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Peeters, M., Sulon, J., Beckers, J. - F., Ledoux, D., & Vandenheede, M. (2011). Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge. Equine Veterinary Journal, 43(4), 487–493.
Abstract: Reasons for performing study: In horses, serum cortisol concentration is considered to provide an indirect measurement of stress. However, it includes both free and bound fractions. The sampling method is also invasive and often stressful. This is not the case for salivary cortisol, which is collected using a more welfare-friendly method and represents a part of the free cortisol fraction, which is the biologically active form. Objectives: To compare salivary and serum cortisol assays in horses, in a wide range of concentrations, using an adrenocorticotropic hormone (ACTH) stimulation test, in order to validate salivary cortisol for stress assessment in horse. Methods: In 5 horses, blood samples were drawn using an i.v. catheter. Saliva samples were taken using swabs. Cortisol was assayed by radioimmunoassay. All data were treated with a regression method, which pools and analyses data from multiple subjects for linear analysis. Results: Mean ± s.d. cortisol concentrations measured at rest were 188.81 ± 51.46 nmol/l in serum and 1.19 ± 0.54 nmol/l in saliva. They started increasing immediately after ACTH injection and peaks were reached after 96 ± 16.7 min in serum (356.98 ± 55.29 nmol/l) and after 124 ± 8.9 min in saliva (21.79 ± 7.74 nmol/l, P<0.05). Discharge percentages were also different (225% in serum and 2150% in saliva, P<0.05). Correlation between serum and salivary cortisol concentrations showed an adjusted r2= 0.80 (P<0.001). The strong link between serum and salivary cortisol concentrations was also estimated by a regression analysis. Conclusions: The reliability of both RIAs and regression found between serum and salivary cortisol concentrations permits the validation of saliva-sampling as a noninvasive technique for cortisol level assessment in horses.
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Stull, C. L., Spier, S. J., Aldridge, B. M., Blanchard, M., & Stott, J. L. (2004). Immunological response to long-term transport stress in mature horses and effects of adaptogenic dietary supplementation as an immunomodulator. Equine Veterinary Journal, 36(7), 583–589.
Abstract: Reasons for performing study: Little information exists on the immunological effects of transport or the use of supplements to minimise transport stress. Objectives: To establish baseline ranges and evaluate immunophenotypic and functional changes associated with transport and a nutritional ‘adaptogen’ supplement. Methods: Horses received either supplement (n = 10) or placebos (n = 9) during the 30 day study. After 28 days in stalls, 12 horses (6 supplement; 6 placebo) were transported for 24 h, then unloaded and recovered. Venous blood samples were collected on Days 1, 14 and 28 to establish baselines, and on Days 28, 29 and 30 to examine changes during transport and recovery. Results: Transport prompted elevations (P<0.05) in cortisol concentration, neutrophil count and white blood cell counts, while lymphocyte subpopulation counts (CD3+, CD4+, CD8+, CD21+) decreased (P<0.05). Normal phenotypic lymphocyte profiles returned within 24 h of recovery. Supplement effects on immunophenotype (CD21+ and CD8+) were observed in stabled horses (P<0.05), but not in transported horses. Conclusions: These results provide insights into the immunological mechanisms associated with long-term transport. Potential relevance: The existence of a small window of immunological uncertainty follows long-term transportation, enhancing the potential risk of infectious disease in susceptible individuals.
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Schmidt, A., Aurich, J., Möstl, E., Müller, J., & Aurich, C. (2010). Changes in cortisol release and heart rate and heart rate variability during the initial training of 3-year-old sport horses. Horm Behav, 58(4), 628–636.
Abstract: Based on cortisol release, a variety of situations to which domestic horses are exposed have been classified as stressors but studies on the stress during equestrian training are limited. In the present study, Warmblood stallions (n = 9) and mares (n = 7) were followed through a 9 respective 12-week initial training program in order to determine potentially stressful training steps. Salivary cortisol concentrations, beat-to-beat (RR) interval and heart rate variability (HRV) were determined. The HRV variables standard deviation of the RR interval (SDRR), RMSSD (root mean square of successive RR differences) and the geometric means standard deviation 1 (SD1) and 2 (SD2) were calculated. Nearly each training unit was associated with an increase in salivary cortisol concentrations (p < 0.01). Cortisol release varied between training units and occasionally was more pronounced in mares than in stallions (p < 0.05). The RR interval decreased slightly in response to lunging before mounting of the rider. A pronounced decrease occurred when the rider was mounting, but before the horse showed physical activity (p < 0.001). The HRV variables SDRR, RMSSD and SD1 decreased in response to training and lowest values were reached during mounting of a rider (p < 0.001). Thereafter RR interval and HRV variables increased again. In contrast, SD2 increased with the beginning of lunging (p < 0.05) and no changes in response to mounting were detectable. In conclusion, initial training is a stressor for horses. The most pronounced reaction occurred in response to mounting by a rider, a situation resembling a potentially lethal threat under natural conditions.
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Gröschl, M., Wagner, R., Rauh, M., & Dörr, H. G. (2001). Stability of salivary steroids: the influences of storage, food and dental care. Steroids, 66(10), 737–741.
Abstract: We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (17OHP) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN3) were stored at room temperature and at 4°C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN3 treated samples. After repeated freezing and re-thawing 17OHP and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.
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Suagee-Bedore, J. K., Linden, D. R., & Bennett-Wimbush, K. (2021). Effect of Pen Size on Stress Responses of Stall-Housed Horses Receiving One Hour of Daily Turnout. J. Equine Vet. Sci., 98, 103366.
Abstract: Group turnout provides important socializing opportunities for horses, particularly those that are primarily stalled. A high percentage of equine injuries occur during group turnout, which could partly be due to the physical constraints of fencing. To investigate appropriate paddock sizes for group turnouts, horses (n = 12) from a single herd were divided into groups of 4, stalled for 24 hours, and then turned out for 1 hour into one of three differently sized pens: 342, 263, and 184 m2 per horse. Groups rotated through pens across 3 days, receiving one treatment per day. Blood was sampled for cortisol concentrations at 08:00 hours each morning, and then at 15 and 60 minutes into the turn out sessions, and 60 minutes after return to individual stalls. Groups rotated through three turnout times: 09:00, 12:00, 14:00 hours. Counts of agonistic behaviors (chasing, contact biting, and kicking) and low-level threats (pinned ears, tail swishing, bite and kick threats) were recorded. When turned out in pens that provided 342 m2 per horse, horses exhibited reduced plasma cortisol concentrations by 15 minutes after turnout and at 1 hour after return to their stalls (P < .05). Horses in pens providing 184 m2 per horse exhibited greater agonistic (P < .001) and low-level threat (P < .01) behaviors than horses in larger pens. These data provide insight into appropriate pen sizes for horses from established herds. Providing at least 342 m2 per horse may reduce the chance of injury in horses accustomed to group turnout.
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