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Evaluation of new biomarkers of stress in saliva of sheep

  • M. D. Contreras-Aguilar (a1), D. Escribano (a1) (a2), A. Quiles (a3), M. López-Arjona (a1), J. J. Cerón (a1), S. Martínez-Subiela (a1), M. L. Hevia (a3) and F. Tecles (a1)...


Some routine handling procedures can produce stress in farm animals, and an adequate control of these stressors is important to avoid the negative effects on animal health and production. The measurement of biomarkers in saliva can be a suitable tool for the evaluation and control of stress. In this report, lipase, butyrylcholinesterase (BChE), total esterase (TEA) and adenosine deaminase (ADA) activities in the saliva of sheep were evaluated as biomarkers of stress. For this purpose, they were measured after inducing stress by facing a dog (experiment 1) and shearing (experiment 2), and comparing them to other stress salivary biomarkers such as α-amylase (sAA) and cortisol, as well as heart rate (HR). Each analyte was measured at the basal time, and during and just after the end of the stressful stimulus, and at various times for the first hour after the period of stress induction. Values were compared with those obtained from a control group. Lipase was the only analyte that showed significant changes between the stress and the control group in both experiments. Although TEA and ADA increased after stress, no significant differences were seen compared with the control group. Lipase was correlated highly with sAA and HR, in experiment 1; and correlated moderately with cortisol and HR in experiment 2. Lipase showed the greatest percentage increase after the stressful stimuli and less overlap with the control group in the two experiments. From the results of this study it can be concluded that lipase, TEA, BChE and ADA are enzymes present in the saliva of sheep and that they can be measured by using simple and fast colorimetric methods. Further studies should be undertaken with regard to the possible application of lipase as a biomarker of stress in sheep.


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Adams, A and Harkness, RA 1976. Adenosine deaminase activity in thymus and other human tissues. Clinical and Experimental Immunology 26, 647649.
Archana, E, Pai, P, Prabhu, BK, Shenoy, RP, Prabhu, K and Rao, A 2012. Altered biochemical parameters in saliva of pediatric attention deficit hyperactivity disorder. Neurochemical Research 37, 330334.
Baganha, MF, Pêgo, A, Lima, MA, Gaspar, EV and Cordeiro, AR 1990. Serum and pleural adenosine deaminase – correlation with lymphocytic populations. Chest 97, 605610.
Carcangiu, V, Vacca, GM, Parmeggiani, A, Mura, MC, Pazzola, M, Dettori, ML and Bini, PP 2008. The effect of shearing procedures on blood levels of growth hormone, cortisol and other stress haematochemical parameters in Sarda sheep. Animal 2, 606612.
Claus Henn, B, McMaster, S and Padilla, S 2006. Measuring cholinesterase activity in human saliva. Journal of Toxicology and Environmental Health A 69, 18051818.
Cook, CJ and Jacobson, LH 1995. Salivary cortisol as an indicator of stress in sheep (Ovis ovis) . New Zealand Veterinary Journal 43, 248.
Fedorova, T, Soendersoe Knudsen, C, Mouridsen, K, Nexo, E and Borghammer, P 2015. Salivary acetylcholinesterase activity is increased in Parkinson’s disease: a potential marker of parasympathetic dysfunction. Parkinson’s Disease 2015, 156479.
Ferguson, DM, Bruce, HL, Thompson, JM, Egan, AF, Perry, D and Shorthose, WR 2001. Factors affecting beef quality – farm gate to chilled carcass. Australian Journal of Experimental Agriculture 41, 879891.
Fuentes-Rubio, M, Fuentes, F, Otal, J, Quiles, A and Hevia, ML 2016. Validation of an assay for quantification of alpha-amylase in saliva of sheep. Canadian Journal of Veterinary Research 80, 197202.
Graca, R, Messick, J, McCullough, S, Barger, A and Hoffmann, W 2005. Validation and diagnostic efficacy of a lipase assay using the substrate 1,2-o-dilauryl-rac-glycero glutaric acid-(6’ methyl resorufin)-ester for the diagnosis of acute pancreatitis in dogs. Veterinary Clinical Pathology 34, 3943.
Grandin, T 1998. Review: reducing handling stress improves both productivity and welfare. The Professional Animal Scientist 14, 110.
Hamosh, M 1978. Rat Lingual Lipase - factors affecting enzyme-activity and secretion. American Journal of Physiology 235, E416E421.
Hamosh, M and Burns, WA 1977. Lipolytic-activity of human lingual glands (Ebner). Laboratory Investigation 37, 603608.
Hargreaves, AL and Hutson, GD 1990. The stress response in sheep during routine handling procedures. Applied Animal Behaviour Science 26, 8390.
Hild, S, Coulon, M, Schroeer, A, Andersen, IL and Zanella, AJ 2011. Gentle vs aversive handling of pregnant ewes: I. Maternal cortisol and behaviour. Physiology & Behavior 104, 384391.
Hinkle, DE, Wiersma, W and Jurs, SG 2003. Applied statistics for the behavioural sciences, 5th edition. Houghton Mifflin, Boston, MA, USA.
Imperato, A, Puglisi-Allegra, S, Casolini, P and Angellucci, L 1991. Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitary-adrenocortical axis. Brain Research 538, 111117.
Kaufer, D, Friedman, A, Seidman, S and Soreq, H 1998. Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393, 373377.
Lee, PC, Purcell, ES, Borysewicz, R, Klein, RM and Werlin, SL 1992. Developmental delay of lingual lipase expression after guanethidine-induced sympathectomy. Proceedings of the Society for Experimental Biology and Medicine 199, 192198.
Meshorer, E, Erb, C, Gazit, R, Pavlovsky, L, Kaufer, D, Friedman, A, Glick, D, Ben-Arie, N and Soreq, H 2002. Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science 295, 508512.
Rai, B, Kaur, J, Jacobs, R and Anand, SC 2011. Adenosine deaminase in saliva as a diagnostic marker of squamous cell carcinoma of tongue. Clinical Oral Investigations 15, 347349.
Rohleder, N and Nater, UM 2009. Determinants of salivary α-amylase in humans and methodological considerations. Psychoneuroendocrinology 34, 469485.
Romeyer, A and Bouissou, MF 1992. Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions. Applied Animal Behaviour Science 34, 93119.
Sánchez-Amate, M, Flores, P and Sánchez-Santed, F 2001. Effects of chlorpyrifos in the plus-maze model of anxiety. Behavioural Pharmacology 12, 285292.
Sayer, R, Law, E, Connelly, PJ and Breen, KC 2004. Association of a salivary acetylcholinesterase with Alzheimer’s disease and response to cholinesterase inhibitors. Clinical Biochemistry 37, 98104.
Sine, J-P, Ferrand, R and Colas, B 1988. Acetylcholinesterase and butyrylcholinesterase in the gut mucosal cells of various mammal species: distribution along the intestine and molecular forms. Comparative Biochemistry and Physiology 91, 597602.
Tagliari, B, dos Santos, TM, Cunha, AA, Lima, DD, Delwing, D, Sitta, A, Vargas, CR, Dalmaz, C and Wyse, AT 2010. Chronic variable stress induces oxidative stress and decreases butyrylcholinesterase activity in blood of rats. Journal of Neural Transmission 117, 10671076.
Tecles, F and Cerón, JJ 2001. Determination of whole blood cholinesterase in different animal species using specific substrates. Research in Veterinary Science 70, 233238.
Tecles, F, Escribano, D, Martínez-Miró, S, Hernández, F, Contreras, MD and Cerón, JJ 2016a. Cholinesterase in porcine saliva: analytical characterization and behavior after experimental stress. Research in Veterinary Science 106, 2328.
Tecles, F, Contreras-Aguilar, MD, Martínez-Miró, S, Tvarijonaviciute, A, Martínez-Subiela, S, Escribano, D and Cerón, JJ 2017. Total esterase measurement in saliva of pigs: validation of an automated assay, characterization and changes in stress and disease conditions. Research in Veterinary Science 114, 170176.
Tecles, F, Rubio, CP, Contreras-Aguilar, MD, López-Arjona, M, Martínez-Miró, S, Martínez-Subiela, S and Cerón, JJ 2018. Adenosine deaminase activity in pig saliva: analytical validation of two spectrophotometric assays. Journal of Veterinary Diagnostic Investigation 30, 175179.
Tecles, F, Tvarijonaviciute, A, De Torre, C, Carrillo, JM, Rubio, M, García, M, Cugat, R and Cerón, JJ 2016b. Total esterase activity in human saliva: validation of an automated assay, characterization and behaviour after physical stress. Scandinavian Journal of Clinical Laboratory Investigation 76, 324330.
Wittwer, A, Krummenacher, P, La, MR and Ehlert, U 2016. Methodology, mechanisms & salivary alpha-amylase correlates with subjective heat pain perception. Pain Medicine 17, 11311136.
Yamanouchi, K, Hayashida, N, Kuba, S, Sakimura, C, Kuroki, T, Togo, M, Katayama, N, Takamura, N and Eguchi, S 2015. Increase in operator’s sympathetic nerve activity during complicated hepatobiliary surgery: evidence for surgeons’ mental stress. The Tohoku Journal of Experimental Medicine 237, 157162.
Yardimci, M, Sahin, EH, Cetingul, IS, Bayram, I, Aslan, R and Sengor, E 2013. Stress responses to comparative handling procedures in sheep. Animal 7, 143150.
Yates, DT, Ross, TT, Hallford, DM, Yates, LJ and Wesley, RL 2010. Technical note: comparison of salivary and serum cortisol concentrations after adrenocorticotropic hormone challenge in ewes. Journal of Animal Science 88, 599603.


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