- Email: [email protected]
Influence of transrectal palpation training on cortisol levels and heart rate variability in cows
Accepted Manuscript Influence of transrectal palpation training on cortisol levels and heart rate variability in cows Hannah Giese, Marc Dilly, Yasmin Gundelach, Gundula Hoffmann, Marion Schmicke PII:
S0093-691X(18)30513-2
DOI:
10.1016/j.theriogenology.2018.07.016
Reference:
THE 14630
To appear in:
Theriogenology
Received Date: 12 March 2018 Revised Date:
12 July 2018
Accepted Date: 16 July 2018
Please cite this article as: Giese H, Dilly M, Gundelach Y, Hoffmann G, Schmicke M, Influence of transrectal palpation training on cortisol levels and heart rate variability in cows, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.07.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT 1
Influence of transrectal palpation training on cortisol levels and heart rate variability in cows
2
Hannah Giesea, Marc Dillya1, Yasmin Gundelachb, Gundula Hoffmannc, Marion Schmickeb
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a
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[email protected] b
6 7
[email protected]; [email protected] c
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Leibniz
Institute
for
Agricultural
Engineering
and
Bioeconomy,
[email protected] Abstract
Potsdam,
Germany;
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Clinic for Cattle, University of Veterinary Medicine Hanover, Hannover, Germany;
RI PT
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Clinical Skills Lab, University of Veterinary Medicine Hanover, Hannover, Germany;
Transrectal palpation of cows is a day-one competence for veterinary students, and it is an essential
11
skill for the diagnosis of pregnancy as well as reproductive disorders. We hypothesized that
12
transrectal palpation induces a stress response in cows, and this stress response may vary with the
13
training students receive before their first transrectal palpation. Therefore, 52 Holstein-Friesian cows
14
were used at the University of Veterinary Medicine Hanover. The experimental group (n = 26) was
15
subjected to transrectal palpations by first and second-year students. Salivary and serum cortisol
16
levels were assessed before and after the intervention. A control group (n = 26) was only restrained
17
and tested for changes in salivary and serum cortisol.
18
A total of 12 cows of the experimental group were examined by two groups of students with
19
different training on two days. The examination was performed one day by students who were
20
theoretically prepared for transrectal palpation in cows (NO-SBT, n = 12). The other day, students
21
who underwent a simulator-based training (SBT, n = 12) performed the examination. The cortisol
22
concentrations, as well as heart rate (HR) and heart rate variability (HRV), were measured in the
23
examined cows. Blood and saliva samples were collected 25 min and immediately before (0 min) and
24
25 min and 85 min after the end of the examination in the experimental group. Serum cortisol levels
25
between 0 min and 25 min were increased by Δ2.6 ng/ml in the cows in the experimental group
26
compared to Δ-0.3 ng/ml in the control group (P = 0.001). The increases in cortisol in saliva (P =
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0.033) and serum (P = 0.013) after transrectal palpation were higher in the NO-SBT group Δ0.32
28
ng/ml saliva, Δ5.8 ng/ml serum than in the SBT group Δ0.03 ng/ml saliva, Δ2.1 ng/ml serum. For HR
29
and HRV analysis values recorded 30 min before the transrectal palpation (-30 min) were set as the
30
baseline concentrations the sequence recorded during the transrectal examination started at 0 min.
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While the mean HR did not change significantly during the transrectal palpation (80 to 83 bpm SBT
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students; 81 to 79 bpm NO-SBT students), the HRV parameter square root of the mean squared
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scil animal care company, Viernheim, Germany; [email protected]
ACCEPTED MANUSCRIPT differences of successive R-R intervals (RMSSD) decreased in the NO-SBT group (P = 0.034) during
34
transrectal palpation compared to the baseline values (17.47 to 5.07 ms). These findings reflect an
35
activation of the hypothalamic-pituitary-adrenal axis as well as a reduction in vagal tone during the
36
teaching and practice of transrectal palpation by students. Moreover, the results indicate that a
37
transrectal palpation is less stressful for cows when the examination is performed by students that
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were previously prepared by simulator-based training.
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Keywords
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Transrectal palpation; Teaching; Cows; Stress; HRV; Cortisol
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1.
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In dairy farming, pregnancy diagnosis has become a central issue for profitable (herd) management
43
[1]. Transrectal palpations are widely used in large animal practices for pregnancy diagnosis and
44
fertility management. In the case when infertility is suspected, further gynecological examinations
45
are needed [2]. The first step in pregnancy and fertility diagnosis is transrectal palpation. Therefore,
46
it is still referred to as a basic diagnostic skill and day-one-competence for veterinary students and
47
veterinarians in large animal practices [3].
48
Previous studies have emphasized the high number of repetitions (up to 200) required to obtain the
49
skill required to determine pregnancy in cows from a transrectal palpation [4, 5]. It has been
50
suggested that training on cows and with a variety of gynecological conditions are necessary to
51
develop proficiency in this skill [4, 5]. Nagel et al. [6] showed that students who had repeated
52
training sessions on horses (four training sessions vs. one training session) scored significantly better
53
when asked to palpate the uterus and ovaries in horses during a practical test. Nevertheless, the
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opportunities for repetitive transrectal palpation training are limited for several reasons (e.g., animal
55
welfare issues, high number of students, etc.). At the University of Veterinary Medicine Hanover,
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Foundation (TiHo) students often perform their first transrectal examination of cows during
57
mandatory training in the first or second year of study. During the teaching of transrectal palpation, a
58
couple of challenges should be considered. From a student’s point of view, identifying structures
59
through feces and the rectum wall is very challenging [7]. At the same time, the instructor has no
60
chance to control the results of the student’s palpation. Thus, the instructor cannot help any student
61
that faces difficulties during the examination.
62
Apart from difficulties during teaching, animal welfare and ethical considerations have led to a
63
reduction in animal use in veterinary education. It has been shown that transrectal palpation of
64
horses and cattle can induce stress. An increase in the blood cortisol levels of cows after transrectal
65
palpation of the reproductive tract is indicative of stress [8, 9]. In mares, Schönbom et al. [10] found
66
a significant increase in salivary cortisol levels after transrectal ultrasonography (whereas
67
transabdominal ultrasonography did not significantly influence the salivary cortisol levels). Increased
68
blood cortisol levels occur due to an activation of the hypothalamic-pituitary-adrenal (HPA) axis.
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Cortisol passes into saliva through passive intracellular diffusion. Salivary cortisol has been shown to
70
correlate to blood cortisol levels, as demonstrated in horses and cattle [11, 12]. Hernandez et al. [13]
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found that there is a 10-minute lag between the peak cortisol concentrations in the saliva and plasma
72
of cattle. Cortisol secretion underlies weak circadian and rather strong ultradian rhythms with
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pulsatile secretion and great individual variation [14]. The mentioned physiological variations must
74
be considered when the cortisol changes during experiments are interpreted.
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Introduction
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ACCEPTED MANUSCRIPT Other parameters used to measure stress-related reactions of an organism are heart rate and heart
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rate variability (HRV). Heart rate variability describes the time intervals between consecutive heart
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beats (R-R intervals) [15]. While changes in mean heart rate (mHR) reflect reactions of both the
78
sympathetic and parasympathetic nervous system, HRV parameters allow for a more accurate
79
interpretation of which autonomic nervous system (ANS) component induced the observed changes.
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The high-frequency component of HRV (HF) and the square root of mean squared differences of
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successive R-R intervals (RMSSD) are associated with changes in the parasympathetic nervous system
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(PNS) [16]. Both parameters have been examined during stressful and/or painful events and it has
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been shown that stress leads to a reduction in vagal tone and thus to a decrease of HF and RMSSD
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[17]. Furthermore, changes in mHR and different HRV parameters were found during transrectal
85
examinations and reflected the stress-induced changes in the activity of the ANS [18, 19]. Last but
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not least the perception of the handling and veterinary procedures and their stress response varies
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among individuals. A combination of genetic disposition, previous experiences and habituation
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influence the (perceived) stress load [18, 20].
89
The use of animals in education and humane teaching methods have been of interest in recent years
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[21-25]. Simulators for teaching transrectal palpation are available and have previously been
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evaluated as effective for teaching and learning [26, 27]. These simulators are considered a useful
92
alternative to training on cows [5, 28, 29]. Comparing different simulators and theoretical
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preparation, students benefit equally from simulator-based training before they perform their first
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transrectal examination on live animals in terms of self-efficacy as well as correct identification of
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organs [30]. Simulator-based training prior to the first transrectal examination of cows has not been
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implemented yet for first-year students at the University of Veterinary Medicine Hanover. This may
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be hindered by a lack of research on the effect of teaching methods on the stress induced in the
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animal. Therefore, we have examined different stress parameters in cows undergoing a transrectal
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palpation after students received simulator-based training versus a theoretical lesson.
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We hypothesized that the stress response of the cows undergoing a transrectal examination by
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veterinary students is lower when the students previously received simulator-based training.
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2.
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2.1
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A group of loose-housed Holstein-Friesian dairy cows (n = 52) was used. The cows were clinically
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healthy, lactating and pluriparous, the ages, number of births or days in milk were not considered.
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Cows in the experimental group were empty, cows in the control group were not tested for
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reproductive state. The study occurred between March and October 2014 on the Farm for Education
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and Research in Ruthe (University of Veterinary Medicine Hanover, Foundation). On this farm, first 4
Materials and Methods Animals
ACCEPTED MANUSCRIPT and second-year veterinary students receive mandatory practical training that includes practicing
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veterinary procedures on cows. Thus, the cows are accustomed to being restrained in the feed fence
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as well as being approached and handled in different manners. The entire experiment took no longer
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than four hours per day, and the cows were fed according to usual practice during the experiment.
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All procedures were approved by the Animal Welfare Committee of the Lower Saxony State Office
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for Consumer Protection and Food Safety, Oldenburg, Germany (AZ: 33.4-42502-05-13A411).
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2.2
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Cows in the experimental group (EG, n = 26) were subjected a transrectal examination by first-year
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veterinary students without previous experience. A control group (CG, n = 26) was housed and
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restrained in feed fences in the same way and over the same period as the EG. The study occurred in
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the morning after milking between 07:45 and 11:45 am. Cows in the experimental group were
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examined once each day, and the examination took a minimum of 5:00 min and a maximum of 7:30
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min due to the individual time required by students to fulfill the assigned task (i.e., finding the cervix
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and uterine horns). The blood and saliva sample collection of one cow in the CG was combined with
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the point in time of the sampling of one animal in the EG. Two samples were taken before the
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examination (in the EG) to measure the possible effects of blood and saliva sample collection.
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Another two samples were collected after the end of the examination to measure the effects of the
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examination and possible normalization of cortisol levels and to compare the effects to those of
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animals in the CG.
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Furthermore, 12 cows from the EG were examined by students from two groups, who received
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different trainings in preparation for the transrectal examination (Fig. 1). All students in this study
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had no hands-on experience with transrectal examinations of cows. Cows were examined by one
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student from each group on different days in alternating order. Students were randomly divided into
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two groups: One group received simulator-based training (SBT, n = 12), and the other group received
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a theoretical lecture (NO-SBT, n = 12). The teaching was scheduled one week before the experiment.
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Duration and theoretical content of the trainings were identical. During the SBT students were taught
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the basic skills of locating and identifying the cervix and uterine horns with the help of a model.
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Students in the NO-SBT group received a demonstration of the transrectal examination using a pelvis
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bone and uterus model, but were not allowed any hands-on experience. Cows were equipped with a
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device to measure and record HR and HRV in addition to saliva and blood sample collection for
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cortisol measurements.
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2.3
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Experimental design
Salivary and serum cortisol
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ACCEPTED MANUSCRIPT Samples were taken 25 min before (−25 min) and immediately before (0 min) the examination in the
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EG and at the same times in cows in the CG. Another two samples were collected 25 min and 85 min
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after the end of the examination in both groups. Salivary samples were taken with synthetic swabs
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(Salivette® Code blue; Sarstedt AG & Co. KG, Nümbrecht, Germany) held with an Allis forceps (25 cm)
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that were placed inside the cows’ cheek and held there for 60 seconds. Blood samples for cortisol
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analysis were taken by puncturing the coccygeal vessels (20G x 40 mm, BD Microlance™, Becton
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Dickinson GmbH, Heidelberg, Germany), and the samples were collected with sterile tubes (S-
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Monovette® Clotting Activator, Sarstedt AG & Co. KG, Nümbrecht, Germany).
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The saliva collection tubes were centrifuged at 1000 X g for 10 min, and the saliva samples were
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transmitted into Eppendorf microtubes, cooled on ice and then stored at -20°C until analysis. Blood
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samples were centrifuged after coagulation at 3000 X g for 10 min; serum was transmitted into
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Eppendorf microtubes and stored at −20°C until analysis.
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Salivary cortisol was determined using a commercially available ELISA (Cortisol free in Saliva ELISA
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DES6611, Demeditec Diagnostics GmbH, Kiel, Germany) following the manufacturer instructions. The
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detection limit was 0.125 ng/ml. The test was validated for bovine saliva by determining the linearity
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of dilution, which could be confirmed. The intra assay CV was determined by measuring 20 times one
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bovine saliva sample. The intra assay CV was 7.4 %. Cortisol in serum was determined using an
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automated, competitive chemiluminescence immunoassay (LKCO1, Immulite™1000, Siemens
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Diagnostics, USA). The detection limit of the assay was 2.0 ng/ml. The intra-assay CV was 4.5% and
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inter-assay imprecision was 8.8%. Cross-reactivity was observed at approximately 49% with
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prednisolone, 21% with methylprednisolone, 8.6% with corticosterone, 5.9% with prednisone and
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0.2% with fludrocortisone as previously described by Gundlach et al. 2017[31].
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2.4
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Heart rate and HRV were recorded with a portable recording system (eMotion, Mega Electronics,
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Kuopio, Finland). The day (24 hours) before the assessment, animals in the experimental group were
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restrained in the feed fence to prepare the spots for electrode attachment. Two self-adhesive skin
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electrodes (Ambu® Blue Sensor VL, Ballerup, Denmark) were attached to the left side of the cow. The
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electrodes were positioned next to the heart at elbow height and approximately two hands under
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the withers. To ensure a strong attachment of the electrodes to the skin, the skin was shaved and
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cleaned with 70% ethanol, and an adhesive spray (Curavet, WDT, Garbsen, Germany) was applied
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immediately before the electrodes were attached. The two electrodes were connected via a cable to
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the recording device, which stored the data for an individual cow. An elastic belt was fixed around
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the thorax of each cow to cover the electrodes and recording device and secure their positions.
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Heart rate and heart rate variability
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ACCEPTED MANUSCRIPT The data recordings started at least 40 min before the students began the transrectal palpation and
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stopped at least 40 min after the examination.
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Data from the recording devices were transferred via USB connection to a computer (software
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eMotion LAB, version 1.2.3.4, Mega Electronics, Kuopio, Finland). HR and HRV were analyzed in
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sequences of 5 min with the Kubios HRV software (version 2.1, Biomedical Analysis and Medical
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Imaging Group, Department of Applied Physics, University of Eastern Finland, Kuopio, Finland).
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Values 30 min before the transrectal palpation (-30 min) were set as the baseline concentrations. The
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sequence recorded during the transrectal examination started at 0 min. Data were detrended
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(detrending method: smooth priors) and an artifact correction (level: 300 ms) was conducted. The
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analyzed HRV variables were mHR, RMSSD and HF (HF; 0.25 to 0.58 Hz), whereby HF was calculated
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by an autoregressive model and in normalized units [15].
185
2.5
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For statistical analysis, SPSS Statistics 24 (IBM Corporation, New York, USA) was used. Since not all
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data were equally distributed, nonparametric tests were used throughout all analyses. Comparisons
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between the two groups (experimental and control group) were made by a Mann-Whitney-U test.
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Comparisons between different times in the same animal or between different students examining
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the same animal were compared by a Wilcoxon-Signed-Rank test. The time needed for transrectal
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palpation is presented as the mean ± standard deviation. All other data are presented as median ±
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median absolute deviation. A P-value < 0.05 was considered to be significant.
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3.
Results
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3.1
Salivary and serum cortisol
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Salivary and serum cortisol levels increased in the control group and the experimental group over the
196
entire period (-25 min to 85 min). Salivary cortisol levels increased in the control group (P = 0.002)
197
and the experimental group (P = 0.004). Serum cortisol levels increased in the control group (P =
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0.0005) and the experimental group (P = 0.002) (Fig. 2).
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In animals that were subject to a transrectal palpation, serum cortisol levels were higher 25 min after
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the transrectal palpation (P = 0.001) while salivary cortisol levels did not increase significantly (P =
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0.054) (Fig. 2).
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The increase in serum cortisol between 0 min and 25 min was significantly higher in the cows that
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underwent a transrectal palpation (Δ2.6 ng/ml) than in those in the control group (Δ-0.3 ng/ml) (Fig.
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3 A and B).
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ACCEPTED MANUSCRIPT The examinations by the SBT students took an average of 6:24 ± 1:18 min (minimum 3:30 min,
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maximum 7:30 min), and the examinations by the NO-SBT students took an average of 6:5 ± 0:48 min
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(minimum 5:30 min, maximum 7:30 min).
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The salivary and serum cortisol concentrations in the cows were higher than the basal concentrations
209
(0 min) 25 min after transrectal palpation, regardless of the student group. The cortisol
210
concentrations in saliva (P = 0.008) and serum (P = 0.001) increased when the students had no
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simulator-based training (Fig. 4). In addition, the increases of both salivary and serum cortisol were
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significantly higher when the NO-SBT students performed the transrectal examination (Fig. 3 C and
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D), and these values were Δ0.32 ng/ml saliva and Δ5.8 ng/ml serum in comparison with the values of
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Δ0.03 ng/ml saliva and Δ2.1 ng/ml serum in the cows examined by the SBT students.
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3.2
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The examinations by SBT students took an average of 7:00 ± 0:49 min (minimum 5:00 min, maximum
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7:30 min), and the examinations by the NO-SBT students took an average of 7:05 ± 0:40 min
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(minimum 5:30 min, maximum 7:30 min).
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The recorded mHR changed not significantly from 80 to 83 bpm (P = 0.084) and from 81 to 79 bpm (P
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= 0.084) when the SBT and NO-SBT students performed the examinations, respectively (Fig. 5 A). The
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HRV parameter HF decreased during the transrectal palpation compared to the baseline values, and
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no significant differences were found in either group (NO-SBT P = 0.099, SBT P = 0.814) (Fig. 5 B).
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When the NO-SBT students performed the examination, the RMSSD showed a decrease (P = 0.034)
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when the values at -30 and 0 min were compared (Fig. 5 C). No significant difference in RMSSD
225
occurred when the SBT students performed the examination (P = 0.530). The mean heart rate did not
226
rise significantly from the baseline values (-30 min) during the examination (0 min). No significant
227
differences were found between the two groups in RMSSD (P = 0.480) and HF (P = 0.388).
228
4.
229
The present study was designed to examine the effect of different training approaches for students
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on the stress response of cows undergoing a transrectal palpation.
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In the experimental group, serum cortisol increased significantly compared to the levels immediately
232
before and after transrectal palpation. These results match those obtained in earlier studies, where
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significant increases in plasma cortisol levels of cows were found after transrectal palpation [8, 9]
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and indicated a stress response of the cows during this type of examination.
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We confirmed the hypothesis and demonstrated that simulator-trained students induced
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significantly lower increases in cortisol in the examined cows than the students with only theoretical
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preparation before their first transrectal palpation.
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Heart rate and HRV
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ACCEPTED MANUSCRIPT Increased cortisol levels suggest an activation of the HPA axis as a response to an acute stressor, but
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these responses vary between individuals and are sensitive to external as well as internal factors [32,
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33]. Internal factors are genetic including also a pulsatile secretion and a diurnal cycle,
241
developmental and experiential while external factors include e.g. feeding, temperature and
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humidity. Therefore, maximum attention was paid to ensure uniform conditions during the study
243
(e.g., time of day, in lactation, feeding procedure, etc.); thus, the observed cortisol increase is
244
believed to be due to the transrectal palpation.
245
Hopster et al. [34] found that repeated jugular venipuncture (five samples taken at 15 min intervals)
246
led to a significant increase in the cortisol in the plasma of cows. In contrast, in the current study, no
247
significant increase in cortisol level was found in the experimental group due to sampling alone
248
(comparing samples -25 min and 0 min). In the control group, cortisol levels increased in only the
249
sample taken at the very end of the trial. These differences may be partially explained by the fact
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that the cows used in this study are part of educational classes on a regular basis and are therefore
251
accustomed to handling [18]. Furthermore, the blood sampling in the current study was conducted
252
by puncturing the coccygeal vein not the jugular vein, suggesting that the sampling site influences
253
the stress response.
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In addition, the mHR and HRV were calculated to determine the changes in the activity of the
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autonomic nervous system. In stressful conditions, increases in sympathetic and/or reductions in
256
vagal activities were found in several species [10, 15, 35-37]. While mHR and HF did not change
257
significantly during the transrectal palpation, RMSSD decreased significantly when the students
258
without simulator-based training performed the examination. While the mHR is equally influenced by
259
the sympathetic and parasympathetic nervous systems, both HRV parameters indicate changes in
260
vagal tone only, and a decrease marks a reduction in the vagal tone due to pain or stress.
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The changes in the mHR and HRV parameters detected in the present study are not completely
262
consistent with those of Kovacs et al. [19], who found an increase in the mHR as well as a decrease in
263
the HF and RMSSD during transrectal palpation of cows. This difference may be due to the more
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challenging setting in this study, with the combination of blood and saliva sampling distorting the
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cardiac reactions. The run of the curve of HF and RMSSD seem to support this presumption, where a
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decrease of these HRV parameters in the very beginning was observed with a tendency to
267
normalization after the end of the transrectal palpation. Furthermore, in some cases, the
268
acclimatization period (to the equipment) was shorter than recommended by some researchers [15,
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16]. Hence, the mHR might have already been elevated due to the experimental setting.
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The decrease in RMSSD during the transrectal palpation by NO-SBT students seems to support the
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findings related to the cortisol concentrations, both suggesting that these students induce a greater
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stress response than the SBT students.
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9
ACCEPTED MANUSCRIPT At the beginning and end of the trial, the cortisol levels in saliva and serum were similar in the
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control and experimental groups. Cortisol increased significantly when the levels in the first
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measurements and those in the last measurements, as well as the measurements directly before the
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transrectal palpation and 85 min after the examination, were compared. This increase in cortisol in
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the control group could indicate a stress response to being restrained in the feed fence for the
278
duration of the study. So far, it cannot be excluded that this increase is due to ultradian rhythms,
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which lead to an increase in cortisol levels approximately every two hours, as has been observed in
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cows [14]. On the other hand, taking all assessed stress parameters into account, it is possible that
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the cows used in this study do perceive handling and sampling as stressful but no longer react with
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HPA axis activation [32]. To further investigate this assumption, it is necessary to utilize other means
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of measuring stress that may be more accurate, such as HRV recording (alone) or observation of
284
behavioral reactions [38].
285
Since repeated training is necessary to gain proficiency in transrectal palpation skills [4, 5, 28, 29] and
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animal welfare issues are to be considered, simulator-based practical training should precede
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transrectal palpation in cows.
288
Simulators allow students to practice skills according to their individual pace of learning and as often
289
as they need. In the case of teaching transrectal palpation, a simulator-based training allows teachers
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to control and provide feedback on what students are palpating. Furthermore, where variations in
291
conditions are limited, simulators are a useful addition to teaching on cows [5]. Simulator-based
292
training prior to first the transrectal palpation of cows by veterinary students has been proven to be
293
effective in terms of students’ self-efficacy as well as the correct identification of internal genital
294
organs [26, 28, 30].
295
In conclusion, transrectal palpation leads to an acute activation of the HPA axis. In this study, cows
296
showed a significantly greater increase in cortisol levels in saliva and serum after transrectal
297
palpation by students with a theoretical preparation than by SBT students, and a significant decrease
298
of the HRV parameter RMSSD was observed during the transrectal palpation. These results suggest
299
that transrectal palpation by theoretically prepared students leads to greater stress than transrectal
300
palpation done by simulator-trained students.
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5.
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The authors would like to thank Dr. Christian Sürie and the staff of the educational farm in Ruthe for
303
the kind assistance during the trials. We also thank the staff of the Clinical Skills Lab who helped
304
before, during and after the trials. Thanks to all the students who spent extra time in the Skills Lab
305
and Ruthe to participate in this study.
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Acknowledgements
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ACCEPTED MANUSCRIPT 306
We kindly thank the Leibniz Institute for Agricultural Engineering and Bioeconomy for providing the
307
heart rate recording systems.
308 Author contributions: Marion Schmicke, Marc Dilly and Hannah Giese designed the study. Marc Dilly,
310
Yasmin Gundelach and Hannah Giese collected the research data. Hannah Giese analyzed and
311
interpreted the data. All authors were involved in drafting this manuscript and revising it critically for
312
important intellectual content. All authors read and approved the final manuscript.
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Funding: This work was supported by the German Federal Ministry of Education and Research.
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Conflicts of interest: none.
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References
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[1] Inchaisri C, Jorritsma R, Vos PLAM, van der Weijden GC, Hogeveen H. Economic consequences of reproductive performance in dairy cattle. Theriogenology. 2010;74:835-46. [2] Hanzen CH, Pieterse M, Scenczi O, Drost M. Relative Accuracy of the Identification of Ovarian Structures in the Cow by Ultrasonography and Palpation Per Rectum. The Veterinary Journal. 2000;159:161-70. [3] EAEVE. S.O.P., ANEX IV, LIST OF RECOMMENDED ESSENTIAL COMPETENCES AT GRADUATION:“DAY-ONE SKILLS”. 2012. [4] Lopes G, Rocha A. Teaching bovine rectal palpation with live cows in the slaughterhouse: is it worthwhile? Reproduction in domestic animals = Zuchthygiene. 2006;41:510-3. [5] Bossaert P, Leterme L, Caluwaerts T, Cools S, Hostens M, Kolkman I, et al. Teaching transrectal palpation of the internal genital organs in cattle. Journal of veterinary medical education. 2009;36:451-60. [6] Nagel C, Ille N, Aurich J, Aurich C. Teaching of diagnostic skills in equine gynecology: Simulatorbased training versus schooling on live horses. Theriogenology. 2015;84:1088-95. [7] Baillie S, Crossan A, Reid S, Brewster S. Preliminary development and evaluation of a bovine rectal palpation simulator for training veterinary students. Cattle Practice. 2003;11:101-6. [8] Cingi CC, Baser DF, Karafakioglu YS, Fidan AF. Stress response in dairy cows related to rectal examination. Acta Scientiae Veterinariae. 2012;40:1-7. [9] Nakao T, Sato T, Moriyoshi M, Kawata K. Plasma Cortisol Response in Dairy Cows to Vaginoscopy, Genital Palpation per Rectum and Artificial Insemination. Journal of Veterinary Medicine Series A. 1994;41:16-21. [10] Schönbom H, Kassens A, Hopster-Iversen C, Klewitz J, Piechotta M, Martinsson G, et al. Influence of transrectal and transabdominal ultrasound examination on salivary cortisol, heart rate, and heart rate variability in mares. Theriogenology. 2015;83:749-56. [11] Negrão JA, Porcionato MA, de Passillé AM, Rushen J. Cortisol in Saliva and Plasma of Cattle After ACTH Administration and Milking. Journal of dairy science. 2004;87:1713-8. [12] Pell SM, McGreevy PD. A study of cortisol and beta-endorphin levels in stereotypic and normal Thoroughbreds. Applied Animal Behaviour Science. 1999;64:81-90. [13] Hernandez CE, Thierfelder T, Svennersten-Sjaunja K, Berg C, Orihuela A, Lidfors L. Time lag between peak concentrations of plasma and salivary cortisol following a stressful procedure in dairy cattle. Acta veterinaria Scandinavica. 2014;56:61. [14] Lefcourt AM, Bitman J, Kahl S, Wood DL. Circadian and Ultradian Rhythms of Peripheral Cortisol Concentrations in Lactating Dairy Cows. Journal of dairy science. 1993;76:2607-12.
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[15] Mohr E, Langbein J, Nurnberg G. Heart rate variability: a noninvasive approach to measure stress in calves and cows. Physiology & behavior. 2002;75:251-9. [16] von Borell E, Langbein J, Despres G, Hansen S, Leterrier C, Marchant-Forde J, et al. Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals -- a review. Physiology & behavior. 2007;92:293-316. [17] Kovács L, Jurkovich V, Bakony M, Szenci O, Póti P, Tőzsér J. Welfare implication of measuring heart rate and heart rate variability in dairy cattle: literature review and conclusions for future research. Animal : an international journal of animal bioscience. 2013;8:316-30. [18] Waiblinger S, Menke C, Korff J, Bucher A. Previous handling and gentle interactions affect behaviour and heart rate of dairy cows during a veterinary procedure. Applied Animal Behaviour Science. 2004;85:31-42. [19] Kovács L, Tőzsér J, Szenci O, Póti P, Kézér F, Ruff F, et al. Cardiac responses to palpation per rectum in lactating and nonlactating dairy cows. Journal of dairy science. 2014;97:6955-63. [20] Visser EK, van Reenen CG, van der Werf JTN, Schilder MBH, Knaap JH, Barneveld A, et al. Heart rate and heart rate variability during a novel object test and a handling test in young horses. Physiology & behavior. 2002;76:289-96. [21] King L. Ethics and welfare of animals used in education: An overview. Animal Welfare. 2004;13:221-7. [22] de Boo J, Knight A. “Concepts in animal welfare”: a syllabus in animal welfare science and ethics for veterinary schools. Journal of veterinary medical education. 2005;32:451-3. [23] Knight A. The effectiveness of humane teaching methods in veterinary education. Altex. 2007;24:91. [24] Martinsen S, Jukes N. Towards a Humane Veterinary Education. Journal of veterinary medical education. 2005;32:454-60. [25] Smeak DD. Teaching veterinary students using shelter animals. Journal of veterinary medical education. 2008;35:26-30. [26] Baillie S, Crossan A, Brewster S, Mellor D, Reid S. Validation of a bovine rectal palpation simulator for training veterinary students. Studies in health technology and informatics. 2005;111:336. [27] Kinnison T, Forrest ND, Frean SP, Baillie S. Teaching bovine abdominal anatomy: use of a haptic simulator. Anatomical sciences education. 2009;2:280-5. [28] Baillie S, Crossan A, Brewster SA, May SA, Mellor DJ. Evaluating an automated haptic simulator designed for veterinary students to learn bovine rectal palpation. Simulation in healthcare : journal of the Society for Simulation in Healthcare. 2010;5:261-6. [29] Baillie S, Mellor DJ, Brewster SA, Reid SW. Integrating a bovine rectal palpation simulator into an undergraduate veterinary curriculum. Journal of veterinary medical education. 2005;32:79-85. [30] Giese H, Ehlers JP, Gundelach Y, Geuenich K, Dilly M. Untersuchungen zur Effektivität verschiedener Unterrichtsmethoden der transrektalen gynäkologischen Untersuchung beim Rind auf Lernerfolg und Selbstevaluierung von Studierenden. Berliner und Münchener Tierärztliche Wochenschrift. 2016;129:216-24. [31] Gundlach NH, Feldmann M, Gundelach Y, Gil MA, Siebert U, Hoedemaker M, et al. Dehydroepiandrosterone and cortisol/dehydroepiandrosterone ratios in dairy cattle with postpartum metritis. Research in veterinary science. 2017;115:530-3. [32] Mormède P, Andanson S, Aupérin B, Beerda B, Guémené D, Malmkvist J, et al. Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare. Physiology & behavior. 2007;92:317-39. [33] Alam MG, Dobson H. Effect of various veterinary procedures on plasma concentrations of cortisol, luteinising hormone and prostaglandin F2 alpha metabolite in the cow. The Veterinary record. 1986;118:7-10. [34] Hopster H, Van der Werf J, Erkens J, Blokhuis HJ. Effects of repeated jugular puncture on plasma cortisol concentrations in loose-housed dairy cows. Journal of animal science. 1999;77:708-14.
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[35] Jong Id, Sgoifo A, Lambooij E, Korte SM, Blokhuis HJ, Koolhaas JM. Effects of social stress on heart rate and heart rate variability in growing pigs. Canadian Journal of Animal Science. 2000;80:273-80. [36] Geverink NA, Schouten WGP, Gort G, Wiegant VM. Individual differences in behavioral and physiological responses to restraint stress in pigs. Physiology & behavior. 2002;77:451-7. [37] Langbein J, Nürnberg G, Manteuffel G. Visual discrimination learning in dwarf goats and associated changes in heart rate and heart rate variability. Physiology & behavior. 2004;82:601-9. [38] Pilz M, Fischer-Tenhagen C, Grau M, Heuwieser W. Behavioural and physiological assessment of stress reactions during vaginal examination in dairy cows. Tierarztliche Praxis Ausgabe G, Grosstiere/Nutztiere. 2014;42:88-94.
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Fig. 1. Flow chart of study design. In total 52 cows were used in the experiment. Cows in the EG were ACCEPTED MANUSCRIPT examined by first and second-year students. 12 cows from the EG were used for two examinations on different days by students who received different trainings beforehand. SBT students received simulator-based training, NO-SBT students were prepared by theoretical instruction and demonstration only. Fig. 2. Salivary and serum cortisol curve (A, B) of animals in the experimental and the control group. Data are median ± median absolute deviation (MAD). Shaded bars indicate time of examination of animals in the experimental group. * Cortisol increased significantly comparing points 0 and 25 minutes (after the transrectal palpation) in the experimental group (n = 26). # significant increase of salivary and serum cortisol comparing points -25 and 85 in the control group (n = 26). ° significant increase in salivary and serum cortisol in the experimental group (n = 21 and n = 26, respectively). Level of significance P < 0.05.
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Fig. 3 Increase of salivary and serum cortisol from point 0 to 25 minutes, i.e. after the transrectal palpation in the experimental group (A, B). ⃰ Increase of cortisol in serum of animals in the experimental group (n = 26) is significantly higher than of animals in the control group (n = 26). Increase of salivary and serum cortisol in the SBT (n = 12) and NO-SBT (n = 12) group (C, D). ⃰ Salivary and serum cortisol increase from 0 to 25 minutes after the transrectal palpation is significantly higher in the NO-SBT group. Level of significance P < 0.05.
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Fig. 4. Salivary and serum cortisol curve (A, B) of animals in the SBT (n = 12) and the NO-SBT (n = 12) group. Data are median ± median absolute deviation (MAD). Shaded bars indicate time of transrectal palpation. * Cortisol increased significantly comparing points 0 and 25 minutes (after the transrectal palpation) in the NO-SBT group. Level of significance P < 0.05.
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Fig. 5. A Mean heart rate (MHR) in beats/min of cows (n = 12) before, during and after transrectal palpation by SBT (n = 12) and NO-SBT students (n = 12). B High frequency component (HF) of heart rate variability in Hz. No significant differences between baseline and during examination or SBT and NO-SBT students. C Root mean squared differences of inter-beats intervals (RMSSD) in ms. # Significant decrease from baseline values in the NO-SBT group. Data are median ± median absolute deviation. Level of significance P < 0.05.
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Teaching transrectal palpation causes symptoms of stress reactions in the animals. Compared to a control group cortisol levels in examined cows increased significantly. A reduction of vagal tone during transrectal palpation was observed as well Cows examined by previously trained students showed lower cortisol increase.
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• • • •
S0093-691X(18)30513-2
DOI:
10.1016/j.theriogenology.2018.07.016
Reference:
THE 14630
To appear in:
Theriogenology
Received Date: 12 March 2018 Revised Date:
12 July 2018
Accepted Date: 16 July 2018
Please cite this article as: Giese H, Dilly M, Gundelach Y, Hoffmann G, Schmicke M, Influence of transrectal palpation training on cortisol levels and heart rate variability in cows, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.07.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
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Influence of transrectal palpation training on cortisol levels and heart rate variability in cows
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Hannah Giesea, Marc Dillya1, Yasmin Gundelachb, Gundula Hoffmannc, Marion Schmickeb
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a
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[email protected] b
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[email protected]; [email protected] c
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Leibniz
Institute
for
Agricultural
Engineering
and
Bioeconomy,
[email protected] Abstract
Potsdam,
Germany;
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Clinic for Cattle, University of Veterinary Medicine Hanover, Hannover, Germany;
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Clinical Skills Lab, University of Veterinary Medicine Hanover, Hannover, Germany;
Transrectal palpation of cows is a day-one competence for veterinary students, and it is an essential
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skill for the diagnosis of pregnancy as well as reproductive disorders. We hypothesized that
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transrectal palpation induces a stress response in cows, and this stress response may vary with the
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training students receive before their first transrectal palpation. Therefore, 52 Holstein-Friesian cows
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were used at the University of Veterinary Medicine Hanover. The experimental group (n = 26) was
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subjected to transrectal palpations by first and second-year students. Salivary and serum cortisol
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levels were assessed before and after the intervention. A control group (n = 26) was only restrained
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and tested for changes in salivary and serum cortisol.
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A total of 12 cows of the experimental group were examined by two groups of students with
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different training on two days. The examination was performed one day by students who were
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theoretically prepared for transrectal palpation in cows (NO-SBT, n = 12). The other day, students
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who underwent a simulator-based training (SBT, n = 12) performed the examination. The cortisol
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concentrations, as well as heart rate (HR) and heart rate variability (HRV), were measured in the
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examined cows. Blood and saliva samples were collected 25 min and immediately before (0 min) and
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25 min and 85 min after the end of the examination in the experimental group. Serum cortisol levels
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between 0 min and 25 min were increased by Δ2.6 ng/ml in the cows in the experimental group
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compared to Δ-0.3 ng/ml in the control group (P = 0.001). The increases in cortisol in saliva (P =
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0.033) and serum (P = 0.013) after transrectal palpation were higher in the NO-SBT group Δ0.32
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ng/ml saliva, Δ5.8 ng/ml serum than in the SBT group Δ0.03 ng/ml saliva, Δ2.1 ng/ml serum. For HR
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and HRV analysis values recorded 30 min before the transrectal palpation (-30 min) were set as the
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baseline concentrations the sequence recorded during the transrectal examination started at 0 min.
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While the mean HR did not change significantly during the transrectal palpation (80 to 83 bpm SBT
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students; 81 to 79 bpm NO-SBT students), the HRV parameter square root of the mean squared
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scil animal care company, Viernheim, Germany; [email protected]
ACCEPTED MANUSCRIPT differences of successive R-R intervals (RMSSD) decreased in the NO-SBT group (P = 0.034) during
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transrectal palpation compared to the baseline values (17.47 to 5.07 ms). These findings reflect an
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activation of the hypothalamic-pituitary-adrenal axis as well as a reduction in vagal tone during the
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teaching and practice of transrectal palpation by students. Moreover, the results indicate that a
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transrectal palpation is less stressful for cows when the examination is performed by students that
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were previously prepared by simulator-based training.
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Keywords
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Transrectal palpation; Teaching; Cows; Stress; HRV; Cortisol
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1.
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In dairy farming, pregnancy diagnosis has become a central issue for profitable (herd) management
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[1]. Transrectal palpations are widely used in large animal practices for pregnancy diagnosis and
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fertility management. In the case when infertility is suspected, further gynecological examinations
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are needed [2]. The first step in pregnancy and fertility diagnosis is transrectal palpation. Therefore,
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it is still referred to as a basic diagnostic skill and day-one-competence for veterinary students and
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veterinarians in large animal practices [3].
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Previous studies have emphasized the high number of repetitions (up to 200) required to obtain the
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skill required to determine pregnancy in cows from a transrectal palpation [4, 5]. It has been
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suggested that training on cows and with a variety of gynecological conditions are necessary to
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develop proficiency in this skill [4, 5]. Nagel et al. [6] showed that students who had repeated
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training sessions on horses (four training sessions vs. one training session) scored significantly better
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when asked to palpate the uterus and ovaries in horses during a practical test. Nevertheless, the
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opportunities for repetitive transrectal palpation training are limited for several reasons (e.g., animal
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welfare issues, high number of students, etc.). At the University of Veterinary Medicine Hanover,
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Foundation (TiHo) students often perform their first transrectal examination of cows during
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mandatory training in the first or second year of study. During the teaching of transrectal palpation, a
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couple of challenges should be considered. From a student’s point of view, identifying structures
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through feces and the rectum wall is very challenging [7]. At the same time, the instructor has no
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chance to control the results of the student’s palpation. Thus, the instructor cannot help any student
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that faces difficulties during the examination.
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Apart from difficulties during teaching, animal welfare and ethical considerations have led to a
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reduction in animal use in veterinary education. It has been shown that transrectal palpation of
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horses and cattle can induce stress. An increase in the blood cortisol levels of cows after transrectal
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palpation of the reproductive tract is indicative of stress [8, 9]. In mares, Schönbom et al. [10] found
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a significant increase in salivary cortisol levels after transrectal ultrasonography (whereas
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transabdominal ultrasonography did not significantly influence the salivary cortisol levels). Increased
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blood cortisol levels occur due to an activation of the hypothalamic-pituitary-adrenal (HPA) axis.
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Cortisol passes into saliva through passive intracellular diffusion. Salivary cortisol has been shown to
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correlate to blood cortisol levels, as demonstrated in horses and cattle [11, 12]. Hernandez et al. [13]
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found that there is a 10-minute lag between the peak cortisol concentrations in the saliva and plasma
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of cattle. Cortisol secretion underlies weak circadian and rather strong ultradian rhythms with
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pulsatile secretion and great individual variation [14]. The mentioned physiological variations must
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be considered when the cortisol changes during experiments are interpreted.
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Introduction
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rate variability (HRV). Heart rate variability describes the time intervals between consecutive heart
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beats (R-R intervals) [15]. While changes in mean heart rate (mHR) reflect reactions of both the
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sympathetic and parasympathetic nervous system, HRV parameters allow for a more accurate
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interpretation of which autonomic nervous system (ANS) component induced the observed changes.
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The high-frequency component of HRV (HF) and the square root of mean squared differences of
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successive R-R intervals (RMSSD) are associated with changes in the parasympathetic nervous system
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(PNS) [16]. Both parameters have been examined during stressful and/or painful events and it has
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been shown that stress leads to a reduction in vagal tone and thus to a decrease of HF and RMSSD
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[17]. Furthermore, changes in mHR and different HRV parameters were found during transrectal
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examinations and reflected the stress-induced changes in the activity of the ANS [18, 19]. Last but
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not least the perception of the handling and veterinary procedures and their stress response varies
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among individuals. A combination of genetic disposition, previous experiences and habituation
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influence the (perceived) stress load [18, 20].
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The use of animals in education and humane teaching methods have been of interest in recent years
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[21-25]. Simulators for teaching transrectal palpation are available and have previously been
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evaluated as effective for teaching and learning [26, 27]. These simulators are considered a useful
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alternative to training on cows [5, 28, 29]. Comparing different simulators and theoretical
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preparation, students benefit equally from simulator-based training before they perform their first
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transrectal examination on live animals in terms of self-efficacy as well as correct identification of
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organs [30]. Simulator-based training prior to the first transrectal examination of cows has not been
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implemented yet for first-year students at the University of Veterinary Medicine Hanover. This may
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be hindered by a lack of research on the effect of teaching methods on the stress induced in the
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animal. Therefore, we have examined different stress parameters in cows undergoing a transrectal
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palpation after students received simulator-based training versus a theoretical lesson.
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We hypothesized that the stress response of the cows undergoing a transrectal examination by
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veterinary students is lower when the students previously received simulator-based training.
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2.
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2.1
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A group of loose-housed Holstein-Friesian dairy cows (n = 52) was used. The cows were clinically
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healthy, lactating and pluriparous, the ages, number of births or days in milk were not considered.
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Cows in the experimental group were empty, cows in the control group were not tested for
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reproductive state. The study occurred between March and October 2014 on the Farm for Education
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and Research in Ruthe (University of Veterinary Medicine Hanover, Foundation). On this farm, first 4
Materials and Methods Animals
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veterinary procedures on cows. Thus, the cows are accustomed to being restrained in the feed fence
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as well as being approached and handled in different manners. The entire experiment took no longer
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than four hours per day, and the cows were fed according to usual practice during the experiment.
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All procedures were approved by the Animal Welfare Committee of the Lower Saxony State Office
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for Consumer Protection and Food Safety, Oldenburg, Germany (AZ: 33.4-42502-05-13A411).
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2.2
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Cows in the experimental group (EG, n = 26) were subjected a transrectal examination by first-year
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veterinary students without previous experience. A control group (CG, n = 26) was housed and
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restrained in feed fences in the same way and over the same period as the EG. The study occurred in
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the morning after milking between 07:45 and 11:45 am. Cows in the experimental group were
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examined once each day, and the examination took a minimum of 5:00 min and a maximum of 7:30
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min due to the individual time required by students to fulfill the assigned task (i.e., finding the cervix
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and uterine horns). The blood and saliva sample collection of one cow in the CG was combined with
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the point in time of the sampling of one animal in the EG. Two samples were taken before the
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examination (in the EG) to measure the possible effects of blood and saliva sample collection.
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Another two samples were collected after the end of the examination to measure the effects of the
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examination and possible normalization of cortisol levels and to compare the effects to those of
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animals in the CG.
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Furthermore, 12 cows from the EG were examined by students from two groups, who received
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different trainings in preparation for the transrectal examination (Fig. 1). All students in this study
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had no hands-on experience with transrectal examinations of cows. Cows were examined by one
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student from each group on different days in alternating order. Students were randomly divided into
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two groups: One group received simulator-based training (SBT, n = 12), and the other group received
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a theoretical lecture (NO-SBT, n = 12). The teaching was scheduled one week before the experiment.
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Duration and theoretical content of the trainings were identical. During the SBT students were taught
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the basic skills of locating and identifying the cervix and uterine horns with the help of a model.
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Students in the NO-SBT group received a demonstration of the transrectal examination using a pelvis
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bone and uterus model, but were not allowed any hands-on experience. Cows were equipped with a
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device to measure and record HR and HRV in addition to saliva and blood sample collection for
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cortisol measurements.
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Salivary and serum cortisol
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EG and at the same times in cows in the CG. Another two samples were collected 25 min and 85 min
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after the end of the examination in both groups. Salivary samples were taken with synthetic swabs
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(Salivette® Code blue; Sarstedt AG & Co. KG, Nümbrecht, Germany) held with an Allis forceps (25 cm)
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that were placed inside the cows’ cheek and held there for 60 seconds. Blood samples for cortisol
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analysis were taken by puncturing the coccygeal vessels (20G x 40 mm, BD Microlance™, Becton
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Dickinson GmbH, Heidelberg, Germany), and the samples were collected with sterile tubes (S-
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Monovette® Clotting Activator, Sarstedt AG & Co. KG, Nümbrecht, Germany).
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The saliva collection tubes were centrifuged at 1000 X g for 10 min, and the saliva samples were
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transmitted into Eppendorf microtubes, cooled on ice and then stored at -20°C until analysis. Blood
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samples were centrifuged after coagulation at 3000 X g for 10 min; serum was transmitted into
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Eppendorf microtubes and stored at −20°C until analysis.
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Salivary cortisol was determined using a commercially available ELISA (Cortisol free in Saliva ELISA
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DES6611, Demeditec Diagnostics GmbH, Kiel, Germany) following the manufacturer instructions. The
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detection limit was 0.125 ng/ml. The test was validated for bovine saliva by determining the linearity
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of dilution, which could be confirmed. The intra assay CV was determined by measuring 20 times one
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bovine saliva sample. The intra assay CV was 7.4 %. Cortisol in serum was determined using an
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automated, competitive chemiluminescence immunoassay (LKCO1, Immulite™1000, Siemens
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Diagnostics, USA). The detection limit of the assay was 2.0 ng/ml. The intra-assay CV was 4.5% and
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inter-assay imprecision was 8.8%. Cross-reactivity was observed at approximately 49% with
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prednisolone, 21% with methylprednisolone, 8.6% with corticosterone, 5.9% with prednisone and
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0.2% with fludrocortisone as previously described by Gundlach et al. 2017[31].
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2.4
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Heart rate and HRV were recorded with a portable recording system (eMotion, Mega Electronics,
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Kuopio, Finland). The day (24 hours) before the assessment, animals in the experimental group were
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restrained in the feed fence to prepare the spots for electrode attachment. Two self-adhesive skin
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electrodes (Ambu® Blue Sensor VL, Ballerup, Denmark) were attached to the left side of the cow. The
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electrodes were positioned next to the heart at elbow height and approximately two hands under
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the withers. To ensure a strong attachment of the electrodes to the skin, the skin was shaved and
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cleaned with 70% ethanol, and an adhesive spray (Curavet, WDT, Garbsen, Germany) was applied
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immediately before the electrodes were attached. The two electrodes were connected via a cable to
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the recording device, which stored the data for an individual cow. An elastic belt was fixed around
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the thorax of each cow to cover the electrodes and recording device and secure their positions.
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Heart rate and heart rate variability
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stopped at least 40 min after the examination.
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Data from the recording devices were transferred via USB connection to a computer (software
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eMotion LAB, version 1.2.3.4, Mega Electronics, Kuopio, Finland). HR and HRV were analyzed in
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sequences of 5 min with the Kubios HRV software (version 2.1, Biomedical Analysis and Medical
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Imaging Group, Department of Applied Physics, University of Eastern Finland, Kuopio, Finland).
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Values 30 min before the transrectal palpation (-30 min) were set as the baseline concentrations. The
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sequence recorded during the transrectal examination started at 0 min. Data were detrended
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(detrending method: smooth priors) and an artifact correction (level: 300 ms) was conducted. The
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analyzed HRV variables were mHR, RMSSD and HF (HF; 0.25 to 0.58 Hz), whereby HF was calculated
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by an autoregressive model and in normalized units [15].
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2.5
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For statistical analysis, SPSS Statistics 24 (IBM Corporation, New York, USA) was used. Since not all
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data were equally distributed, nonparametric tests were used throughout all analyses. Comparisons
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between the two groups (experimental and control group) were made by a Mann-Whitney-U test.
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Comparisons between different times in the same animal or between different students examining
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the same animal were compared by a Wilcoxon-Signed-Rank test. The time needed for transrectal
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palpation is presented as the mean ± standard deviation. All other data are presented as median ±
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median absolute deviation. A P-value < 0.05 was considered to be significant.
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Results
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Salivary and serum cortisol
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Salivary and serum cortisol levels increased in the control group and the experimental group over the
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entire period (-25 min to 85 min). Salivary cortisol levels increased in the control group (P = 0.002)
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and the experimental group (P = 0.004). Serum cortisol levels increased in the control group (P =
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0.0005) and the experimental group (P = 0.002) (Fig. 2).
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In animals that were subject to a transrectal palpation, serum cortisol levels were higher 25 min after
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the transrectal palpation (P = 0.001) while salivary cortisol levels did not increase significantly (P =
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0.054) (Fig. 2).
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The increase in serum cortisol between 0 min and 25 min was significantly higher in the cows that
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underwent a transrectal palpation (Δ2.6 ng/ml) than in those in the control group (Δ-0.3 ng/ml) (Fig.
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3 A and B).
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maximum 7:30 min), and the examinations by the NO-SBT students took an average of 6:5 ± 0:48 min
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(minimum 5:30 min, maximum 7:30 min).
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The salivary and serum cortisol concentrations in the cows were higher than the basal concentrations
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(0 min) 25 min after transrectal palpation, regardless of the student group. The cortisol
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concentrations in saliva (P = 0.008) and serum (P = 0.001) increased when the students had no
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simulator-based training (Fig. 4). In addition, the increases of both salivary and serum cortisol were
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significantly higher when the NO-SBT students performed the transrectal examination (Fig. 3 C and
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D), and these values were Δ0.32 ng/ml saliva and Δ5.8 ng/ml serum in comparison with the values of
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Δ0.03 ng/ml saliva and Δ2.1 ng/ml serum in the cows examined by the SBT students.
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3.2
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The examinations by SBT students took an average of 7:00 ± 0:49 min (minimum 5:00 min, maximum
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7:30 min), and the examinations by the NO-SBT students took an average of 7:05 ± 0:40 min
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(minimum 5:30 min, maximum 7:30 min).
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The recorded mHR changed not significantly from 80 to 83 bpm (P = 0.084) and from 81 to 79 bpm (P
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= 0.084) when the SBT and NO-SBT students performed the examinations, respectively (Fig. 5 A). The
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HRV parameter HF decreased during the transrectal palpation compared to the baseline values, and
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no significant differences were found in either group (NO-SBT P = 0.099, SBT P = 0.814) (Fig. 5 B).
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When the NO-SBT students performed the examination, the RMSSD showed a decrease (P = 0.034)
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when the values at -30 and 0 min were compared (Fig. 5 C). No significant difference in RMSSD
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occurred when the SBT students performed the examination (P = 0.530). The mean heart rate did not
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rise significantly from the baseline values (-30 min) during the examination (0 min). No significant
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differences were found between the two groups in RMSSD (P = 0.480) and HF (P = 0.388).
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4.
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The present study was designed to examine the effect of different training approaches for students
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on the stress response of cows undergoing a transrectal palpation.
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In the experimental group, serum cortisol increased significantly compared to the levels immediately
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before and after transrectal palpation. These results match those obtained in earlier studies, where
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significant increases in plasma cortisol levels of cows were found after transrectal palpation [8, 9]
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and indicated a stress response of the cows during this type of examination.
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We confirmed the hypothesis and demonstrated that simulator-trained students induced
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significantly lower increases in cortisol in the examined cows than the students with only theoretical
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preparation before their first transrectal palpation.
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these responses vary between individuals and are sensitive to external as well as internal factors [32,
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33]. Internal factors are genetic including also a pulsatile secretion and a diurnal cycle,
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developmental and experiential while external factors include e.g. feeding, temperature and
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humidity. Therefore, maximum attention was paid to ensure uniform conditions during the study
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(e.g., time of day, in lactation, feeding procedure, etc.); thus, the observed cortisol increase is
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believed to be due to the transrectal palpation.
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Hopster et al. [34] found that repeated jugular venipuncture (five samples taken at 15 min intervals)
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led to a significant increase in the cortisol in the plasma of cows. In contrast, in the current study, no
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significant increase in cortisol level was found in the experimental group due to sampling alone
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(comparing samples -25 min and 0 min). In the control group, cortisol levels increased in only the
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sample taken at the very end of the trial. These differences may be partially explained by the fact
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that the cows used in this study are part of educational classes on a regular basis and are therefore
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accustomed to handling [18]. Furthermore, the blood sampling in the current study was conducted
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by puncturing the coccygeal vein not the jugular vein, suggesting that the sampling site influences
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the stress response.
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In addition, the mHR and HRV were calculated to determine the changes in the activity of the
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autonomic nervous system. In stressful conditions, increases in sympathetic and/or reductions in
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vagal activities were found in several species [10, 15, 35-37]. While mHR and HF did not change
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significantly during the transrectal palpation, RMSSD decreased significantly when the students
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without simulator-based training performed the examination. While the mHR is equally influenced by
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the sympathetic and parasympathetic nervous systems, both HRV parameters indicate changes in
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vagal tone only, and a decrease marks a reduction in the vagal tone due to pain or stress.
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The changes in the mHR and HRV parameters detected in the present study are not completely
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consistent with those of Kovacs et al. [19], who found an increase in the mHR as well as a decrease in
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the HF and RMSSD during transrectal palpation of cows. This difference may be due to the more
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challenging setting in this study, with the combination of blood and saliva sampling distorting the
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cardiac reactions. The run of the curve of HF and RMSSD seem to support this presumption, where a
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decrease of these HRV parameters in the very beginning was observed with a tendency to
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normalization after the end of the transrectal palpation. Furthermore, in some cases, the
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acclimatization period (to the equipment) was shorter than recommended by some researchers [15,
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16]. Hence, the mHR might have already been elevated due to the experimental setting.
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The decrease in RMSSD during the transrectal palpation by NO-SBT students seems to support the
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findings related to the cortisol concentrations, both suggesting that these students induce a greater
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stress response than the SBT students.
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control and experimental groups. Cortisol increased significantly when the levels in the first
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measurements and those in the last measurements, as well as the measurements directly before the
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transrectal palpation and 85 min after the examination, were compared. This increase in cortisol in
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the control group could indicate a stress response to being restrained in the feed fence for the
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duration of the study. So far, it cannot be excluded that this increase is due to ultradian rhythms,
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which lead to an increase in cortisol levels approximately every two hours, as has been observed in
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cows [14]. On the other hand, taking all assessed stress parameters into account, it is possible that
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the cows used in this study do perceive handling and sampling as stressful but no longer react with
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HPA axis activation [32]. To further investigate this assumption, it is necessary to utilize other means
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of measuring stress that may be more accurate, such as HRV recording (alone) or observation of
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behavioral reactions [38].
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Since repeated training is necessary to gain proficiency in transrectal palpation skills [4, 5, 28, 29] and
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animal welfare issues are to be considered, simulator-based practical training should precede
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transrectal palpation in cows.
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Simulators allow students to practice skills according to their individual pace of learning and as often
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as they need. In the case of teaching transrectal palpation, a simulator-based training allows teachers
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to control and provide feedback on what students are palpating. Furthermore, where variations in
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conditions are limited, simulators are a useful addition to teaching on cows [5]. Simulator-based
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training prior to first the transrectal palpation of cows by veterinary students has been proven to be
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effective in terms of students’ self-efficacy as well as the correct identification of internal genital
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organs [26, 28, 30].
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In conclusion, transrectal palpation leads to an acute activation of the HPA axis. In this study, cows
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showed a significantly greater increase in cortisol levels in saliva and serum after transrectal
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palpation by students with a theoretical preparation than by SBT students, and a significant decrease
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of the HRV parameter RMSSD was observed during the transrectal palpation. These results suggest
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that transrectal palpation by theoretically prepared students leads to greater stress than transrectal
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palpation done by simulator-trained students.
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5.
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The authors would like to thank Dr. Christian Sürie and the staff of the educational farm in Ruthe for
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the kind assistance during the trials. We also thank the staff of the Clinical Skills Lab who helped
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before, during and after the trials. Thanks to all the students who spent extra time in the Skills Lab
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and Ruthe to participate in this study.
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Acknowledgements
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We kindly thank the Leibniz Institute for Agricultural Engineering and Bioeconomy for providing the
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heart rate recording systems.
308 Author contributions: Marion Schmicke, Marc Dilly and Hannah Giese designed the study. Marc Dilly,
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Yasmin Gundelach and Hannah Giese collected the research data. Hannah Giese analyzed and
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interpreted the data. All authors were involved in drafting this manuscript and revising it critically for
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important intellectual content. All authors read and approved the final manuscript.
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Funding: This work was supported by the German Federal Ministry of Education and Research.
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Conflicts of interest: none.
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Fig. 1. Flow chart of study design. In total 52 cows were used in the experiment. Cows in the EG were ACCEPTED MANUSCRIPT examined by first and second-year students. 12 cows from the EG were used for two examinations on different days by students who received different trainings beforehand. SBT students received simulator-based training, NO-SBT students were prepared by theoretical instruction and demonstration only. Fig. 2. Salivary and serum cortisol curve (A, B) of animals in the experimental and the control group. Data are median ± median absolute deviation (MAD). Shaded bars indicate time of examination of animals in the experimental group. * Cortisol increased significantly comparing points 0 and 25 minutes (after the transrectal palpation) in the experimental group (n = 26). # significant increase of salivary and serum cortisol comparing points -25 and 85 in the control group (n = 26). ° significant increase in salivary and serum cortisol in the experimental group (n = 21 and n = 26, respectively). Level of significance P < 0.05.
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Fig. 3 Increase of salivary and serum cortisol from point 0 to 25 minutes, i.e. after the transrectal palpation in the experimental group (A, B). ⃰ Increase of cortisol in serum of animals in the experimental group (n = 26) is significantly higher than of animals in the control group (n = 26). Increase of salivary and serum cortisol in the SBT (n = 12) and NO-SBT (n = 12) group (C, D). ⃰ Salivary and serum cortisol increase from 0 to 25 minutes after the transrectal palpation is significantly higher in the NO-SBT group. Level of significance P < 0.05.
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Fig. 4. Salivary and serum cortisol curve (A, B) of animals in the SBT (n = 12) and the NO-SBT (n = 12) group. Data are median ± median absolute deviation (MAD). Shaded bars indicate time of transrectal palpation. * Cortisol increased significantly comparing points 0 and 25 minutes (after the transrectal palpation) in the NO-SBT group. Level of significance P < 0.05.
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Fig. 5. A Mean heart rate (MHR) in beats/min of cows (n = 12) before, during and after transrectal palpation by SBT (n = 12) and NO-SBT students (n = 12). B High frequency component (HF) of heart rate variability in Hz. No significant differences between baseline and during examination or SBT and NO-SBT students. C Root mean squared differences of inter-beats intervals (RMSSD) in ms. # Significant decrease from baseline values in the NO-SBT group. Data are median ± median absolute deviation. Level of significance P < 0.05.
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Highlights
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Teaching transrectal palpation causes symptoms of stress reactions in the animals. Compared to a control group cortisol levels in examined cows increased significantly. A reduction of vagal tone during transrectal palpation was observed as well Cows examined by previously trained students showed lower cortisol increase.
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