Theriogenology 84 (2015) 667–673

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Theriogenology
journal homepage: www.theriojournal.com

Endothelin 1 in healthy foals and in foals affected

by neonatal diseases
A. Giordano a, b, C. Castagnetti c, S. Panzani d, *, S. Paltrinieri a, b, F. Freccero c,
M.C. Veronesi d
a
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy
b
Veterinary Teaching Hospital, Polo Veterinario di Lodi, University of Milan, Lodi, Italy
c
Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, Bologna, Italy
d
Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In newborn babies, endothelin 1 (ET-1), a potent vasoconstrictor, increases during septi-
Received 17 March 2015 cemia and severe respiratory syndromes. Because equine neonatal sepsis (ENS) and
Received in revised form 27 April 2015 perinatal asphyxia syndrome (PAS) are major causes of morbidity and mortality in
Accepted 28 April 2015
newborn foals and because no information on the concentration of ET-1 in healthy and sick
foals has been reported yet, the aims of this study were (1) to define the serum concen-
Keywords:
tration of Big ET-1 in healthy neonatal foals during the first week of age; (2) to prelimi-
Endothelin 1
narily explore the diagnostic and prognostic role of Big ET-1 during ENS and PAS. Six
Equine neonatal sepsis
Perinatal asphyxia syndrome healthy and 23 sick foals affected by ENS and/or PAS were enrolled in the study. In healthy
Newborn foal foals, Big ET-1 concentration increased in the first hours of life until 24 hours after birth,
and it remained constant during the first 3 days, then gradually decreased becoming
significantly lower from Day 4 onward (P < 0.05). In sick foals, only 26.1% of animals
showed higher values of Big ET-1 than controls at admission, and no difference between
surviving and nonsurviving foals was found. Because in nonsurviving foals, Big ET-1
remained over the maximum value recorded in clinically healthy horses or, when
normal at admission, increased over time; this study suggested that repeated measure-
ment of Big ET-1 during hospitalization may be helpful in monitoring the course of the
disease. In conclusion, possible prognostic information may be obtained by repeated
analysis of Big ET-1 during hospitalization, but further studies are needed.
Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction myocardiocytes. Moreover, ET-1 modulates the interaction

Endothelin 1 (ET-1) is a potent vasoconstrictor synthe-
sized mainly by endothelial cells in response to several
stimuli that include proinflammatory cytokines, hormones,
angiotensin II, hypoxia, and vasodilation [1]. Endothelin 1
contributes to maintain the baseline vascular tone or
to increase systemic pressure [1,2] by stimulating
specific receptors expressed on smooth muscle cells or on
* Corresponding author. Tel.: þ39 02 50318149; fax: þ39 02 50318148.
E-mail address: sara.panzani@unimi.it (S. Panzani).
between endothelial cells and leukocytes [3] and the pro-
duction of cytokines [4]. In turn, sepsis and endotoxemia
may damage endothelial cells and induce overproduction
of ET-1 [5]. Endothelin 1 is considered a useful marker in
human neonatology because its serum concentration in
newborns is particularly high compared to adults, increases
in newborns with sepsis, and correlates with the severity of
the disease [6]. Additionally, high serum concentrations of
ET-1 have been reported in asphyctic newborns [7] or in
newborns with persistent pulmonary hypertension [8].
Endothelin 1 can be measured in domestic animals
using reagents designed for human ET-1 because of a high

0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.04.026
668 A. Giordano et al. / Theriogenology 84 (2015) 667–673
interspecies homology of ET-1 [9]. Measurement of the
precursor Big ET-1 that has a longer half-life and well cor-
relates with the concentration of ET-1 is usually preferred
[10]. Big ET-1 has been measured in cardiopathic dogs and
cats [10,11], in dogs with chronic kidney disease [12] or
endotoxic shock [13].
In horses, ET-1 has been studied during laminitis [14,15],
joint diseases [16], colic syndromes [17], and respiratory
disorders [18–20].
Equine neonatal sepsis (ENS) and perinatal asphyxia
syndrome (PAS) are the major causes of morbidity and
mortality in newborn foals [21].
Equine neonatal sepsis occurs in the first 2 weeks of life
and is characterized by the release of proinflammatory
cytokines that induce a systemic inflammatory reaction
syndrome, which in turn causes endothelial damage and
release of vasoactive mediators [22] and may progress to
septic shock and death. Vasoactive mediators induce
pulmonary hypertension, followed by a hypotensive status
typical of the endotoxic shock [23].
Perinatal asphyxia syndrome may depend on maternal,
placental, or fetal [24] causes. Hypoxia affects primarily
the central nervous system and induces a variety of clinical
signs that depend on the type and duration of hypoxia.
Moreover, tissue hypoxia may induce systemic inflam-
matory reaction syndrome [25]. Both conditions (ENS and
PAS) can, therefore, lead to a possible overproduction of
ET-1. The early diagnosis of ENS and PAS is essential for a
successful outcome; thus, the availability of biomarkers
that support a clinical diagnosis or provide prognostic
information may be useful in the management of sick
foals.
Because the authors are not aware of other studies on
the concentration of ET-1 in clinically healthy foals or in
foals with ENS or PAS, the aims of this study were to define
the serum concentration of Big ET-1 in healthy neonatal
foals of different ages and to explore the potential of Big
ET-1 as a diagnostic and prognostic biomarker of neonatal
septicemia and asphyxia.
2. Materials and methods
The study has been conducted during the breeding
seasons 2013. All procedures on the animals were carried
out with the approval of the Ethical Committee of the
University of Bologna, in accordance with DL 116/92,
approved by the Ministry of Health. The owners gave oral
informed consent.
2.1. Control group
Six light horse foals (Equus caballus), two females, and
four males, born by spontaneous delivery at the Equine
Perinatology Unit of the Department of Veterinary Medical
Sciences of the University of Bologna, were included in the
study. All foals should fulfill some requirements: they
should be full term, with normal size, coat, and fetlock joint
extension. The mean Apgar index within 10 minutes of
birth should be greater than eight, and all foals must show
normal physical and behavioral characteristics, as previ-
ously described to assess maturity and viability [26,27].
No clinical signs or laboratory changes consistent with
ENS, PAS, or with any other disease should be found during
the period of observation.
For the study purposes, blood was collected from of
each foal via the jugular vein, with the following schedule:
3 hours from birth (T0); 12 hours from birth (T1); 24 hours
from birth (T2); 2 days (T3); 3 days (T4); 4 days (T5); 5 days
(T6); 6 days (T7); 7 days (T8).
Blood was collected in plain tubes and, immediately
after clotting, serum was separated by centrifugation
(2500  g for 10 minutes) and stored at 20  C until
analyses.
2.2. Sick foals
Twenty-three light breed sick foals, aged less than 4
days old (mean age: 28.4  25 hours), admitted to the
Equine Perinatology Unit of the Department of Veterinary
Medical Sciences of the University of Bologna were
included in the sick group.
The inclusion criterion for the pathologic group was the
diagnosis of ENS and/or PAS requiring level 2 or 3 of
intensive care [27]. Level 2 care is provided to neonates that
are quite severely affected; they may be unable to stand or
to nurse from the mare and need round-the-clock care.
Usually this level of care involves separation of the foal
from the dam. Level 3 care is specific for extremely
compromised foals that usually have multisystem
dysfunction; they need round-the-clock care and must be
assisted by specialists.
Foals were classified as affected by ENS when showed
a positive blood culture and/or a sepsis score greater
than 11 [28], whereas PAS was diagnosed on the basis of
history and clinical signs, such as the presence of neuro-
logic dysfunction without other neurologic diseases or
trauma [29].
Blood culture was performed regardless of antibiotic
therapy before referral using 10 mL of jugular blood with-
drawn after clipping and aseptic preparation of the skin.
The sampling needle was then discarded, and a new needle
was used to inoculate the blood into the commercially
available culture bottle (Oxoid Signal Blood Culture System;
Oxoid Limited).
Ten foals were classified as affected by ENS, eight by PAS,
whereas five foals suffered both the diseases.
From admission until discharge or death, animals were
constantly monitored by clinical, laboratory, and instru-
mental examinations, according to the severity of the
disease.
Each foal received proper treatments in relation to its
specific clinical presentation; any resuscitation treatment
(that could influence ET-1 concentrations) was recorded.
Sick foals were sampled every 24 hours from the day of
admission until discharge (n ¼ 12) or death (n ¼ 8, four
with ENS, three with ENS and PAS, and one with PAS). The
first sample was always collected before any medical
treatment. Only 20 foals were repeatedly sampled during
hospitalization, since three foals died soon after
admission.
Blood was collected in plain tubes and, immediately
after clotting, serum was separated by centrifugation
 A. Giordano et al. / Theriogenology 84 (2015) 667–673 669

Fig. 1. Values of Big endothelin 1 recorded in six healthy foals during the first week of life. Boxes indicate the I to II interquartile interval, the horizontal line
corresponds to the median, the vertical lines are the limits of outlier distribution according to the Tukey rule. Near outliers are indicated by the symbol “þ”. The
same letter indicates values not statistically different; different letters correspond to P < 0.05.

(2500  g for 10 minutes) and stored at 20  C until
analyses.
2.3. Endothelin 1 measurement
Big ET-1 was measured in all the samples using a
sandwich ELISA (IBL Hamburg). The kit includes a primary
capturing antibody against the C-terminal 22 to 38 amino
acid sequence of rat Big ET-1 which shows closer homology
to the equine amino acid sequence [9] and a secondary
antibody for detection, directed against a different anti-
genic site of the molecule. As a standard material to
generate a calibration curve, human big ET-1 is included in
the kit and the tetramethylbenzidine as a chromogen. The
procedure was performed following the recommendation
of the manufacturer of the kit, and the plates were read
using an automated plate reader (Dasit multiscan, Dasit)
at a wavelength of 450 nm. Intra-assay and interassay
coefficients of variation reported by the manufacturer are
lower than 5% and 13%, respectively at each value. However,
to reduce the interassay variability, all samples were pro-
cessed in the same batch.
2.4. Statistical analysis
Statistical analysis was performed using Analyse-it
software for Microsoft Excel. P values less than 0.05 were
considered to be statistically significant. Results recorded in
healthy foals at different sampling times were compared
using a nonparametric ANOVA test for paired data
(Friedman test) followed by the Wilcoxon signed-rank test
to assess the difference between each time point. The
frequency of abnormal values (i.e., values higher than the
maximum value recorded in healthy foals in the first 3 days
of life) recorded at admission in all sick foals and in foals
grouped on the basis of diagnosis was calculated. Finally, to
achieve preliminary information about the prognostic
value of ET-1, results recorded in nonsurviving foals at
admission were compared with those of survivors using a
nonparametric Student’s t test for unpaired samples (Mann
Whitney U test).
3. Results
All six healthy foals fulfilled the required characteristics,
and in detail: birth weight ranging between 40 and 58 kg,
mean Apgar index within 10 minutes of birth was 9  0.89,
the time to stand up was 55.9  10.27 minutes and to the
first suckle was 101  13.37 minutes. Results collected from
healthy foals in the first 7 days of life are reported in
Figure 1.
As shown in the Figure 1, the serum concentration of
Big ET-1 moderately increases in the first hours of life
(17.4  4.0 pg/mL at 3 hours, 22.9  5.5 pg/mL at 12 hours)
until 24 hours (20.4  6.4 pg/mL) after birth and then
gradually decreases until Days 5, 6, and 7 (mean values
around 12 pg/mL), when values are significantly lower than
those recorded on Days 1, 2, and 3 (mean values around 18
pg/mL). No significant differences were recorded in the first
3 days of life.
On the basis of lack of significant differences in healthy
animals in the first five samples, values collected from sick
foals at admission (before any medical treatment) were
compared with the maximum value recorded in healthy
670 A. Giordano et al. / Theriogenology 84 (2015) 667–673
Fig. 2. Distribution of values recorded in 23 sick foals at admission. The
dotted gray line indicates the upper value recorded in clinically healthy foals
aged less than 4 days. Big-ET-1, Big endothelin 1.
foals from T0 to T4 (35.2 pg/mL). However, considering the
low number of animals involved, only a descriptive analysis
was performed. Such a comparison (Fig. 2) revealed that
only 6 of 23 sick foals (26.1%) had values higher than this
threshold. This group of foals with high Big ET-1 included 3
of 10 foals with ENS (30%), 2 of 8 foals with PAS (25%), and 1
of 5 foals with ENS and PAS (20%).
Six of the sick foals received resuscitation treatment
(four foals were treated with dobutamine and two foals
with dobutamine and norepinephrine): two with PAS, one
with ENS, and three with ENS and PAS. Eight sick foals
(four with ENS, three with ENS and PAS, one with PAS)
died during hospitalization, five of which received the
previously mentioned treatments. Among these, only four
(two with ENS, one with PAS, and one with both the
diseases) had higher ET-1 values than controls at admis-
sion. Nevertheless, the results of nonsurviving foals
(mean  standard deviation: 23.5  5.7 pg/mL; median:
21.8 pg/mL; I to III interquartile range: 19.4–28.6 pg/mL)
were not significantly different (P ¼ 0.123) from those of
survivors (29.9  25.2 pg/mL, 18.6 pg/mL, 13.3–54 pg/mL,
respectively).
The analysis of sequential results (Fig. 3) showed that
only one of eight nonsurviving foal had Big ET-1 values
persistently below the maximum values recorded in
healthy foals. Conversely, the serum concentrations of Big
ET-1 remained persistently high or had only transient
decreases in the four foals that at admission had abnormal
values and increased during hospitalization in the three
foals that had normal levels at admission.
In surviving foals that had normal values of Big ET-1 at
admission, instead, serum levels of Big ET-1 remained
within the normal values during the whole hospitalization,
whereas in the only one foal with high values at admission
that was repeatedly sampled over time, Big ET-1 values
rapidly normalized.
4. Discussion
Despite the frequent occurrence of ENS and PAS in
newborn foals, studies about prognostic markers of both
these conditions are scarce. In human medicine, sepsis
and asphyxia are known to induce increases in the serum
concentration of ET-1 either in adults or in newborns,
where increases of ET-1 or of its precursor Big ET-1 may
provide prognostic information [6,7,30,31]. Conversely, no
information is reported on ET-1 in foals affected by ENS
and/or PAS and, more importantly, no information is
available on the possible age-related changes of the
serum concentration of this biomarker in healthy
neonatal foals, so this represents the first study on the
possible use of ET-1 for health status evaluation in
newborn foals.
Therefore, the first step of the study was focused on
investigating the possible presence of changes in the
serum concentration of Big ET-1 in the first days of life in
healthy foals. Because, unfortunately, this part of the study
was biased by the low number of animals, it was not
possible to define appropriate reference intervals for each
sampling time, using the approaches recommended by the
current guidelines [32,33]. However, the statistical
 A. Giordano et al. / Theriogenology 84 (2015) 667–673
Fig. 3. Values recorded during hospitalization in sick foals. Foals with septicemia are indicated with the squares; foals with asphyxia are indicated with circles;
foals with septicemia and asphyxia are indicated with the triangles. Black symbols indicate nonsurviving foals; white symbols indicate surviving foals. The dotted
gray line indicates the upper value recorded in healthy foals less than 4 days old. Big ET-1, Big endothelin 1.
comparison of the results obtained in healthy foals in the
first week of life revealed a trend that parallels what is
reported in human newborns, where Big ET-1 values in-
crease in the first hours of life and then start to decrease
after 5 days until Day 30 [34,35]. A similar trend has been
also reported in piglets [36]. In our caseload, even on Day 7,
values were still higher than those reported in adult horses
in studies that, however, used a different method for
measuring Big ET-1 [17,18]. Because ET-1 is elevated in fe-
tuses, where it contributes to maintain an adequate pul-
monary vasodilation at birth followed by a progressive
reduction of vascular resistance after birth [37], this age-
related change may depend on an incomplete removal of
circulating ET-1 by the liver of newborns, less functional
than in adults [31]. As already reported for other molecules
involved in inflammation or tissue damages [38,39],
independently from the mechanism responsible for these
changes, the finding of higher serum concentration of Big
ET-1 in newborn foals than in adults evidences the need to
establish, in the future, age-specific reference intervals.
This would avoid to erroneously interpreting as extremely
high Big ET-1 values that actually depend on the young age
of the foals.
Results from sick foals, however, evidenced that the
diagnostic or prognostic role of increased Big ET-1 levels at
admission (before any medical treatment) seems to be
moderate, because approximately one fourth (26.1%) of sick
foals had values higher than the maximum value recorded
in clinically healthy foals. Moreover, increases of serum Big
ET-1 seem not to be associated with a specific condition
because the group of foals with abnormal values included
foals with ENS, PAS, or both.
Additionally, although the majority of foals with
abnormal levels of Big ET-1 (four of six) died during hos-
pitalization, 4 of 8 nonsurviving foals had normal values at
admission and no significant differences were found
671
between survivors and nonsurvivors. All these findings
seem to contrast with what reported in human medicine,
where high ET-1 values correlate with a poor prognosis
either in sepsis or in conditions characterized by hypoxia
[30,31,40]. Also, previous studies on adult horses reported
that ET-1 may be a marker of severity of inflammatory
conditions and negatively correlated with survival [17].
Therefore, it may be postulated that the magnitude of ET-1
responses to endotoxic shock or hypoxia in foals is less
intense than in adult horses or in other species. Alterna-
tively, the lack of differences between sick and healthy foals
or between surviving and nonsurviving foals may depend
on the short time elapsed between the onset of clinical
signs and the first sampling or on different degree of
severity of tissue damages in affected horses. Figueras-Aloy
et al. [6] reported that the highest ET-1 levels in neonatal
sepsis appeared at 3 to 5 days after the diagnosis and later
decreased. Only newborns with severe sepsis presented a
significant increase in ET-1 concentrations from the
beginning of the septicemic process.
However, any conclusion about the possible prognostic
value of a single measurement of serum ET-1 at admission
should be reviewed when age-specific reference intervals
are established on a larger cohort of healthy neonatal foals.
Conversely, this study reported that repeated mea-
surement of Big ET-1 during hospitalization may be helpful
in monitoring the course of the disease: Because of the
heterogeneity of samplings collected during hospitaliza-
tion (the different foals were admitted at different ages,
foals received different therapies, such as resuscitation
treatments, specific for their clinical conditions, and the
length of hospitalization was variable), results recorded
over time were not statistically compared to each other.
However, with a single exception, Big ET-1 values in non-
survivors, independently on the disease (PAS and/or ENS),
remained over the maximum value recorded in healthy
672 A. Giordano et al. / Theriogenology 84 (2015) 667–673
foals or, when normal at admission, increased over time.
Five of the six animals that received resuscitation therapies
did not survive; therefore, treatment was ineffective and
may have contributed to the increase of Big ET-1 during
hospitalization because it is known that in humans, during
septic shock, the release or the production of endothelin
may increase as a consequence of the infusion of cate-
cholamines [41]. It must be stressed that the significance of
high values in sequential samplings is stronger than the
single value at admission. In healthy foals, in fact, the
serum concentration of Big ET-1 decreases with age, and
therefore, the difference between values recorded in
healthy and in sick foals become even more evident as age
increases. These results indicate that, when the disease
progresses despite the administration of any kind of
treatment, stimuli inducing the release of ET-1 from
damaged endothelium persist and maintain the serum
concentration of Big-ET-1 elevated.
4.1. Conclusions
In conclusion, this study evidenced that serum con-
centration of Big ET-1 in foals shares some similarity with
that of human ET-1. In particular, age-related changes
found in the first days of life are the same as recorded in
human newborns. The low number of animals involved in
the study is the most important limitation of the work to
arrive to strong conclusions. However, considering that this
is the first reference of using ET-1 for health status evalu-
ation in foals and the promising results obtained, this work
underlines the need of further investigations to create
specific age-related reference ranges for the equine
newborn. This information will be relevant on a diagnostic
perspective, to avoid erroneously interpreting as high
values that are actually normal in newborns.
Moreover, because in nonsurviving foals, Big ET-1
remained over the maximum value recorded in clinically
healthy horses or, when normal at admission, increased
over time, this study suggested that repeated measurement
of Big ET-1 during hospitalization may be helpful in
monitoring the course of the disease. Prognostic informa-
tion may be obtained by repeated analysis of Big ET-1
during hospitalization, but further studies are needed.
Competing Interests
None of the authors has any financial or personal
relationships that could inappropriately influence or bias
the content of the article.
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