The authors have declared that no competing interests exist.
The hypothalamic-pituitary-adrenal (HPA) axis response to chronic stress is far from straight forward, particularly with regards to animal welfare. There are reports of no effect as well as both decreases and increases in cortisol after chronic stressors. Therefore, the first aim of the present study was to determine how measures of compromised welfare, such as chronic pain and haematological anomalies, related to cortisol levels in domestic horses (
Animals confronted with a physical or emotional stressor rely on an intricate interplay between many biological systems (behavioral, autonomic, neuroendocrine and/or immune) to elicit responses to cope with that stressful situation [
Although HPA axis activation is well described in response to acute stress, its interpretation in cases of chronic stress is far from straight forward [
Moreover, the effects of chronic stress on physiological data and other responses (such as behavior) can also be contradictory. For example, the plasma cortisol levels of pigs submitted to unpredictable and inescapable electric shocks for a month were similar to those of control animals that had received no shocks [
In this study on horses, we wanted to 1) explore the relationship between cortisol levels and additional measures of welfare that may be affected by chronic stressors and 2) determine if fecal cortisol metabolite levels could be used as an appropriate indicator of plasma cortisol and be related to welfare measures. Different definitions have been given for animal welfare, such as a balanced physiological state [
We used a multidimensional approach to investigate the relationship between baseline cortisol levels on the one hand, and selected indicators of chronic compromised welfare, including health-related (vertebral problems, haematological disorders), and postural (ears-backward position) indicators. As mentioned above, a previous study has already shown that apathetic horses have lower cortisol levels while no correlation was found with stereotypic behaviours [
All our experiments complied with current French laws related to animal experimentation and were in accordance with the European directive 86/609/CEE. The project was approved by the local Ethics Committee in Animal Experiments at the University of Rennes 1. Fifty-nine horses (44 geldings and 15 mares), from three riding centres in Brittany, France, were tested. Sixty-eight percent of the horses were French Saddlebreds, equally distributed among the centres. Other horses belonged to a variety of breeds or were unregistered animals. Their ages ranged from 5 to 20 years old (
For plasma samples, blood was collected from the neck vein of each horse while in its box as previously described [
Plasma cortisol levels were measured using Immunotech kits for cortisol determination (Beckmann and Coulter). The kit was modified for equine plasma as follows: 1) the quantity of plasma per dose was 25 μL instead of 50 μL; 2) a two-hour preliminary incubation at 20°C between plasma and antibodies was added; 3) we used two standard curves: the first with increasing cortisol contents in buffer (as indicated by the manufacturer) and the second with increasing cortisol contents in equine plasma (from pooled data for controls totaling less than 2 ng/mL of cortisol). When data did not fit these two standards, they were discarded. These modifications produced linear curves (log B/Bo) between 2 ng/mL and 300 ng/mL. A good linearity was observed for dilution or overload experiments. The statistical reproducibility variation coefficient was 1.37% and the sensitivity was 2 ng/mL. Plasma concentrations of cortisol measures were previously reported in relationship to withdrawn postures in Fureix
To determine the relationship between FCM and plasma cortisol levels, and FCM and welfare measures, fresh fecal samples were collected within 1 minute of defecation, directly from the bedding by an experimenter using sterile gloves. Samples were collected between 12:00 and 13:00, three times for each horse: twice 24h after a day’s work and once after a day’s rest (again taking into account the potential impact of working), based on previous work in ponies [
The two fecal measures after a day’s work were averaged to give one measure and indicate HPA activity during work. Outliers more than 3SD above or below the mean were removed. This occurred once due to technical error with FCM after a day’s rest.
To determine general welfare state and how welfare may be related to cortisol levels, some indicators such as ear positions (while feeding, [
In horses, backward ear position is commonly associated with negative emotional states, such as pain or a negative cognitive bias [
The observer then resumed walking along the midline to the next box. The percentage of scans in each ear position was calculated for each horse. Further analyses categorised horses according to their most used (≥ 50% scans) posture. Here, we focused particularly on backward ears, as this position has been previously shown to reflect chronically impaired welfare in horses [
Evaluation of horses’ spines was performed by a licensed chiropractor with 20 years experience (H. Menguy), who was blind to the results of the observations performed during the behavioral tests, and did not know the horses beforehand. Manual palpation was performed from head to tail. Chiropractic approaches clearly address subclinical conditions (of special interest here) and licensed professionals have an expertise in the evaluation of joints and spinal related disorders [
Examinations were based on bony and soft tissue manual palpation to localise regions of vertebral stiffness based on spinal mobilisation and palpable areas of muscle hypertonicity [
At the same time as the morning cortisol sample was taken a blood sample was collected for each horse, between 08:00 and 09:00 and taken within the hour to a veterinary laboratory for analysis (Laboratoire Vétérinaire Départemental Ille et Vilaine, Rennes, France). Haematological data analysed were numbers of red blood cells (millions / mm3), white blood cells (million / mm3) and platelets (mille / mm3), percentages of neutrophils, eosinophils, basophils, lymphocytes, monocytes, hematocrit and haemoglobin (g/100ml), haemoglobin / hematocrit (g/100ml) and hematocrit / number of red blood cells per L (μm3). These analyses allowed us to identify horses with unusual levels of haematological parameters, such as anaemic animals. Norms provided by Laboratoire Vétérinaire Départemental Ille et Vilaine, Rennes, France and indicated in
Morning | Evening (day of work) | Evening (day of rest) | |
---|---|---|---|
Plasma cortisol (ng/ml) | 28.5 ± 1.4 | 15.2 ± 1.8 | 12.6 ± 1.1 |
FCM (ng/g) | - | 5.0 ± 0.3 | 4.9 ± 0.4 |
Based on our data, horses were further split into ‘compromised welfare’ and ‘normal welfare’ groups based on four measures; ear position, vertebrae disorders, neutrophil levels and anaemia. A ‘compromised welfare’ horse had at least 3 of the 4 measures associated with compromised welfare such as ears back greater than 50% of the time, severe vertebrae issues, neutrophil levels and/or anaemia outside the norms ‘Normal welfare’ horses had 0–1 of these compromised welfare measures. Previous work has shown that vertebrae disorders and haematological data showed no correlation while ears’ chronic backwards position seems to indicate an overall negative affect [
Analyses were conducted using Statistica 13 (Dell Inc). T-tests were run to investigate circadian variations in cortisol levels. ANOVAs were used to calculate differences in fecal and plasma cortisol measures with day (rest vs work) when welfare indicators were divided into nominal categories (
In accordance to circadian variations previously reported in horses [
Plasma cortisol concentrations were significantly lower in the evening, both after a day’s work and after a day’s rest, than in the morning (p < 0.00001, N = 55–56). B) Average evening plasma cortisol levels and average levels of fecal cortisol metabolites were significantly correlated (r = 0.66, p<0.0001, N = 55). * denotes a significant difference.
On average, the horses were observed with ears forward in 44.1% (± 21.8, 0–90) of scans, with their ears backward 53.4% (± 24.0, 0–100) of scans, and with ears in axial position 2.5% (± 5.4, 0–20) of the scans. 52% of horses showed ears backward in a majority of the scans (
(A). Horses with mostly the backward ear position had significantly lower average fecal cortisol measures after work (p = 0.04). Time horses spent with ears in a backwards position was negatively correlated with average evening (B) FCM (p = 0.02) and (C) plasma cortisol measures (p = 0.04). (A) Forward ears N = 19, Backward ears, N = 31; (B) N = 59, (C) N = 55.
Chiropractic examinations indicated, in accordance with the literature [
Severely affected horses (n = 40) had significantly lower evening plasma cortisol levels compared to totally exempt or slightly affected horses (n = 15), regardless of cortisol levels after a day’s work or rest (main effect of vertebral disorder severity F(1,53) = 5.34, p = 0.02;
A) Horses with severely affected vertebral problems had significantly lower evening plasma cortisol levels compared to totally exempt or slightly affected horses (p = 0.02; severely affected horses (n = 40) vs slightly affected horses (n = 15)). B) Anaemic horses had significantly lower evening plasma cortisol concentrations compared to non-anaemic horses (p = 0.02; anaemic horses = 10, non-anaemic horses = 45). C) Horses with levels of neutrophils outside the norm had significantly lower FCM levels compared to horses with normal neutrophil levels (p = 0.03; unusual neutrophils = 19, normal neutrophils = 36). D) Horses with ‘compromised’ global welfare had significantly lower evening plasma cortisol levels compared to ‘normal’ horses (p = 0.001; compromised = 13, normal = 17).). *denotes significant main effect, regardless of day of rest or work.
Our haematological data are summarised in
Mean (±SD) | Observed range ( |
% with unusual levels | |
---|---|---|---|
Red blood cells (millions/mm3) | 7.7 ± 0.9 | 6.3–10.6 ( |
0% |
White blood cells (millions/mm3) | 6.8 ± 1.7 | 3.1–11.7 ( |
0% |
Neutrophils (%) | 58.7 ± 11.8 | 26.0–81.1 (30–65) | 42% (>) |
Eosinophils (%) | 2.0 ± 1.8 | 0.0–10.0 (0–5) | 0% |
Basophils (%) | 0.2 ± 0.4 | 0.0–2.0 (0–3) | 0% |
Lymphocytes (%) | 37.6 ± 11.4 | 18.0–70.0 (25–70) | 11% (<) |
Monocytes (%) | 1.4 ± 0.8 | 1.0–5.0 (0.5–7) | 0% |
Hematocrit (%) | 36.3 ± 4.5 | 34.0–43.0 (32–52) | 0% |
Haemoglobin (g/100ml) | 12.4 ± 1.5 | 9.6–16.4 (11–19) | 18% (<) |
Haemoglobin/hematocrit (g/100ml) | 34.7 ± 1.7 | 24.9–36.7 (34–39) | 0% |
Hematocrit/#red blood cells per L (um3) | 46.7 ± 2.3 | 42.0–52.0 (34–58) | 0% |
Mean (±SD), range, and percent of horses with unusual levels: lower (<) and higher (>) than the norm. N = 55.
Anaemic horses had significantly lower evening plasma cortisol concentrations compared to non-anemic horses (main effect: F(1,53) = 5.8, p = 0.02;
Further distribution of horses into ‘compromised’ welfare states (had 3 or 4 of the following: ears backwards in a majority of the observations (more than 50%), severely affected spine, neutrophils and/or anaemia above or below the normal range, and ‘normal’ welfare states (with 0–1 of the 4 measures) showed that horses with ‘compromised’ welfare had significantly lower average FCM and evening plasma cortisol levels compared to ‘normal’ horses (FCM—F(1,27) = 5.02, p = 0.03; plasma—F(1,28) = 13.1, p = 0.001;
The findings of the present study, comparing horses’ baseline levels of cortisol and several welfare indicators, revealed that horses whose welfare was clearly compromised (as indicated by an unusual ears backward position, presence of vertebral problems or haematological anomalies,
Our results expand earlier findings evidencing low or no change in cortisol levels after chronic training and racing of thoroughbred fillies [
Research to date in horses has shown that compromised welfare is associated with decreased cortisol levels, and/or blunted HPA axis activity [
Although we have shown that low cortisol levels are associated with compromised welfare in horses, it has often been reported, in other species, that different types of chronic stressors, associated with general wellbeing, can also elevate glucocorticoid levels. These differences in effects may be due to the species studied, the type of stressor or welfare measure, duration of the stress, and techniques used to analyse glucocorticoid levels [
We also report a positive correlation between evening plasma cortisol levels and fecal cortisol metabolite concentrations in horses, extending our previous work [
The present study shows that horses living in compromised welfare conditions have low evening plasma cortisol levels and low levels of fecal cortisol metabolites (FCM). These lower levels of cortisol are associated with behavioral measures, such as ear positions (present study) and withdrawn posture [
We are grateful to the directors and staff of the riding centers for their help with data collection;C. Fureix, L. Dupont and C. Lavertue at the University of Rennes 1 and E Klobetz-Rassam at the University of Veterinary Medicine, Vienna, who aided with sample collection and analyses. This work was funded by the Caisse Centrale de la Mutualité Agricole, University of Rennes 1 and the Le Centre national de la recherche scientifique (CNRS).