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Alexander, F., & Nicholson, J. D. (1968). The blood and saliva clearances of phenobarbitone and pentobarbitone in the horse. Biochem Pharmacol, 17(2), 203–210.
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Alexander, F., Horner, M. W., & Moss, M. S. (1967). The salivary secretion and clearance in the horse of chloral hydrate and its metabolites. Biochem Pharmacol, 16(7), 1305–1311.
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Alexander, F. (1966). A study of parotid salivation in the horse. J Physiol, 184(3), 646–656.
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Shanahan, S. (2003). Trailer loading stress in horses: behavioral and physiological effects of nonaversive training (TTEAM). J Appl Anim Welf Sci, 6(4), 263–274.
Abstract: Resistance in the horse to trailer loading is a common source of stress and injury to horses and their handlers. The objective of this study was to determine whether nonaversive training based on the Tellington-Touch Equine Awareness Method (TTEAM; Tellington-Jones &Bruns, 1988) would decrease loading time and reduce stress during loading for horses with a history of reluctance to load. Ten horses described by their owners as “problem loaders” were subjected to pretraining and posttraining assessments of loading. Each assessment involved two 7-min loading attempts during which heart rate and saliva cortisol were measured. The training consisted of six 30-min sessions over a 2-week period during which the horse and owner participated in basic leading exercises with obstacles simulating aspects of trailering. Assessment showed heart rate and saliva cortisol increased significantly during loading as compared to baseline (p <.001 and p <.05, respectively). Reassessment after training showed a decrease in loading time (p <.02), reduced heart rate during loading (p <.002), and reduced saliva cortisol as compared to pretraining assessments. Seven “good loaders” also were subject to loading assessment for physiological comparison. Increases in heart rate during loading were significantly higher in the good loaders (p <.001). Nonaversive training simulating aspects of loading may effectively reduce loading time and stress during loading for horses with a history of resistance to trailer loading.
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Elsaesser, F., Klobasa, F., & Ellendorff, F. (2001). ACTH stimulation test for the determination of salivary cortisol and of cortisol responses as markers of the training status/fitness of warm-blooded sports horses]. Dtsch Tierarztl Wochenschr, 108(1), 31–36.
Abstract: Previous work (Marc et al., 2000) suggested that plasma cortisol responses to treadmill exercise or ACTH injection are a reliable marker for performance evaluation in warmblood horses. For practical purposes blood sample collections and treadmill exercise tests are somewhat troublesome and time consuming. The goal of this study was thus to evaluate the use of saliva for cortisol determination (by direct EIA) as a marker for performance and to investigate the reliability and repeatability of plasma cortisol responses to a single i.v. injection of ACTH (50 micrograms or 250 micrograms). Furthermore, the effect of training horses for 8 weeks 3 times per week covering the same distance (increasing from 3.5 km during the first week to 8 km during the last week) either by trotting (approximately 240 m/min) or by cantering (375 m/min) was investigated. For this purpose initially ten four-year-old Hannovarian geldings, all reared in the same State stud, were used. Mean overall correlation between salivary cortisol and plasma cortisol concentrations was 0.64 when samples of various points of time were used. However, in spite of attempts to standardize saliva sample collection, correlation between salivary cortisol levels and plasma cortisol levels at distinct points of time in different tests were low and significant (r = 0.85, p < 0.02) only in one test. Thus, salivary cortisol measurements for diagnostic purposes are not reliable or useful. The repeatability of plasma cortisol responses to ACTH for untrained and trained horses were r = 0.86 and r = 0.8 respectively (p < or = 0.01 and p < or = 0.05 respectively). Training horses either by trotting or cantering did not affect the cortisol response either to treadmill exercise or to stimulation by ACTH. It is concluded that the relationship between salivary cortisol levels and plasma cortisol levels is not close enough to allow the use of salivary cortisol determination as marker of the training status/fitness of horses. The repeatability of the cortisol response to ACTH is similar to the cortisol response to treadmill exercise. Based on plasma cortisol responses to ACTH or treadmill exercise training horses by cantering at low speed is not superior to training by trotting for the fitness of horses.
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Stahl, F., & Dorner, G. (1982). Responses of salivary cortisol levels to stress-situations. Endokrinologie, 80(2), 158–162.
Abstract: A procedure is described for determining salivary cortisol levels by a competitive protein-binding assay using horse transcortin. The collection of saliva was performed by means of filter paper-strips. Filter paper samples are more than 5 days stable after air-drying. In this form, the samples could be stored without refrigerator or deep-freezer and, if necessary, sent by post to the laboratory without any special precaution. Stressful situation of either painful or anxious origin were associated with an adequate increase of salivary cortisol levels. The increases were 157 to 230% of the initial or normal values dependent on the kind of stress. The mean values in 4 cases of Cushing's syndrome were 380% and 1 hour after 25 I.U. ACTH 690% higher than those in normal persons. In normal persons, a well-defined circadian rhythm has been observed.
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van der Kolk, J. H., Nachreiner, R. F., Schott, H. C., Refsal, K. R., & Zanella, A. J. (2001). Salivary and plasma concentration of cortisol in normal horses and horses with Cushing's disease. Equine Vet J, 33(2), 211–213.
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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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Dreschel, N. A., & Granger, D. A. (2009). Methods of collection for salivary cortisol measurement in dogs. Horm. Behav., 55(1), 163–168.
Abstract: Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human–animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented.
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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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