Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN SCIENZE VETERINARIE Ciclo XXXIII Settore Concorsuale: 05/E3 Settore Scientifico Disciplinare: BIO/12 GIRAFFE’S URINE: FROM URINALYSIS TO PROTEOMICS AND METABOLOMICS Presentata da Dr.ssa Sabrina Fasoli Coordinatore Dottorato Supervisore Prof. Arcangelo Gentile Prof.ssa Giulia Andreani Co-supervisore Prof.ssa Gloria Isani Esame finale anno 2021 Table of contents ABSTRACT ............................................................................................................... 5 LITERATURE REVIEW ...................................................................................... 6 1. Urine of non-domestic species ...................................................................................... 6 1.1. Urinalysis ........................................................................................................................... 6 1.1.1. Physical and chemical evaluation of urines ................................................................. 8 1.1.2. Urine specific gravity ...................................................................................................... 9 1.1.3. Urine microscopic sediment evaluation ....................................................................... 9 1.2. Urine protein:creatinine ratio ...................................................................................... 10 1.3. Urine proteomics .......................................................................................................... 11 1.4. Urine metaBolomics ..................................................................................................... 13 2. Giraffe ............................................................................................................................ 14 2.1. Giraffe and zoo animal welfare .................................................................................. 14 2.2. Kidneys and urine values of giraffes .......................................................................... 17 2.2.1. Anatomy ......................................................................................................................... 19 2.2.2. Physiology ...................................................................................................................... 21 2.2.3. Urine ............................................................................................................................... 22 3. Aims and OBJectives .................................................................................................... 23 EXPERIMENT 1 ................................................................................................... 25 Introduction ................................................................................................................. 26 Material and methods .................................................................................................. 28 1. Urine collection ............................................................................................................. 28 2. Urinalysis ........................................................................................................................ 28 3. Urine total protein and urine creatinine .................................................................... 30 3.1. Urine total protein ........................................................................................................ 30 3.2. Urine creatinine ............................................................................................................. 31 4. 1D-SDS-PAGE electrophoresis ................................................................................ 31 5. Statistical analysis .......................................................................................................... 32 Results ......................................................................................................................... 33 i 1. Cows ............................................................................................................................... 33 2. Giraffe ............................................................................................................................ 36 Discussion ................................................................................................................... 38 Conclusions .................................................................................................................. 39 EXPERIMENT 2 ................................................................................................... 40 Introduction ................................................................................................................. 41 Material and methods .................................................................................................. 43 1. Animal studied .............................................................................................................. 43 2. Selection of reference individuals .............................................................................. 45 3. Pre-analytical procedures ............................................................................................. 45 4. Analytical procedures ................................................................................................... 46 4.1 Urinalysis ........................................................................................................................ 46 4.2 Urine total protein and creatinine determination .................................................... 48 4.3 1D-SDS-PAGE electrophoresis ................................................................................ 48 4.4 Protein identification By mass spectrometry ............................................................ 48 5. Statistical analysis .......................................................................................................... 49 Results ......................................................................................................................... 51 1. Animal studied .............................................................................................................. 51 2. Selection of reference individuals .............................................................................. 52 3. Urinalysis ........................................................................................................................ 53 3.1. Physical-chemical urine evaluation ............................................................................ 53 3.2. Urine microscopic sediment evaluation .................................................................... 53 4. Reference intervals ....................................................................................................... 54 4.1. Sex and age .................................................................................................................... 56 5. 1D-SDS-PAGE electrophoresis ................................................................................ 59 5.1. Band NumBer ............................................................................................................... 61 5.2. Molecular Mass protein Bands .................................................................................... 62 5.2.1. Sex ................................................................................................................................... 66 5.2.2. Age .................................................................................................................................. 67 6. Protein identification by Mass Spectrometry ........................................................... 69 ii Discussion ................................................................................................................... 74 1. Reference intervals ....................................................................................................... 74 2. Urinalysis and urinary reference values ..................................................................... 74 3. Urinary proteome ......................................................................................................... 76 3.1. 1D-SDS-PAGE electrophoresis ................................................................................ 76 3.2. Protein identification By mass spectrometry ............................................................ 77 3.2.1. Acidic mammalian chitinase ....................................................................................... 79 3.2.2. Actin cytoplasmic 1 ...................................................................................................... 79 3.2.3. Alpha-1B-glycoprotein ................................................................................................ 80 3.2.4. Apolipoprotein D ......................................................................................................... 80 3.2.5. Cathelicidin-1 ................................................................................................................ 81 3.2.6. Clusterin ......................................................................................................................... 82 3.2.7. DeoxyriBonuclease-1 .................................................................................................... 82 3.2.8. HaptogloBin .................................................................................................................