AGEING IN DOGS (CANIS FAMILIARIS) AND ITS RELATIONSHIP TO THEIR ENVIRONMENT LUÍSA MASCARENHAS LADEIA DUTRA PhD Thesis 2019 Ageing in dogs (Canis Familiaris) and its rela- tionship to their environment Luísa Mascarenhas Ladeia Dutra This thesis is presented to the School of Environmental & Life Science, University of Salford, in fulfilment of the requirements for the degree of Ph.D. Supervised by Professor Robert J. Young Co-supervised by Professor Jean Phillippe Boubli March 2019 Table of contents Chapter 1 General Introduction ......................................................................... 19 1.1 Animal Welfare Assessment ................................................................... 19 1.2 Evaluation of stress levels as a measure of animal welfare .................... 21 1.3 Telomeres as biological clocks ................................................................ 25 1.4 Apparent age as a measure of life experience ........................................ 29 1.5 Canids as a model group ........................................................................ 31 1.6 Thesis structure ....................................................................................... 32 Chapter 2 Validating the use of oral swabs for telomere length assessment in dogs 34 2.1 Introduction ............................................................................................. 34 2.2 Objective ................................................................................................. 37 2.3 Justification ............................................................................................. 37 2.4 Methods .................................................................................................. 38 2.4.1 Ethical statement: ............................................................................. 38 2.4.2 Samples ............................................................................................ 38 2.4.3 DNA extraction.................................................................................. 40 2.4.4 DNA quantification ............................................................................ 40 2.4.5 Primer validation ............................................................................... 41 2.4.6 qPCR assay ...................................................................................... 42 2.4.7 Measuring relative telomere length (RTL) ......................................... 44 2.4.8 Statistical Analysis ............................................................................ 44 2.5 Results .................................................................................................... 45 2.5.1 DNA quantity and quality .................................................................. 45 2.5.2 Swab and Dried Blood Spot rTL were significantly correlated .......... 46 i 2.6 Discussion ............................................................................................... 47 Chapter 3 Validating telomere length as a marker for animal welfare in domestic dogs 49 3.1 Introduction ............................................................................................. 49 3.2 Objectives ............................................................................................... 54 3.3 Justification ............................................................................................. 54 3.4 Methods .................................................................................................. 54 3.4.1 Subjects ............................................................................................ 54 3.4.2 Dog housing and husbandry ............................................................. 55 3.4.3 Collection of faeces: ......................................................................... 56 3.4.4 Hormone Extraction .......................................................................... 56 3.4.5 Enzyme Immunoassay ..................................................................... 57 3.4.6 Swab collection ................................................................................. 57 3.4.7 DNA extraction and quantification ..................................................... 57 3.4.8 qPCR assay ...................................................................................... 58 3.4.9 Measuring relative telomere length (RTL) ......................................... 58 3.4.10 Statistical analysis ......................................................................... 58 3.5 Results .................................................................................................... 59 3.5.1 Model 1: Factors that impact on a dog’s relative Telomere Length ... 59 3.5.2 Model 2: Factors that impact on a kennel dog’s relative Telomere Length 60 3.5.3 Model 3: Factors that impact on a laboratory dog’s relative Telomere Length 61 3.6 Discussion ............................................................................................... 63 Chapter 4 Evaluating dogs’ lives through relative Telomere Length (rTL) ......... 67 ii 4.1 Introduction ............................................................................................. 67 4.2 Objective ................................................................................................. 72 4.3 Justification ............................................................................................. 72 4.4 Methods .................................................................................................. 72 4.4.1 Ethical statement .............................................................................. 72 4.4.2 Subjects’ background ....................................................................... 73 4.4.3 Subjects ............................................................................................ 75 4.4.4 Swab collection ................................................................................. 82 4.4.5 DNA Extraction ................................................................................. 82 4.4.6 DNA Quantification ........................................................................... 82 4.4.7 qPCR assay ...................................................................................... 82 4.4.8 Measuring relative telomere length (RTL) ......................................... 83 4.4.9 Data collection and statistical analyses ............................................ 83 4.5 Results .................................................................................................... 86 4.5.1 DNA extraction kits - Pilot Study ....................................................... 86 4.5.2 Grouping Dogs.................................................................................. 87 4.5.3 Relative Telomere Length and Age in dogs’ groups ......................... 88 4.5.4 Association of rTL and Age ............................................................... 88 4.5.5 Model 1: Association of rTL and two-year-old dogs .......................... 92 4.5.6 Model 2: Association of rTL and dogs’ breed .................................... 94 4.5.7 Model 3: Factors that impact on a dog’s relative Telomere Length ... 95 4.5.8 Investigating significant factors from Model 3 ................................... 97 4.5.9 Model 4: Factors that impact on working dogs’ relative Telomere Length 102 4.5.10 Investigating significant factors from Model 4 .............................. 104 iii 4.6 Discussion ............................................................................................. 108 Chapter 5 Evaluating laboratory dogs’ relative Telomere Length over one year 115 5.1 Introduction ........................................................................................... 115 5.2 Objectives ............................................................................................. 118 5.3 Justification ........................................................................................... 118 5.4 Methods ................................................................................................ 119 5.4.1 Laboratory Dogs ............................................................................. 119 5.4.2 Dog housing and husbandry from 2017 and 2018 .......................... 120 5.4.3 Swab collection ............................................................................... 121 5.4.4 DNA extraction and quantification ................................................... 121 5.4.5 qPCR assay .................................................................................... 121 5.4.6 Measuring relative telomere length (RTL) ....................................... 122 5.4.7 Statistical Analysis .......................................................................... 122 5.5 Results: ................................................................................................. 122 5.5.1 Descriptive statistics for years 17 and 18 ....................................... 122 5.5.2 Swab rTL were significantly different .............................................. 123 5.6 Discussion ............................................................................................. 125 Chapter 6 Evaluating Dogs’ Welfare through ‘apparent age’ ........................... 127 6.1 Introduction ........................................................................................... 127 6.2 Objectives ............................................................................................