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Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Physiological adjustments of haematological prole during

the last trimester of pregnancy and the early post partum
period in mares
Marilena Bazzano, Claudia Giannetto, Francesco Fazio, Maria Rizzo,
Elisabetta Giudice, Giuseppe Piccione
Department of Veterinary Sciences, University of Messina, Polo Universitario dellAnnunziata, 98168 Messina, Italy

a r t i c l e

i n f o

Article history:
Received 12 May 2014
Received in revised form 2 July 2014
Accepted 4 July 2014
Available online xxx
Keywords:
Haematology
Mare
Pregnancy
Post partum
Leucocyte differential count

a b s t r a c t
The aim of the study was to assess changes in some haematological parameters of periparturient mares from the last three months of pregnancy until the third week after foaling. The
study was carried out on 15 healthy pregnant mares (Group A) and 7 healthy non-pregnant
non-lactating mares (Group B). Blood samples were collected by jugular venipuncture into
EDTA tubes and tested for Haematocrit (Hct), Red Blood Cell (RBC), Haemoglobin (Hb),
Platelets (Plt) and White Blood Cell (WBC). Peripheral blood smears were also performed
to assess changes in the leucocyte differential count. Group A showed a progressive reduction in Hct and Hb levels over the experimental period, whereas Plt and WBC (P < 0.05)
peaked at parturition. Among WBC, neutrophils increased until parturition when lymphocytes reached the lowest values. The statistical analysis also revealed signicant interactions
time pregnancy on Hb (P < 0.001), WBC (P < 0.001), neutrophils (P < 0.001) and lymphocytes (P < 0.001). Analysing the last trimester of pregnancy and the rst post partum period
the present study provides suitable information about the inuence of peripartum on mares
haematological parameters.
2014 Elsevier B.V. All rights reserved.

1. Introduction
During pregnancy maternal body system is adapted to
ensure growth and development of the foetus (Hadden
and McLaughlin, 2009). Despite homeostatic mechanisms
function to maintain blood parameters within physiologic
levels, some changes are like to occur (Harvey et al., 2005).
In effect, perinatal mares are physiologically unstable (Aoki
et al., 2013). At this stage they are susceptible to a number of
abnormalities including uterine torsion and haemorrhage,
retained foetal membranes, metritis and necrotic vaginitis

Corresponding author. Tel.: +39 0903503584; fax: +39 0903503975.

E-mail address: giuseppe.piccione@unime.it (G. Piccione).

(LeBlanc, 2008). Recent studies dealt with several aspects of

mares physiology during pregnancy, some authors focused
on immunologic (Aoki et al., 2013), haemostatic (Bazzano
et al., 2014), biochemical (Satu and Montesinos, 2013)
and haematological (Aoki and Ishii, 2012; Mariella et al.,
2014) proles around parturition. It is well known that the
equine species has various haematological differences as
a result of age (foals or adults), breed (horses or ponies),
sex (male or female) and reproductive status (Weiss and
Wardrop, 2010). The haematological prole represents a
useful screening and diagnostic test that can promptly
provide valuable information about mares health status.
Although mares haematology has been investigated, these
studies concerned with few data points and a limited
observation time. However, reproductive problems can

http://dx.doi.org/10.1016/j.anireprosci.2014.07.005
0378-4320/ 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Bazzano, M., et al., Physiological adjustments of haematological prole during the last trimester of pregnancy and the early post partum period in mares. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.07.005

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Table 1
Gestation length (days) and parity (+ multiparous, primiparous)
recorded from pregnant mares (Group A).
Group A

Parity

Gestation length (days)

Horse 1
Horse 2
Horse 3
Horse 4
Horse 5
Horse 6
Horse 7
Horse 8
Horse 9
Horse 10
Horse 11
Horse 12
Horse 13
Horse 14
Horse 15

+
+
+

+
+
+
+

+
+
+

337
350
346
341
350
356
335
323
333
344
345
328
338
331
339

adversely affect mares health since the last trimester of

pregnancy (LeBlanc, 2008).
On the basis of this knowledge, we aimed to assess
changes in some haematological parameters of periparturient mares from the last three months of pregnancy until
the third week after foaling. Furthermore, we focused on
the determination of leucocyte differential cell counts, in
order to analyze whether modications in peripheral blood
leukocytes occurred at this time.
2. Materials and methods
2.1. Animals and sampling
The study was carried out on 15 healthy pregnant
mares (Group A) and 7 healthy non-pregnant non-lactating
mares (Group B). Animals of different breed (Italian Saddle,
Thoroughbred, Standardbred) and age (417 years) were
enrolled in the study with the informed owner consent.
Animals were housed in individual straw-bedded boxes
(4.0 m 3.5 m) at the same breeding centre (latitude 37.46
N; longitude 14.93 E) under natural environmental conditions (mean temperature 14 8 C; mean relative humidity
70%). Animals were fed twice a day (7.30 am; 5.00 pm) and
water was available ad libitum. Pregnant mares received
6 1 kg/day hay and 5 0.5 kg/day concentrates (crude
protein 16%, crude fat 6%, crude bre 7.35%, ash 10.09%,
sodium 0.46%, lysine 0.85%, methionine 0.35%, omega-3
0.65%), non-pregnant mares received 5 0.5 kg/day hay
and 2 0.5 kg/day concentrates. Animals were allowed to
go to pasture during the day (from 10.00 am to 4.00 pm).
Group A was monitored from the 34th week of pregnancy until the 3rd week after foaling. In the rst three days
after parturition animals were subjected to daily clinical
examination. During the post partum period, transrectal ultrasound exams were weekly performed to ensure
the normal involution of the uterus using the M-Turbo
ultrasound system (FUJIFILM SonoSite, London, United
Kingdom). All the pregnant mares delivered between
March and mid-May, the mean gestation length was
340 9 days (Table 1). Mares from Group A showed the

post partum oestrus 8 1 days after foaling although none

of them was bred at the foal heat. Blood samples were
weekly collected on the same day of the week in the morning (7.00 am) until the time of parturition. For each mare
we recorded 12 time-points (P-12 to P-1) before delivery
occurred. Additional samples were taken within 24 12 h
from parturition (P) and then 7, 14 and 21 days after foaling (P + 1, P + 2, P + 3). Blood samples were collected by
jugular venipuncture into 3 ml vacutainer tubes containing
EDTA (Terumo Corporation, Tokyo, Japan). After collection
all samples were delivered to the laboratory and analyzed
within an hour.
All treatments, housing and animal care were carried
out in accordance with the standards recommended by the
EU Directive 2010/63/EU for animal experiments.
2.2. Haematological analysis
On blood samples Haematocrit (Hct), Red Blood Cell
(RBC), Haemoglobin (Hb), Platelets (Plt) and White Blood
Cell (WBC) were assessed using an automated haematology
analyzer (HeCo Vet C, SEAC, Florence, Italy). Furthermore, two peripheral blood smears were performed for
each sample. After air-drying, the slides were stained
through Dif-Stain kit (Titolchimica srl, Rome, Italy). The
same laboratory professional has later performed the
microscopic analysis of blood lms by using an optical
microscope (Nikon Eclipse e200, Nikon Instruments Europe
BV, Amsterdam, Netherlands). A manual 200-cell differential count was performed on each blood lm. For each mare,
the leucocyte differential count was calculated by averaging of the data recorded from each blood lm of the same
sample.
2.3. Statistical analysis
Data, expressed as means standard error of the mean
(SEM), were tested for normality using the ShapiroWilk
normality test. Two-way repeated measure ANOVA was
applied to assess signicant effects of the experimental
condition on haematological parameters. When signicant
differences were found Bonferronis post hoc comparison
was applied. P values <0.05 were considered statistically
signicant. Statistical analysis was performed using the
STATISTICA software package (STATISTICA 7 Stat Software
Inc., Tulsa, Oklahoma).
3. Results
Animals included in the study showed no clinical signs
of disease during the experimental period. All pregnant
mares delivered healthy, viable foals at full term gestation.
The statistical analysis revealed signicant changes on
Hct, Hb, Plt and WBC values recorded in pregnant mares
over the experimental period (Table 2). The ShapiroWilk
analysis showed a normal distribution of the data. A progressive reduction in Hct and Hb levels was observed
only in Group A over the experimental period, with the
lowest values recorded at the time-points subsequent
to parturition. Signicant changes were observed in Plt
count (P < 0.05) that reached the highest concentration

Please cite this article in press as: Bazzano, M., et al., Physiological adjustments of haematological prole during the last trimester of pregnancy and the early post partum period in mares. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.07.005

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Table 2
Mean values standard error of the mean (SEM) of Haematocrit (Hct), Red Blood Cell (RBC), Haemoglobin (Hb), Platelets (Plt) and White Blood Cell (WBC)
recorded in experimental group A. Statistical signicances related to each haematological parameter (within the same column) are indicated by superscripts.
Time was expressed as weeks before () and after (+) parturition (P).
Time (week)
12
11
10
9
8
7
6
5
4
3
2
1
P
+1
+2
+3

RBC (106 /L)

Hct (%)
38.14
38.05
37.99
38.03
37.55
37.32
36.78
36.08
35.81
35.75
36.67
35.83
35.97
34.64
34.06
33.55

cd

0.94
0.80cd
0.92cd
0.72cd
0.80cd
0.65
0.70
1.00
0.78
0.91
1.11
1.21
1.08
0.89
1.02
0.91

7.88
7.85
7.82
7.89
7.82
7.79
7.74
7.70
7.52
7.51
7.76
7.61
7.60
7.34
7.19
7.22

0.17
0.14
0.17
0.13
0.15
0.13
0.12
0.09
0.11
0.11
0.20
0.16
0.19
0.13
0.16
0.14

Plt (103 /L)

Hb (g/dL)
14.98
15.04
15.11
14.95
14.38
14.18
13.76
13.26
12.90
12.95
12.98
12.02
10.81
10.76
10.04
10.17

abcde

0.37
0.35abcde
0.42abcde
0.36abcde
0.58ab
0.55ab
0.58ab
0.76ab
0.68cd
0.79cd
1.07cd
0.94
0.75
0.76
0.74
0.78

141.0
141.0
142.1
145.2
148.5
149.4
149.8
147.1
145.7
146.4
141.0
148.4
170.8
159.9
143.7
140.8

WBC (103 /L)

a

8.67
8.46a
6.91a
6.51a
8.15a
8.37a
7.67a
8.23a
9.62a
6.48a
7.38a
8.74a
6.23
6.13a
4.95a
6.88a

6.61
6.62
6.67
6.74
6.80
6.86
6.91
7.11
7.04
7.46
7.66
7.88
8.89
7.80
7.35
7.31

0.23abef
0.21abef
0.24abef
0.25abef
0.28abcd
0.23abcd
0.22abcd
0.22abcd
0.24abcd
0.19abcd
0.19abcd
0.24
0.33
0.22
0.28
0.29

Signicances (P < 0.05): a vs P; b vs +1; c vs +2; d vs +3; e vs 1; f vs 2.

Fig. 1. Mean values standard error of the mean (SEM) of neutrophils and lymphocytes recorded in pregnant (a) and control (b) mares throughout the
study. Time was expressed as weeks before (12 to 1) and after (+1 to +3) parturition (P). Signicances: * vs 12; vs 1; vs P;  vs +1, +2, +3.

Please cite this article in press as: Bazzano, M., et al., Physiological adjustments of haematological prole during the last trimester of pregnancy and the early post partum period in mares. Anim. Reprod. Sci. (2014),
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at parturition in pregnant mares. Signicant effects of

pregnancy and time (P < 0.05) were found on WBC that
started to increase from the 4th week preceding foaling
(P-4) and peaked at parturition. During pregnancy, the
percentage of neutrophils and lymphocytes signicantly
changed in Group A showing an opposite way. Neutrophils
gradually increased peaking at parturition, while lymphocytes tended to decrease reaching the lowest values at P
(Fig. 1).
The statistical analysis also revealed signicant interactions time pregnancy on Hb (P < 0.001), WBC (P < 0.001),
neutrophils (P < 0.001) and lymphocytes (P < 0.001).
4. Discussion
The present study shows how mares haematological parameters changed during the last three months
of pregnancy and the rst three weeks after parturition. Signicant changes on Hct, Hb, Plt, WBC, neutrophil
and lymphocyte levels occurred within the experimental
group during the study. Starting from the 8th week before
parturition, a progressive decrease in Hct was observed
throughout the experimental period. A recent review by
Tan and Tan (2013) showed that plasma volume increases
3050% in pregnancy (Bernstein et al., 2001). The fall
in Hct in pregnant women is always lower compared
to non-pregnant women. The increase in plasma volume
depends on the increased activity of the renin-angiotensinaldosterone system that is a physiological condition of
pregnancy both in women (Irani and Xia, 2008) and mares
(Satu and Domingo, 2011). This results in increased
sodium reabsorption from the kidneys and water retention. The increase in blood volume together with the drop in
blood viscosity represent important adjustments in order
to keep up with the increase in blood ow to organs like
uterus and kidneys (Harm et al., 2012). The hypervolemia
of pregnancy is also necessary to protect the mother and the
foetus from the harmful effects of decreased venous return
and to prevent the mother from suffering the adverse
effects of blood loss during delivery (McMullin et al., 2003).
The Hb concentrations progressively decreased in pregnant
mares, reaching the lowest values in the post partum
period. A similar Hb prole was observed in Brasileiro
de Hipismo and Breton mares (Orozco et al., 2007), as
well as in Standardbred mares (Mariella et al., 2014). Our
results also agree with studies carried out in pregnant
women (Bailit et al., 2007), dairy cows (Steinhardt et al.,
1994) and sows (Zvorc et al., 2006). Because of no signicant change was found on RBC values the disproportionate
rise in plasma volume might have determined a dilutional
anaemia. Effectively, mares water needs start increasing in
the last trimester of pregnancy because of placental uids
and the foetus. A further increase in water consumption
occurs during lactation when mares requirement rise up
to 5080% above that required for maintenance (Lewis,
1995). Another physiological cause of the decline in Hb is
the reduced lifespan of circulating erythrocytes in pregnant
females compared to non-pregnant ones (Lurie, 1993). Plt
count stayed within the physiological range indicated for
the horse (100350 103 /L) (Weiss and Wardrop, 2010).
However, the peak in Plt recorded at foaling might reect a

hypercoagulable state that physiologically occurs in mares

around parturition (Bazzano et al., 2014). As previously
observed in Standardbred (Mariella et al., 2014) and heavy
draft mares (Aoki et al., 2013), we found a signicant
increase of WBC count around parturition. Our results also
agree with a study by Harvey et al. (1994) that showed the
highest leucocyte count after foaling. An adrenocorticoidmediated leukocytosis was found in pregnant women as
well. This results in an increase of white cell count that
is particularly noticeable during labour and the puerperium (Tan and Tan, 2013). The leucocyte differential
cell count revealed signicant modications in peripheral blood neutrophil and lymphocyte percentage that
comply with the studies by Aoki et al. (2013) on heavy
draft mares. A progressive increase in neutrophil percentage was found starting from the 4th week before foaling,
although the highest values were recorded at the time
of delivery. Oppositely the lymphocyte percentage gradually decreased around parturition. Similar results were
recorded by Harvey et al. (1994) who found a significant increase in neutrophil concentration shortly after
foaling. Effectively, in the advanced stage of gestation an
endogenous adrenaline release occur inducing a greater
mobilization of neutrophils in the circulation resulting in
an increase in total leucocyte count (Kramer, 2000). A signicant rise in blood cortisol concentrations was observed
in pregnant mares in the imminence of parturition as well
(Nagel et al., 2012). Cortisol has a considerable inuence
on blood cells, including a reduction in the number of lymphocytes and an increase in the number of neutrophils and
in the total number of leukocytes (Kaneko et al., 1997).
In addition, during the peripartum the hormonal prole
signicantly changes as the mare quickly restores a normal
cyclicity and fertility following parturition (Cupps, 1991).
Although ovarian hormones like oestradiol and progesterone have been associated with a number of physiological
changes in the female, studies on cycling mares (Roberto
da Costa et al., 2003) and cows (Ahmadi et al., 2006) found
no signicant change in studied haematological parameters at either phase of the oestrus cycle. According to this
knowledge we considered irrelevant the inuence of the
oestrous cycle on mares haematological prole during the
post partum period.
5. Conclusion
The haematological prole represents one of the most
common investigations performed in the clinical practice
that promptly provide suitable information about animals
health status. During a specic physiological status like
pregnancy, it is important for the clinician to have the best
knowledge about physiological modications at this stage.
Our study analyzed the last trimester of pregnancy and
the rst post partum period providing suitable information
about the inuence of peripartum on mares haematological parameters.
Conict of interest statement
None.

Please cite this article in press as: Bazzano, M., et al., Physiological adjustments of haematological prole during the last trimester of pregnancy and the early post partum period in mares. Anim. Reprod. Sci. (2014),
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Please cite this article in press as: Bazzano, M., et al., Physiological adjustments of haematological prole during the last trimester of pregnancy and the early post partum period in mares. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.07.005