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J Vet Med SciJ. Vet. Med. SciJVMSThe Journal of Veterinary Medical Science0916-72501347-7439The Japanese Society of Veterinary Science 2978007618-006610.1292/jvms.18-0066PhysiologyFull PaperThe influence of the duration of the preoperative time spent in the veterinary clinic without the owner on the psychogenic and oxidative stress in dogs JUODŽENTĖ Dalia 12*KARVELIENĖ Birutė 1RIŠKEVIČIENĖ Vita 21) Dr. L. Kriaučeliūnas Small Animals Clinic, Faculty of Veterinary, Veterinary Academy, Lithuanian University of Health Sciences, Tilžės str. 18, LT-47181,
Kaunas, Lithuania2) Department of Veterinary Pathobiology, Faculty of Veterinary, Veterinary Academy, Lithuanian University of Health Sciences, Tilžės str. 18, LT-47181, Kaunas,
Lithuania* Correspondence to: Juodžentė, D.: dalia.kacinkeviciute@lsmuni.lt21 5 2018 7 2018 80 7 1129 1133 08 2 2018 09 5 2018 ©2018 The Japanese Society of Veterinary Science2018This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:  https://creativecommons.org/licenses/by-nc-nd/4.0/)The objective of this study was to evaluate the influence of the dog’s long-term separation from its owner in the novel environment on the occurrence of psychogenic and oxidative stress.
Group I dogs (n=9) were brought to the veterinary clinic and stayed in a kennel room for 12 hr before the surgery, and group II dogs (n=9) −for 10 min before the surgery. Physiological
parameters (heart rate (HR) (beats/min) and respiratory rate (fR) (breaths/min)) were measured and blood sampling was done 12 hr before the surgery (T0) for group
I dogs and 10 min before the surgery (T1) for both groups dogs. Oxidative stress index (OSI) was determined using spectrophotometer and Rel Assay Diagnostics kits by measuring TAS ant TOS in
blood plasma. The cortisol level was measured using AIA-360 Automated Immunoassay Analyzer and ST AIA-pack Cortisol assays. Group I dogs’ HR and fR were elevated
at T0 and T1, and group II dogs’−at T1 compared to physiological range. OSI and cortisol levels in group I dogs was higher at T1 compared to T0 (P<0.05). There was no
significant difference in cortisol level between group I at T0 and group II at T1 (P>0.05). It might be concluded that dogs’ longer stay in the novel environment without
the owner induced significant changes in OSI and cortisol level, which could lead to slow wound healing and the occurrence of systemic diseases.

dogoxidative stresspsychogenic stress
==== Body
Psychogenic stress is common in animals and it describes exposure to psychological or social challenges which result in disruption of psychological well-being. Negative psychogenic stress in a
domesticated animal may be induced by separation from the owner, or exposure to a novel environment [37]. Stressful situations can impair animal’s welfare
and can have an influence on various organism systems, especially for hypothalamic-pituitary-adrenal axis (HPA) activation. Biochemical and molecular changes can emerge at the cellular level in
response to exposure to stressful situations. During stress, body releases adrenaline and cortisol hormones. Adrenalin cause increase in heart and respiratory rate and cortisol suppress the
immune system [3, 6, 10, 19, 22, 26, 27, 36]. These changes can affect
reactive oxygen species (ROS) formation in the body what can lead to OS. Excess ROS amount attack biologically relevant molecules such as lipids, DNA, carbohydrates, and proteins, causing cell
membrane and nucleus impairment, and form destructive products, such as lipid peroxides. DNA damage results in carcinogenesis and mutagenesis, while protein damage results in loss of enzyme
activity [10, 19, 22, 29, 41, 42, 47]. Moreover, examination of OS occurrence due to psychogenic stress in
dogs might have practical significance, both for the welfare of confined dogs, and possibly for better understanding of negative clinical outcomes (postoperative anemia, prolonged wound healing
and prolonged postoperative recovery rates) which originate after surgeries. It is crucial to ensure patients safety before and during anesthesia, as OS level also elevates after anesthetics
administration [24, 38, 41, 46].

To our knowledge, there have been numerous studies investigating the effects of novel environment and separation from the owner on the occurrence of psychogenic stress [13, 21, 44]. However, during these studies the authors did not investigate the influence of
psychogenic stress on the occurrence of OS in dogs. We put forward the hypothesis, that long-term psychogenic stress induce significant changes not only in cortisol level but also in OS
parameters (including plasma total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI)) in client-owned dogs. For this reason, the purpose of this study was to
evaluate the influence of long-term separation from the owner in the novel environment on the occurrence of psychogenic stress and OS in dogs.

MATERIALS AND METHODS
Eighteen client-owned dogs, 10 females and 8 males, 14.9 ± 1.5 month, weighing 19.3 ± 0.5 kg, scheduled for ovariohysterectomy or orchiectomy surgery were used in this study. All dogs were
categorized to be in I group according to the American Society of Anesthesiologists (ASA) classification, based on a physical examination, complete blood cell count, analysis of serum
biochemistry, and thoracic radiographs. The study was carried out in compliance with the EU legislation. The procedures complied with the criteria given by the Lithuanian animal welfare
regulations (No. B1-866, 2012; No. XI-2271, 2012) and the decree of the director of the State Food and Veterinary Service, the Republic of Lithuania (No. B6-(1.9)-855, 2017).

Nine dogs (group I) were confined to the kennel room in the veterinary clinic in the evening, 12 hr before the surgery (the dogs previously have never been isolated in the novel environment
and separated from their owners), and other nine dogs (group II) were confined to the kennel room in the veterinary clinic 10 min before the surgery. Both group dogs were brought to the
veterinary clinic 20 min before they were confined to the kennel room. During this time, physical examination, blood sampling for complete blood cell count and analysis of serum biochemistry,
and thoracic radiographs were done. Owners were together with their dogs during these procedures.

Physiological parameters (heart rate (HR) (beats/min) and respiratory rate (fR) (breaths/min)) were observed by auscultation. Dogs were considered to have
tachycardia if their HR was higher than 100 beats/min. Tachypnea was characterized by a fR that was higher than 30 breaths/min. The first observation of HR and
fR was performed 12 hr before the surgery (T0) for group I dogs, and the second observation-10 min before the procedure (T1) for group I and group II dogs. Group
I dogs were taken out of the cage.

The housing in the individual cage in novel environment and separation from the owner were considered as stressors for dogs. Group I dogs were housed in individual cages, without food.
Drinking water was available ad libitum. Group II dogs has been subjected to food restriction 12 hr before being admitted. The temperature of the kennel room was 19 ± 1°C and
the air humidity was 50 ± 10%.

Venous blood samples (3 ml) of the dogs were collected into heparin tubes via venepuncture from the jugular vein. Blood samples were taken 12 hr before the surgery for group
I dogs (T0) and 10 min before the surgery for both group I and group II dogs (T1). Plasma samples were separated by centrifugation at 1,500 rpm for 15 min and were stored at −80°C until their
test. The cortisol level (µg/dl) was measured using AIA-360 Automated Immunoassay Analyzer (Tosoh Bioscience, Inc., South San Francisco, CA, U.S.A.) and ST
AIA-pack Cortisol assays (Tosoh Bioscience, Inc.). The total oxidant status (TOS) as well as the total antioxidant status (TAS) was determined using Lambda 25 UV/Vis spectrophotometer
(PerkenElmer, Waltham, MA, U.S.A.) and Rel Assay Diagnostics kits (Mega Tip, Gaziantep, Turkey), following the manufactures’ instructions [11, 12]. Oxidative stress index (OSI) was calculated as follows: OSI (arbitrary unit)=TOS (µmol H2O2
Eq/l)/TAS x10 (mmol Trolox Eq/l) [2].

The statistical analysis was performed using computer software SPSS 22. Averaged experimental results are reported as means ± standard error of the mean. Cortisol levels, HR and
fR between group I at T0 and group II at T1 were compared by Independent t-test. HR, fR, cortisol levels, TAS, TOS
and OSI parameters in group I between T0 and T1 were analysed using paired t-test. The level of significance was set at P<0.05.

RESULTS
Initial physiological and vital parameters of all dogs were similar and within normal range in both group I and II dogs. Animal age (group I −14.3 ± 1.2 months; group II −14 ± 2.2 months) and
weight (group I −18.7 ± 0.6 kg; group II −19.6 ± 0.8 kg) did not differ between the groups (P>0.05).

In the present study, HR and fR were observed in dogs. All dogs of group I had increased HR and fR compared to physiological range at
T0 and T1 (HR: 135 ± 4 beats/min vs 136 ± 4 beats/min, respectively; and fR 38 ± 2 breaths/min vs 39 ± 2 breaths/min, respectively). There was no significant
difference between T0 and T1 in group I dogs in HR and fR (P>0.05). Group II dogs also had increased HR and fR
compared to physiological range at T1 (HR: 138 ± 8 beats/min; and fR 42 ± 4 breaths/min). There was no significant difference between group I at T0 and group II at
T1 in HR and fR (P>0.05).

The long-term duration of the preoperative time spent in the veterinary clinic was effective in inducing measurable psychogenic (group I and group II dogs) and oxidative stress (group I dogs)
in dogs. TAS, TOS, OSI and cortisol values are represented in Table 1Table 1. Oxidative stress parameters and cortisol level of the dogs
Parameters	Dogs	T0
 (12 hr before procedure)	T1 
(10 min before procedure)	P value	
TAS (mmol Trolox Eq/l)	group I (n=9)	0.47 ± 0.02	0.43 ± 0.02 a)	<0.05	
TOS (µmol H2O2 Eq/l)	group I (n=9)	4.33 ± 0.11	6.13 ± 0.10 a)	<0.05	
OSI (arbitrary unit)	group I (n=9)	0.93 ± 0.03	1.43 ± 0.03 a)	<0.05	
Cortisol (µg/dl)	group I (n=9)	2.50 ± 0.27	6.81 ± 0.34 a)	<0.05	
Cortisol (µg/dl)	group II (n=9)	-	2.65 ± 1.42 b)	>0.05	
The values represent the average ± standard error. a) Statistically difference compared to T0 (n=9) (P<0.05). b) No difference in cortisol levels between group I at
T0 and group II at T1 (P>0.05).

.

In the present study, cortisol level elevated in group I dogs at T1 compared to T0 (P<0.05). There was no significant difference in cortisol level between group I dogs at
T0 and group II dogs at T1 (Table 1) (P>0.05). TOS and OSI levels increased (P<0.05), while TAS level
decreased in group I at T1 compared to T0 (P>0.05). Paired t-test revealed differences in cortisol, TAS, TOS and OSI levels in group I dogs between T0 and
T1 time points (P<0.05).

DISCUSSION
A visit to a veterinary clinic may be stressful for the patient. Stressful situations are associated with physiological changes, increased cortisol and catecholamine production and oxidative
stress [5, 13, 33]. Usually, dogs visit veterinary clinic due to annual
vaccination, routine checks or for elective surgery. There are situations, when a dog can be separated from its owner in the veterinary clinic (before the surgery, etc.). Dogs confined in
veterinary clinic cage room before surgery experience a wide range of psychogenic stressors, including not only separation from the owner and impact of novel environment, but also noise,
unfamiliar people, unpredictability, constrained environment [3, 13, 18,
21, 35, 44]. It is very important to reduce psychogenic (do not leave the
dogs in the veterinary clinic before surgery for long period of time) and oxidative stress of pre-operative hospitalized dogs under veterinary care, because it seems likely that the impact of
these types of stress on clinical outcomes, such as postoperative anemia or prolonged postoperative recovery rates, is underestimated [38, 41].

In the present study we have showed, that long-term separation from the owner in the novel environment induced psychogenic stress and increased cortisol level in group I dogs. Cortisol has
been used as a stress biomarker in both humans and animals [8, 15]. Our data correspond with other studies that had
investigated psychogenic stress in rodents, primates and dogs. The studies with rodents and primates determined that isolation in the novel environment activates HPA axis [17, 30]. Tuber et al. [43], in their study also have proven
that domestic dogs placed into a novel environment for 4 hr showed an increasing of plasma adrenocorticoid levels. According to Hennessy et al. [16], dogs sampled at their homes were found to have much lower plasma cortisol levels than did the comparison group of dogs sampled in the novel environment (county animal
shelter) on the days 1–3 of the trial.

In the present study we have also shown, that stressors, but not daytime had an influence on fluctuations of cortisol levels in group I dogs. We compared group I (blood sample was taken in
the evening) and group II (blood sample was taken in the morning) dogs blood plasma cortisol levels and did not find any significant difference (P>0.05). Kolevska
et al. in their study have not found diurnal rhythms in experimental (dogs used for experimental purposes on a long-term basis) and in working group (dogs used for various
operations) dogs, but circadian rhythm was found in dogs with common daily routine with no physical and emotional load [23]. Takahashi et
al. [40] did not confirm the diurnal cortisol secretion rhythm in their study, as well as Kemppainen and Sartin [20]. However, whether cortisol in dogs’ blood shows circadian rhythm is controversial. Beerda et al. [4] have shown
that cortisol levels change according to a circadian rhythm, having a peak in the morning hours decreasing gradually until the evening. Palazzolo and Quadri [31] have found that circadian rhythm is present in adult dogs only, while in puppies and old dogs no rhythmic fluctuations in cortisol secretion were found.

Reactions to stress are associated with increased secretion of hormones such as glucocorticoids, catecholamines etc., which change physiological parameters−accelerate heart and respiratory
rate, increase cardiac output [34].

In the present study, HR was increased comparing to physiological range. Our findings are in agreement with those of other studies that investigated the effect of stress on the HR. Csoltova
et al. [9], have shown that veterinary examinations produced acute stress responses in dogs with significant increases in HR. Also,
increases in HR (136.4 ± 17.2 beats/min) and cortisol level (179.89 ± 91.87 nmol/l) were observed in dogs, which have been stressed while waiting in the veterinary clinic
waiting room [32].

During our study, fR rate was elevated comparing to physiological range in dogs. Increased fR refers to conditions of higher oxygen
levels than normal partial pressure of oxygen in the lungs or other body tissues. It leads to greater production of ROS [7, 28]. Srithunyarat et al. [39] in their study have shown that dogs came for ovariohysterectomy had elevated
fR (92.6 ± 63.3 breaths/min) and cortisol level (174.6 ± 78.5 nmol/l) due to stress caused by novel environment (veterinary clinic).

In the present study, we also evaluated the effects of novel environment and separation from the owner on the occurrence of oxidative stress in dogs. Usually all types of stresses appear at
the cellular level as oxidative stress that is characterized by decreased activity of antioxidant defense system and increased production of ROS. Our data showed, that long-term separation
from the owner in the novel environment activated antioxidant defense mechanisms in dogs (increase in oxidants (TOS) level and decrease in antioxidants (TAS) level). Elevated
oxidant-antioxidant ratio could occur due to activation of HPA axis, which caused different effects on physiological parameters (HR, fR). According to Maksymchuk
et al. [25] psychogenic stressors significantly altered oxidative stress markers in mice liver. In the human studies, it was shown
that chronic stress exposure promotes oxidative damage through the activation of the HPA axis [1]. Also, there is some evidence, that behavioural stress
increases oxidative damage to cell nuclei in humans [14].

This study provides valuable data on the impact of long-term (chronic) stress on OS in dogs. It might be useful for veterinary surgeons, as during surgery, anesthetics and surgical trauma
also increase OS level, and higher than normal OS can have a negative influence on postoperative recovery rates and wound healing [24, 38, 45].

These findings support the hypothesis that long-term stress induced significant changes in OSI, which could lead to slow wound healing and the occurrence of systemic diseases.

This study was supported by the Science Foundation of the Lithuanian University of Health Sciences (LUHS).
==== Refs
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