
has also been shown that decreased levels of calcium stores
in peripheral blood mononuclear cells precedes measurable
hypocalcemia and that hypocalcemia at parturition further
exacerbates the ability of these cells to release intracellular
calcium in response to intracellular signals therefore impair-
ing these cells’ ability to be activated (8). Tese changes col-
lectively could probably contribute to the impaired immune
system of the periparturient cow and its increased susceptibil-
ity to infectious diseases (5, 6, 7, 8).
It would be reasonable to assume that these cows would
show impaired reproductive performance and decreased
milk production later in their lactation. However, our re-
sults demonstrate that in the Israeli high producing cow
this subclinical hypocalcemia is not a risk factor for the
development of postpartum diseases, furthermore we have
demonstrated that subclinical hypocalcemic cows produce
more milk. Tese results agree with previous studies which
showed that hypocalcemia at calving is not a signifcant
risk factor for decreased milk yield (14, 23). Our results of
higher milk production for the hypoclacemic cows could
be related to a higher genetic merit causing these cows to
produce more milk and subsequently exhibit lower circulating
calcium levels. On the other hand it must be pointed out that
these milk parameters do not rule out the possible damage
caused by these low circulating blood calcium levels. It has
been demonstrated that lame cows produce more milk in
comparison to their non-lame herd mates (25, 26). Despite
these fndings it is clear that lameness is a risk factor for
decreased milk production. When comparing the impact of
these two diseases it could be that the hypocalcemic cows in
our study, although higher in milk than their normoclacemic
herd mates could have produced even more milk had we cor-
rected their circulating blood calcium levels. Furthermore, we
did not analyze the quality of milk and whether calcium level
had any efect on the immunoglobulins or other parameters
of milk quality.
Our study had several limitations. First, due to the num-
ber of cows in the frst study we made a subjective decision on
the time of sampling which could have infuenced the number
of cows being defned as subclinical hypocalcemic. Second,
we could not measure ionized calcium and therefore had to
Figure 2: Cumulative conception until 180 days in milk Figure 3: Milk production (Kg) by month
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 21 Subclinical Hypocalcemia in Dairy Cows
use the correction equation according to albumin levels. It
is possible that this calculation of corrected calcium resulted
in an underestimation of the true level of ionized calcium
in the blood and that some cows in the subclinical calcium
group should have actually been included as normocalcemic
cows. On the other hand our study was done on several farms
feeding from diferent rations, feeds and feed suppliers which
gave substantial power to our results.
Taking into consideration other research done in this
feld, our results should be interpreted with caution and
ideally should be validated by future large scale studies and
further research.
In conclusion, subclinical hypoclacemia in the Israeli
dairy herd does not seem to impair production and repro-
duction parameters. Terefore the authors do not fnd it
reasonable or necessary to incorporate feed additives that
reduce the incidence of this disorder as long as milk fever
incidence remains in the normal range for Israeli dairy herds.
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