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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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., & 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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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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Palm, A. - K. E., Wattle, O., Lundström, T., & Wattrang, E. (2016). Secretory immunoglobulin A and immunoglobulin G in horse saliva. Vet. Immunol. Immunolpathol., 180, 59–65.
Abstract: This study aimed to increase the knowledge on salivary antibodies in the horse since these constitute an important part of the immune defence of the oral cavity. For that purpose assays to detect horse immunoglobulin A (IgA) including secretory IgA (SIgA) were set up and the molecular weights of different components of the horse IgA system were estimated. Moreover, samples from 51 clinically healthy horses were tested for total SIgA and IgG amounts in saliva and relative IgG3/5 (IgG(T)) and IgG4/7 (IgGb) content were tested in serum and saliva. Results showed a mean concentration of 74μg SIgA/ml horse saliva and that there was a large inter-individual variation in salivary SIgA concentration. For total IgG the mean concentration was approx. 5 times lower than that of SIgA, i.e. 20μg IgG/ml saliva and the inter-individual variation was lower than that observed for SIgA. The saliva-serum ratio for IgG isotypes IgG3/5 and IgG4/7 was also assessed in the sampled horses and this analysis showed that the saliva-serum ratio of IgG4/7 was in general approximately 4 times higher than that of IgG3/5. The large inter-individual variation in salivary SIgA levels observed for the normal healthy horses in the present study emphasises the need for a large number of observations when studying this parameter especially in a clinical setting. Moreover, our results also indicated that some of the salivary IgG does not originate from serum but may be produced locally. Thus, these results provide novel insight, and a base for further research, into salivary antibody responses of horses.
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Skandakumar, S., Stodulski, G., & Hau, J. (1995). Salivary IgA: a Possible Stress Marker In Dogs. In Animal Welfare (Vol. 4, pp. 339–350).
Abstract: Stress in humans has been reported to be associated with a decrease in the salivary immunoglobulin A (s-IgA) levels enabling the possible use of s-IgA to assess stress. Prolonged stress, if reliably assessed in a non-invasive manner, may be used to assess animal welfare. This study analysed groups of dogs undergoing physical and temperamental training and s-IgA levels were measured by rocket immunoelectrophoresis in prospective samples. Behavioural assessment was carried out and cortisol levels in saliva were measured by ELISA. A significant negative correlation (P < 0.007) between the logarithmic cortisol concentrations and s-IgA levels in saliva was recorded. The behavioural assessment of the dogs agreed well with the biochemical markers. It is concluded that IgA levels in saliva may be a useful marker of dog well-being and that stress results in decreased s-IgA levels.
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Jafarzadeh A., Sadeghi M., Karam G.A., & Vazirinejad R. (2010). Salivary IgA and IgE levels in healthy subjects: relation to age and gender. Braz. Oral Res., 24(1), 21–27.
Abstract: It has been reported that the immune system undergoes age and gender changes. The aim of this study was to investigate the age- and gender-dependent changes of salivary IgA and IgE levels among healthy subjects. A total of 203 healthy individuals (aged 1-70 years) were enrolled in the study. Two milliliters of saliva were collected from all participants, and salivary IgA and IgE levels were measured by the ELISA technique. Mean salivary IgA levels were significantly higher in subjects aged 11-20 years as compared to subjects aged 1-10 years (P < 0.01). Mean salivary IgA levels increased with age up to the age of 60 years, and then slightly decreased in subjects aged 61-70 years. The frequency of subjects with detectable levels of salivary IgE and mean salivary IgE levels gradually increased with age, with maximum levels being observed in the 31-40 years age group and not changing significantly thereafter. The mean levels of salivary IgA and IgE in adults were significantly higher than those observed in children (P < 0.00001 and P < 0.05, respectively). No significant differences were observed between men and women regarding both salivary immunoglobulins. These results showed age-dependent changes of the salivary IgA and IgE levels. Gender had no effect on the salivary levels of IgA and IgE.
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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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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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Tiefenbacher, S., Lee, B., Meyer, J. S., & Spealman, R. D. (2003). Noninvasive technique for the repeated sampling of salivary free cortisol in awake, unrestrained squirrel monkeys. Am. J. Primatol., 60(2), 69–75.
Abstract: The use of noninvasive measures of hypothalamic-pituitary-adrenal (HPA) axis function is of growing interest among preclinical and clinical investigators. This report describes a method for the repeated assessment of salivary free cortisol in awake, unrestrained squirrel monkeys (Saimiri sciureus) based on a saliva sampling technique previously developed for rhesus monkeys. Individually housed adult male squirrel monkeys were trained to chew on dental rope attached to a pole, from which saliva was extracted by centrifugation and analyzed for cortisol by radioimmunoassay (RIA). Eight of nine monkeys readily acquired the task, reliably providing adequate saliva samples for the assay. Salivary free cortisol levels were examined in these subjects under basal conditions and in response to two types of neuroendocrine challenge. Levels of salivary free cortisol showed relatively low intra- and interindividual variability, with mean individual morning levels ranging between 17.1 and 37.9 µg/dl. Squirrel monkeys demonstrated a consistent daily rhythm in salivary free cortisol ranging from a high of 27.4 ± 5.2 µg/dl (mean ± SEM) at 12 P.M. to a low of 7.5 ± 1.6 µg/dl at 6 P.M.. Intravenous (IV) challenges with 1 µg/kg ACTH, or 10 and 50 µg/kg CRF resulted in significant increases in salivary free cortisol. The described sampling technique provides a reliable and sensitive means for repeated measurement of HPA activity in unrestrained, awake squirrel monkeys. In addition, our findings illustrate several features of HPA system rhythmicity and reactivity using salivary cortisol instead of blood plasma or serum. Am. J. Primatol. 60:69–75, 2003. © 2003 Wiley-Liss, Inc.
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