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The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Computerised analysis of semen in equids Sabrina Gacem Equine Reproduction Service Department of Animal Medicine and Surgery Faculty of Veterinary Sciences, Universitat Autònoma Barcelona, Spain PhD Thesis supervised by Dr. Jordi Miró Roig Dr. Carles Soler Vázquez Jordi Miró Roig, Profesor Titular del Departamento de Medicina y Cirugía Animal de la Universidad Autónoma de Barcelona, y Carles Soler Vázquez, Professor Titular del Departament de Biologia Celular, Biologia Funcional i Antropologia Física de la Facultat de Ciències Biològiques de la Universitat de València. CERTIFICAN: Que la tesis titulada: “Computerised analysis of semen in equids.” presentada por Sabrina Gacem para optar por el grado de Doctor en Medicina y Sanidad Animales por la Universidad Autónoma de Barcelona, se ha realizado bajo nuestra dirección y, considerándola terminada y cumpliendo los requisitos para poder optar por la Mención Internacional, autorizamos su presentación para ser juzgada por la comisión correspondiente. Y, para que así conste a los efectos oportunos, firman este documento Dr. Jordi Miró Roig Dr. Carles Soler Vázquez, Bellaterra (Cerdanyola del Vallès), 18 de diciembre del 2020. 3 A mon ange Zahraa A vous mes très chers parents, et à ma Fatma « Le poisson rouge ne peut ramener la complexité des océans à la quiétude de son bocal ». Yasmina Khadra, Index Acknowledgements ..................................................................................................................................... 11 Abbreviations .............................................................................................................................................. 13 Abstract ....................................................................................................................................................... 15 Introduction ................................................................................................................................................. 23 1.1. How the sperm is generated and shaped? ................................................................................. 23 1.1.1. Development of male gonads ............................................................................................. 23 1.1.2. Spermatozoa formation ...................................................................................................... 25 1.1.3. Ejaculate .............................................................................................................................. 29 1.1.4. Sperm structure .................................................................................................................. 29 1.1.5. Sperm morpho-abnormalities ............................................................................................. 32 1.2. How do the sperm move? ........................................................................................................... 35 1.2.1. Sperm transport .................................................................................................................. 35 1.3. Breeding soundness examination and semen analysis. .............................................................. 37 1.3.1. Ultrasonography of reproductive tract ............................................................................... 38 1.3.2. Computer Assisted Semen Analysis of motility (CASA-Mot)............................................... 39 1.3.2.1. Computer semen analysis and fertility ........................................................................... 41 1.3.2.2. Technical conditions and limitations of CASA technology .............................................. 42 1.3.3. Automated sperm morphology analysis (ASMA) ................................................................ 44 1.3.3.1. Trumorph® technique ..................................................................................................... 46 1.3.4. Sperm subpopulation concept ............................................................................................ 47 Aims and objectives .................................................................................................................................... 49 2.2. Overview of thesis parts ............................................................................................................. 50 2.2. Research output .......................................................................................................................... 52 2.2.1. Publications .............................................................................................................................. 52 2.2.2. Conference proceedings ........................................................................................................... 52 Manuscripts ................................................................................................................................................ 54 1. Examination of a jackass (Equus asinus) accessory sex glands by B‐mode ultrasound and of testicular artery blood flow by colour pulsed‐wave Doppler ultrasound: Correlations with semen production. .......................................................................................................................................... 55 2. Optimal frame rate when there were stallion sperm motility evaluations and determinations for kinematic variables using CASA-Mot analysis in different counting chambers .................................. 63 5 3. Optimization of CASA-Mot Analysis of Donkey Sperm: Optimum Frame Rate and Values of Kinematic Variables for Different Counting Chamber and Fields ........................................................ 74 4. A new approach of sperm motility subpopulation structure in stallion and donkey ..................... 89 5. Equine sperm morphology analysis: alive vs dead stained sperms technique ............................ 105 6. Atlas of sperm morpho-abnormalities using Trumorph® in equids ............................................. 117 Discussion.................................................................................................................................................. 133 Conclusions ............................................................................................................................................... 140 References ................................................................................................................................................ 141 List of tables Introduction Table 1: Comparison of testis parameters in sexually mature donkeys and horses (a: Neves et al., 2014; b: Swiestra et al., 1974). .................................................................................................................................. 26 Manuscript 1 Table 1: Sperm motility descriptors and the CASA settings used. Table 2: Ultrasound measurements of the Catalonian donkey accessory sex glands. Table 3: Testicular artery blood flow at the pampiniform plexus (PPP), in the supratesticular rea (ST) and at the level of the capsular artery (CA). Table 4: Results for CASA variables. Table 5: Kendal correlations τ (p < 0.05) between testicular artery blood flow variables and those of the accessory sex glands/sperm analysis variables at different points along the artery. Manuscript 2 Table 1: Determining the optimal frame rate to be used for stallion sperm when there were evaluations using different counting chamber types and depths; One ejaculate from 11 stallions was analysed using each chamber; Curvilineal velocity of stallion sperm for the optimal frame rate (VCLα) and with different frames rates 25, 50,100,150, 200 and 250 were calculated based on α and β values. Table 2: Motility characteristics (mean ± SD) of stallion spermatozoa determined using the CASA system utilising the different counting chambers at 250 f/s. Table 3: Values for stallion sperm kinematic variables in different chamber fields of the Spermtrack chambers at 250 f/s (mean ± SD). Table 4: Values for stallion sperm kinematic variables in different chamber fields of the D4C chambers at 250 f/s (mean ± SD). Table 5: Values for stallion sperm kinematic variables in different chamber fields of the D4CL20 chamber at 250 f/s (mean ± SD). Manuscript 3 Table 1: Optimum frame rate calculated to have the threshold level for different chambers types and depths. Curvilineal velocity of donkey sperm for the optimal frame rate (VCL. μm/s) and with different frames rates (25–250) calculated based on α and β values. Table 2: Motility parameters (mean ± SEM) of donkey spermatozoa determined by CASA system using disposable and reusable chambers obtained at 250 frames. Table 3: CASA motility parameters (mean ± SEM) in different fields of disposable chamber ISAS®D4C10 and 20 µm depth. 7 Table 4: CASA motility parameters (mean±SEM) in different fields of disposable chamber ISAS®D4C20L.