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Further Approaches on Sperm Cryopreservation Protocol Towards the Establishment of the Sperm Bank from Endangered Native Catalan Ram Breeds (Xisqueta and Aranesa)

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Further Approaches on Sperm Cryopreservation Protocol Towards the Establishment of the Sperm Bank from Endangered Native Catalan Ram Breeds (Xisqueta and Aranesa) ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. 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 Further approaches on sperm cryopreservation protocol towards the establishment of the sperm bank from endangered native Catalan ram breeds (Xisqueta and Aranesa) Uchebuchi Ike Osuagwuh Doctoral Thesis Facultat de Veterinària Departament de Medicina i Cirurgia Animal Universitat Autònoma de Barcelona 2017 Dra. María Jesús Palomo Peiró, Profesora Titular del Departament de Medicina i Cirurgia Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona Certifica: Que la tesis titulada “Further approaches on sperm cryopreservation protocol towards the establishment of the sperm bank from endangered native Catalona ram breeds (Xisqueta and Aranesa)”, presentada por Uchebuchi Ike Osuagwuh para optar al grado de Doctor en Veterinaria se ha realizado bajo mi direccion y, considerándola acabada, autorizo su presentación para que sea juzgada por la comisión correspondiente. Bellaterra (Cerdanyola del Vallés), Septiembre de 2017. Dra. María Jesús Palomo Peiró Sr. Uchebuchi Ike Osuagwuh Directora Candidato TABLE OF CONTENT SUMMARY RESUMEN CHAPTER I. INTRODUCTION 1 CHAPTER II. OBJECTIVES 5 CHAPTER III. LITERATURE REVIEW 9 3.1 Factors influencing sperm production and quality on rams 11 3.1.1 Donor Age 11 3.1.2 Size of testis 12 3.1.3 Method of collection 12 3.1.4 Breed 12 3.1.5 Season and environment 13 3.2 Semen cryobank and sperm cryopreservation factors 14 3.2.1 Specie and individual male factor 15 3.2.2 Semen collection method 16 3.2.3 Seminal plasma effect 16 3.2.4 Season 17 3.2.5 Freezing extender and cryoprotectant 18 3.3.6 Refrigeration 19 3.3.7 Freezing and thawing rates 20 3.3 Semen Analysis 22 3.3.1 Evaluation of Sperm Motility 22 3.3.2 Sperm analysis through flow cytometry 24 3.3.3 Analysis of the sperm plasma membrane integrity 25 3.3.4 Analysis of mitochondrial functionality 26 3.3.5 Analysis of acrosomal integrity 26 3.3.6 Oxidative stress analysis 27 3.3.7 Analysis of free-cysteine residues in sperm nucleoproteins 28 3.3.8 Thermal resistance test 29 CHAPTER IV. RESULTS 31 Study 1. Influence of melatonin implantation during a non-breeding season 33 to semen donor rams on semen parameters held for 24h at 5ºC Study 2. A Comparison of Semen Collection Methods (electroejaculation 55 vs. artificial vagina) on the Quality of Pre- and Post-thawed Aranesa Ram Spermatozoa Study 3. Effect of semen washing on thawed ram spermatozoa subjected to 75 a four hours post-thawing thermal evaluation test Study 4. Assessment of post-thawed (washed vs. unwashed) ram sperm 83 quality after incubation with autologous whole seminal plasma obtained by electroejaculation and artificial vagina CHAPTER V. GENERAL DISCUSSION 101 CHAPTER VI. CONCLUSSIONS 111 CHAPTER VII. REFERENCES 115 SUMMARY The new interest towards the characterization and conservation of Aranesa and Xisqueta ram breeds was constituted by ex situ programs using assisted reproductive techniques (ARTs) such as semen cryopreservation and the development of sperm banks. Therefore, the most commonly used application in the development of sperm banks are semen collection and freezing, and this requires the knowledge of the reproductive physiology for these species. Besides semen collection and freezing, its utilization towards artificial insemination (AI) as well as the suitable assisted reproductive technologies is also a key aspect in order to prevent the disappearance of these breeds and maintain their genetic diversity. However, there are still concerns due to issues regarding frozen-thawed sperm quality. With the present technological advances made in recent years on sperm evaluating techniques such as the use of computer-assisted sperm analysis (CASA) and flow cytometry, a wealth of information on their sperm characteristics may be achieved. Furthermore, as the application of refrigerated and frozen-thawed semen technology increases, more information on sperm quality and advances are required. To this regard, the general aim of this thesis was to provide some basic information on Aranesa and Xisqueta sperm processes for cryopreservation and to determine the efficacy of some strategies towards improving post-thawed sperm quality prior to testing its usefulness for further application. In this doctoral thesis, we evaluated the seasonal effect and the melatonin implantation on scrotal circumference, time of semen emission and characteristics of fresh ram ejaculates collected by electroejaculation. In addition, the sperm qualitative characteristics stored at 50C and held for 24h in a cryopreservation media from the various treated males during non- breeding was determined, and directly compared with same parameters from a breeding season. The goal was to provide some useful information on holding spermatozoa below body temperature to various time periods prior to cryopreservation or as an alternative to frozen-thawed semen for AI. In this study, semen was collected (n=8) from ten rams (Aranesa and Xisqueta breeds) via electro-ejaculation during the non-breeding season; five males were implanted with Melovine®, while the other five rams remained untreated. Thereafter, semen was collected from six rams (n=8) during the breeding season. Results from this study showed no difference in any sperm parameter between melatonin treated and breeding season groups but both groups differed significantly (P < 0.05) when compared with the untreated/non-breeding semen donor group except on scrotal circumference. Also, despite all parameters being negatively influenced by the chilled liquid storage time, melatonin treated group showed better sperm quality parameters when compared to other groups. This study demonstrates that melatonin implantation during the non-breeding season improved almost all qualitative sperm parameters studied during chilled liquid semen storage. This thesis also addresses the efficacy of collection methods electroejaculation (EE) vs. artificial vagina (AV)) on fresh and post-thawed sperm quality of Aranesa ram sperm towards the establishment of a sperm bank. The aim being that for the successful sperm cryobank establishment towards conservation, information on semen recovery methods on pre- and post-thawed sperm quality is essential. This may lead to rapid constitution of the sperm bank especially from a large number of untrained males within a short period of time. Results in this study on fresh semen showed no significant difference between methods except for volume, concentration, total sperm count and functional sperm membrane integrity. Motility parameter was significantly higher in EE samples than those obtained via AV in pre-freeze samples but none was observed after thawing. Post-thawed samples evaluated by flow cytometry showed that semen sample obtained by EE had lower percentage of viable cells with damaged acrosome and active mitochondria (cryo- capacitation) and total acrosome damage than those via AV. Results herein supports the inclusion of EE method as a viable alternative and quicker method for semen collection towards the ongoing conservation program and establishment of Aranesa sperm cryobank. Furthermore, in this thesis, we evaluated the sperm motility and qualitative characteristics of ram spermatozoa cryopreserved with or without seminal plasma under a controlled condition and subjected to a 4 h post-thawing thermal evaluation test. The aim being that exposing these frozen-thawed sperm to thermal resistance test will provide some knowledge on their survivability in the female genital tract towards fertilizing the ovum. This was achieved by collecting semen from 5 males (5 years old) via artificial vagina, and split into two aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600 × g for 10 min, while the other aliquot was kept unwashed. Thereafter, washed and unwashed sperm were extended in a TCG-based powdered egg yolk media and frozen. Frozen sperm were thawed and incubated at 37oC for 4 h. Results from this work showed that unwashed samples generally had better results than washed samples as regards to sperm motility characteristics irrespective of incubation. Also, post-thawing incubation time had a significant effect on acrosome integrity and mitochondria functionality irrespective of sperm treatment. This study demonstrates that the presence of seminal plasma prior to cryopreservation was beneficial in maintaining post thawed sperm motility, and as such, may be useful for ex situ ram sperm cryopreservation towards its use for artificial insemination. The addition of seminal plasma to frozen-thawed sperm has been suggested as an alternative measure to improving its sperm quality. Therefore, we evaluated the effect of removing seminal plasma and freezing media by washing via centrifugation and subsequently supplementing with autologous whole seminal plasma (20%) collected by EE from a non- breeding and breeding season or by AV from a breeding
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