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Ageing in Dogs (Canis Familiaris) and Its Relationship to Their Environment

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Ageing in Dogs (Canis Familiaris) and Its Relationship to Their Environment 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 ............................................................................................
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