Avances en el conocimiento de la cromogranina A y otros biomarcadores salivares de estrés en cerdos

Résumé

This PhD thesis, for compendium of articles, was designed in order to produce advances in the measurement of salivary biomarkers of stress and to develop and validate a panel of salivary biomarkers to evaluate the different physiological systems (sympatho-adrenomedullary (SAM), hypothalamic-pituitary-adrenal (HPA), hypothalamus-pituitary-gonadal (HPG) and immune system) that are taking part in stress mechanism. To this end, the objectives of this thesis are: to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour as a potential biomarkers of SAM system in an acute stress model. In addition, to study if salivary CgA concentrations exhibit any circadian pattern during the daytime, and to evaluate its stability under different storage conditions. Moreover, validate commercially available immunoassays for cortisol (HPA system biomarker), testosterone (HPG system biomarker) and IgA (immune biomarker) quantification on the saliva of pigs. To evaluate the response of a panel of several salivary biomarkers both of immune system as well as of the neuroendocrine system to repeated administration of Escherichia coli lipopolysaccharide (LPS) and, finally, to investigate the responses of this group of salivary biomarkers after applying a psychosocial stressor model in pigs based on isolation and regrouping. Regarding the methodology, polyclonal antibodies specific for the development of TR-IFMA were produced in rabbits immunized with a recombinant protein peptide of CgA porcine. Precision, accuracy and analytical sensitivity of the TR-IFMA developed and of the commercial immunoassays used were evaluated by the coefficients of variation intra- and inter-assay, linearity under dilution and the percentage of recovery, limits of detection and limits of quantitation. To study the stress response of salivary biomarkers, models in which the animals were immobilized with a nose-snare or transported to slaughterhouse were used. Saliva samples were taken before and after of applying these stressful stimuli. On the other hand, in the case of LPS administrations, the pigs were selected to receive three doses of LPS at 48 h intervals. Saliva samples were taken prior to any LPS administration and at time points corresponding to 3 h after each injection. Moreover, in the model of isolation and regrouping all animals were adapted to group living for 5 days, after which time, pigs in one group were subjected to the isolation procedure, where each animal had not physical contact with other pigs during 5 days. After, test pigs were regrouped again during 3 days returning to the initial conditions. Control pigs were remained in the same housing condition during the entire experimental period. Saliva samples were taken daily, at the same time, and one sampling was carried out at 30 min after of isolation and regrouping events. In overall, we can conclude of this work that: the TR-IFMA developed allows the quantification of CgA in porcine saliva samples in a precise, accurate and sensitive way and its concentrations increase after an acute stress situation. In addition, no circadian pattern has been detected for salivary CgA and this biomarker was fairly stable being able to be stored till 1 year at -20ºC or -80ºC. Moreover, the commercial immunoassays validated for cortisol, testosterone and IgA concentrations can be suitable for its use in saliva samples of pigs with a good precision, sensitivity and accuracy. In relation to LPS administrations, a single administration in pigs produces increases of salivary IgA and cortisol. However, repeated administrations only produced increases in IgA whereas cortisol returned to baseline values. No variation in CgA concentrations were observed neither in single nor in repeated LPS administration. Finally, the use of a panel of salivary biomarkers would be essential in stress research, since these react differently to various types of stressors. In the case of isolation, CgA and IgA appear to be more sensitive markers than cortisol and testosterone, whereas after the process of regrouped, cortisol, testosterone and CgA seem to be more sensitive markers than IgA.