Corso di Dottorato di Ricerca in Scienze e Biotecnologie Agrarie in convenzione con Università degli Studi di Udine Dipartimento di Scienze AgroAlimentari, Ambientali e Animali Ciclo XXX Coordinatore: prof Giuseppe Firrao TESI di DOTTORATO di RICERCA Development of biomarkers in non-invasive biological matrices in order to assess dog well-being. Dottoranda Supervisore Alice Colussi Bruno Stefanon Anno Accademico 2017/2018 Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT SUMMARY Chapter 1 OVERALL INTRODUCTION …………………………………………………………….. 3 1.1_AIMS OF THIS RESEARCH ................................................................................... 5 1.2_LITERATURE REVIEW .......................................................................................... 6 1.2.1_SALIVA: A NON-INVASIVE, READILY AVAILABLE MATRIX FOR MANY BIOMARKERS ................................................................................................. 6 1.2.1.1_Salivary Cortisol: a hormone used to evaluate HPA axis activity ............................................................................................... 7 1.2.2_MICROBIOME: AN IMPORTANT MARKER FOR ANIMAL WELFARE ...... 11 1.2.2.1_Metagenomics as an evaluation tool of microbiome composition ....................................................................................... 14 1.2.3_HAIR, A NON-INVASIVE MATRIX USEFUL FOR MEASURING SOME BIOMARKERS ................................................................................................. 16 1.2.3.1_Hair Cortisol: a useful matrix for long term monitoring ....... 16 1.2.3.2_Heavy metals in hair as biomarkers of health state............. 18 1.3_OUTLINE OF THE THESIS .................................................................................... 19 1.4_REFERENCES …………………………………………………………………………………………………. 23 Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT Part 1 SALIVARY CORTISOL: AN EFFICIENT BIOINDICATOR OF HYPOTHALAMIC–PITUITARY–ADRENAL AXIS ACTIVATION Chapter 2 SALIVARY CORTISOL: A MARKER OF THE ADAPTIVE RESPONSE OF THE ORGANISM TO ENVIRONMENTAL STIMULI ............................... 36 2.1_ABSTRACT .......................................................................................................... 37 2.2_THE PLEIOTROPIC ROLE OF CORTISOL ............................................................... 37 2.3_ASSAYING CORTISOL IN BIOLOGICAL MATRICES ............................................... 40 2.3.1_Sampling ............................................................................................. 40 2.3.2_Factors that influence salivary cortisol levels in the dog .................... 42 2.3.3_Potential applications of salivary cortisol assays ................................ 46 2.3.3.1_Animal-assisted activities ..................................................... 47 2.3.3.2_Genetics and breeding .......................................................... 48 2.3.3.3_Diseases ................................................................................ 49 2.4_CONCLUSIONS ................................................................................................... 50 2.5_REFERENCES …………………………………………………………………………………………………. 51 Chapter 3 SALIVARY CORTISOL CONCENTRATION IN HEALTHY DOGS IS AFFECTED BY SIZE, SEX, AND HOUSING CONTEXT ............................ 57 3.1_ABSTRACT .......................................................................................................... 58 3.2_INTRODUCTION ................................................................................................. 59 3.3_MATERIALS AND METHODS ............................................................................... 60 3.3.1_Animal selection .................................................................................. 60 Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT 3.3.2_Salivary sampling ................................................................................ 60 3.3.3_Statistical analysis ............................................................................... 61 3.4_RESULTS ............................................................................................................. 61 3.5_DISCUSSION ....................................................................................................... 66 3.6_CONCLUSIONS ................................................................................................... 68 3.7_REFERENCES …………………………………………………………………………………………………. 69 Chapter 4 VARIATIONS OF SALIVARY CORTISOL IN DOGS EXPOSED TO DIFFERENT COGNITIVE AND PHYSICAL ACTIVITIES ………………………… 73 4.1_ABSTRACT ……………………………………………………………………………………………………… 74 4.2_INTRODUCTION …………………………………………………………………………………………….. 75 4.3_MATERIALS AND METHODS ………………………………………………………………………….. 76 4.3.1_Recruitment of dogs ……………………………………………………………………….. 76 4.3.1.1_Study 1: Baseline value of salivary cortisol ……………………….. 76 4.3.1.2_Study 2: Variation of salivary cortisol during activity ………… 76 4.4_RESULTS ………………………………………………………………………………………………………… 81 4.4.1_Study 1: Baseline value of salivary cortisol ……………………………………… 81 4.4.2_Study 2: Variation of salivary cortisol during activity ………………………. 81 4.5_DISCUSSION ....................................................................................................... 84 4.5.1_Study 1: Baseline value of salivary cortisol ……………………………………… 84 4.5.2_Study 2: Variation of salivary cortisol during activity ………………………. 84 4.6_CONCLUSIONS ………………………………………………………………………………………………. 86 4.7_REFERENCES …………………………………………………………………………………………………. 87 Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT Part 2 NUTRIONAL EFFORTS, FAECAL MICROBIOME, HPA AXIS Chapter 5 PRELIMINARY STUDY: FAECAL MICROBIOME AS BIOINDICATOR OF DOG WELL-BEING AND POSSIBLE RELATION WITH HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS ........................... 91 5.1_ABSTRACT ……………………………………………………………………………………………………… 92 5.2_INTRODUCTION …………………………………………………………………………………………….. 93 5.3_MATERIALS AND METHODS ………………………………………………………………………….. 95 5.3.1_Animal selection .................................................................................. 95 5.3.2_Diet ...................................................................................................... 96 5.3.3_Experimental design ............................................................................ 97 5.3.4_Sample collection ................................................................................ 98 5.3.4.1_Faeces collection .................................................................. 98 5.3.4.2 Salivary sampling .................................................................. 98 5.3.5_Faeces analysis .................................................................................... 99 5.3.5.1_Faecal DNA extraction and sequencing ................................ 99 5.3.5.2_Faecal score, pH, nitrogen and fatty acids analysis .............. 99 5.3.6_Saliva analysis ..................................................................................... 101 5.3.7_Statistical Analysis .............................................................................. 101 5.4_RESULTS AND DISCUSSIONS .............................................................................. 103 5.4.1_Microbiome analysis ........................................................................... 103 5.4.2_Microbiome, SCFAs, lactate and nitrogen in faeces ........................... 112 5.4.3_Faecal microbiome and salivary cortisol ............................................ 116 Development of biomarkers in non-invasive biological matrices in order to assess dog well-being INDEX OF CONTENT 5.5_CONCLUSIONS ................................................................................................... 117 5.6_REFERENCES ....................................................................................................... 119 Part 3 BIOMARKERS OF DOG WELL-BEING DETECTED IN HAIR Chapter 6 PRELIMINARY STUDY: HEAVY METALS AND CORTISOL IN HAIR, EVALUATED AS POSSIBLE BIOMARKERS OF DOG WELL-BEING ........ 126 6.1_ABSTRACT ……………………………………………………………………………………………………… 127 6.2_INTRODUCTION …………………………………………………………………………………………….. 128 6.3_MATERIALS AND METHODS ………………………………………………………………………….. 131 6.3.1_Animal selection .................................................................................. 131 6.3.2_Diet ...................................................................................................... 131 6.3.3_Experimental design ............................................................................ 131 6.3.4_Sample collection ................................................................................ 132 6.3.4.1_Hair sampling ....................................................................... 132 6.3.5_Samples analysis ................................................................................. 132 6.3.5.1_Cortisol analysis .................................................................... 132 6.3.5.2_Heavy metal analysis ........................................................... 133 6.3.6_Statistical analysis .............................................................................. 134 6.4_RESULTS AND DISCUSSION