Advances in biomarkers for welfare evaluationoxytocin and corticoids

Resum

The main objective of this Doctoral Thesis was to provide an advance in the development of new assays to accurately determine oxytocin concentrations in saliva, hair, and other samples from various species, such as porcine, bovine, canine, and human, as well as methods that can evaluate the cortisol and cortisone concentrations, and the 11β-hydroxysteroid dehydrogenase isoenzyme type 2 activity in the hair of pigs. All this with the overall objective of providing new analytical tools to assess stress and welfare in these species in a non-invasive way. The specific objectives were: (1) To develop and validate immunoassays for oxytocin measurement in the saliva of porcine species and apply them in different stress and positive situations, and to develop an immunoassay for oxytocin measurement in seminal plasma of porcine species, as well as a review on assays for oxytocin measurement. (2) Develop and validate immunoassays for oxytocin measurement in the saliva of canine, bovine, and human species, and apply them in different stress or positive situations. (3) Develop and validate an immunoassay for the determination of oxytocin in the hair of pigs. (4) To develop and validate immunoassays for the determination of cortisol and cortisone as biomarkers of stress in hair of pigs. For the first objective, two new immunoassays based on AlphaLISA technology were developed and validated for oxytocin measurement in the saliva of pigs and its evaluation in different situations, such as farrowing and lactation, ejaculate collection, and transport and lairage at the slaughterhouse. The influence of the extraction procedure and the reduction and alkylation treatment on the saliva samples was also evaluated. In addition, a method for oxytocin measurement in the seminal plasma of pigs was developed, and its relationship with reproductive parameters was evaluated. For the second objective, two new immunoassays based on AlphaLISA technology were developed and validated for oxytocin measurement in the saliva of bovine, canine, and human species to evaluate the changes after stress or positive situations, as well as the evaluation of the reduction and alkylation treatment on the samples. For the third objective, an immunoassay based on AlphaLISA technology was developed and validated for oxytocin measurement in the hair of pigs and to evaluate changes during the reproductive cycle. For the fourth objective, two immunoassays based on AlphaLISA technology were developed and validated, one of them cortisone measurement and the other one for cortisol measurement in the hair of pigs, and the evaluation of the changes during the reproductive cycle, and the evaluation of the 11β-hydroxysteroid dehydrogenase type 2 isoenzyme activity. In conclusion, and globally, all the methods developed in this thesis proved to be adequate from the analytical point of view and allowed the detection of (1) oxytocin in the saliva of porcine, canine, bovine, and human species without the need for prior treatment of the samples, and in seminal plasma and hair of pigs and (2) cortisol, cortisone, and the 11β-hydroxysteroid dehydrogenase isoenzyme type 2 activity in the hair of pigs using the cortisone/cortisol ratio. In addition, all methods were able to detect the variations of the analytes in different stress and welfare situations.