Survey on basal blood plasma catecholamine concentrations in
Martina Franca donkey (Equus asinus)
P. DE PALO, A. MAGGIOLINO* , E. CECI, G. CALZARETTI, P. CENTODUCATI and A. TATEO
Department of Veterinary Medicine, University “A. Moro” of Bari, Italy, Valenzano (Ba), Italy.
*Correspondence email: firstname.lastname@example.org; Received: 12.07.17; Accepted: 02.12.17
Background: Catecholamines are among the most frequently investigated parameters for studying sympathoadrenal activity in response to stress
Objectives: To evaluate basal plasma concentrations of catecholamines (adrenaline, noradrenaline and dopamine) in healthy donkeys.
Study design: Cross-sectional study.
Methods: Catecholamine concentrations from 440 Martina Franca donkeys were determined: 269 females and 171 entire males, aged from 4 months
to 24 years. Animals were subdivided into four age categories: under 12 months old (64 males and 54 females), from 13 to 36 months (56 males and
75 females), from 37 to 120 months (49 males and 80 females) and over 120 months (24 males and 38 females). Indwelling jugular catheters were
inserted at least 12 h before drawing blood. The data set was subjected to analysis of variance considering age, sex and the two-way interaction
between them as independent variables. Pearson’s correlation coefﬁcients between the three catecholamines were evaluated.
Results: Conﬁdence intervals (CI) for noradrenaline concentration ranged between 239.98 and 255.07 ng/L (mean 247.52 ng/L), for adrenaline between
129.27 and 137.90 ng/L (mean 133.59 ng/L), dopamine concentrations between 149.62 and 160.80 ng/L (mean 155.21 ng/L) and noradrenaline/
adrenaline ratio between 1.91 and 2.05 (mean 1.98). Catecholamine plasma concentrations were not inﬂuenced by sex. Donkeys older than 37 months
had lower adrenaline and noradrenaline plasma concentrations (P<0.001) and higher noradrenaline/adrenaline ratios (P<0.01) than younger animals.
Main limitations: Indwelling catheters and blood drawing procedures may have inﬂuenced catecholamine levels.
Conclusions: Catecholamine concentrations were established within a large group of healthy Martina Franca donkeys.
Keywords: horse; donkey catecholamines; adrenaline; noradrenaline; dopamine
Activation of adrenergic system is mediated by neurotransmitters such as
noradrenaline, dopamine and adrenaline [1,2]. The magnitude of these
responses is relative to the type and length of the stimulus, such as the
duration of transport , the intensity and duration of training , and also
interaction with human subjects . Animals’ previous experience, age,
sex, temperament, breed, health status and environmental conditions can
affect their response [6,7]. The absence of stress is a potential indicator of
animal welfare, but there is a need for species-speciﬁc biomarkers.
Currently, studies of the variations in catecholamine levels have been
Thoroughbreds [8–12] and Standardbreds . There are no studies
reporting catecholamine concentrations in donkeys and there is a need for
these to facilitate studies on donkey welfare. The aim of this study was to
evaluate levels of blood plasma catecholamines in healthy Martina Franca
donkeys, with no evident stressors, and to determine if there are
differences associated with age and sex.
Materials and methods
A ﬂow chart illustrating eligible animals, their recruitment and ﬂow through
the study is provided in Supplementary Item 1. Initially, 962 Martina Franca
donkeys were considered eligible. After exclusions, 440 donkeys were
used for this study, 269 females and 171 entire males, aged from
4 months to 24 years. All donkeys were clinically healthy and had not
received any medication during the month before sampling. The animals
were from 11 different farms in the South of Italy in which they were bred
under semi-extensive conditions: kept at pasture during daytime, and in
indoor pens with paddock access at night. Animals were fed daily with a
diet including oat hay and commercial feed (rolled corn 30%, soybean meal
24%, wheat bran 16%, rolled barley 14%, rolled oats 14%, vitamin and
mineral integration 2%), while an automatic drinking trough provided water
Indwelling jugular catheters were placed at least 12 h before the start of
sampling. The 16 Gauge catheters were placed aseptically into the left
jugular vein of each animal, and ﬁxed to the skin with adhesive tape and
covered with bandage. None of the donkeys lost the catheter. The
catheter was heparinised (1000 U/mL). Sampling was conducted from 9.00
to 12.00. Venous blood for analysis was placed in chilled glass tubes
containing EDTA (Vacutainer
. Collection procedures were timed with a
digital stop watch and lasted at most 15 s. If blood drawing lasted more
than 15 s, the sample was excluded from data set. The blood samples
were immediately stored on ice for less than 10 min, then the plasma was
separated by centrifugation at 3500 9 g at 4°Cfor15min.Foreach
sample, duplicate 1 mL aliquots of plasma were collected in cryovials,
immediatelyfrozenondryiceandtransferredtoaÀ80°C freezer for
storage until analysis.
Behavioural patterns were observed during the blood sampling
procedures. All animals showing signs of aversion to sampling (sudden
head deviation, recoiling, attempts to bite, resistance to restraint) were
excluded from the trial.
1100 HPLC system consisted of isopumps (G1310A), an
autosampler(G1313A),adegasser(G1379B) and an FLD detector (G1321A).
A guard column (Inertsil
ODS-3, 5 lm 9 20 mm 9 4.0 mm) and a
reversed-phase C18 column (Genesis ODS-3V,
4 lm 9 150 mm 9
4.6 mm) were also used. The concentrations of dopamine, adrenaline and
noradrenaline in the blood samples were determined by HPLC according to
the guidelines set out on the kit data sheet (Eureka Serotonin and
Catecholamine HPLC detection KITs
). The detection limit was 0.03 nmol/L
Equine Veterinary Journal 50 (2018) 493–497 © 2017 EVJ Ltd
Equine Veterinary Journal ISSN 0425-1644