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Cd Marsia Miranda Phd Thesis Xxx Cycle Agriculture UNIVERSITÀ DEGLI STUDI DEL MOLISE Department of Agricultural, Environmental and Food Sciences International Ph.D Course in: AGRICULTURE TECHNOLOGY AND BIOTECHNOLOGY (C URRICULUM : Welfare, biotechnology and quality of animal production) (C YCLE XXX) Related disciplinary scientific section: AGR/20 (Zoocolture) PhD thesis Semen cryopreservation as a tool for preserving genetic biodiversity of avian and rabbit species: national cryobank launch Coordinator of the Ph.D Course: Prof. Giuseppe Maiorano Supervisor: Ph.D candidate: Prof. Nicolaia Iaffaldano Marsia Miranda-153768 ACADEMIC YEAR 2016/2017 INTRODUCTION The conservation of genetic diversity in livestock species can be related to biological, social, cultural and economic aspects. Animal genetic resources and animal management systems are an integral part of ecosystems and productive landscape in Italy, Europe and all over the world. Local breeds can be considered as a part of the history of some human populations as well as important materials from a scientific point of view. To date there is an urgent need of a specific conservation program for these breeds because of the limited number of their individuals and their risk of extinction. Although the local breeds are usually not competitive for production traits, they may carry valuable features such as disease resistance or distinctive product quality. The higher economic value of typical productions compared to conventional commercial products and the growing consumer preferences towards food quality, could strongly support the need to launch conservation programs of livestock biodiversity. In this regard, efficient in situ and ex situ conservation strategies are obligatory tools in order to implement an appropriate action for the conservation of livestock biodiversity. Conservation of genetic diversity across and within farm animal breeds is an insurance for the future. A broad genetic base is crucial to deal with future changes in environment, consumers demand for animal products and animal production systems. A wide genetic variation is also a safeguard against sanitary outbreak and natural disasters, in the maintenance of agro-ecosystem diversity and conservation of rural cultural diversity. The conservation of livestock variability is also a crucial element in order to preserve and valorize specific nutritional and nutraceutical properties of animal products. In situ conservation strategies are effective in reaching all these objectives except for the safeguard against emerging diseases, political instability and natural disaster. In contrast, ex situ conservation strategy is the method of choice to safeguard farm Animal Genetic Resources (AnGRs) against natural calamities and it is a valuable option when socio- economic, cultural and ecological values linked to a breed, are missing or of no interest. Therefore, the field of ex situ conservation is getting more attention with its wider application in the field of AnGRs management and conservation. To this aim, the cryopreservation technique is commonly used in the storage of genetic resources including oocytes, embryos, somatic cells, primordial germ cells (PGCs) and sperm. This technique offers important opportunities for the conservation and utilization of farm animal genetic I resources by the creation of a cryobank supported by government, institutional breeding organizations and private breeders. Some European countries such as France, The Netherlands and others, outside of Europe such as America and Japan, have been involved in specific conservation programs for AnGRs and include poultry (e.g. chicken and turkey) and rabbit breeds. Currently also in Italy there is an urgent need of national actions aiming at specific conservation programs for Italian poultry and rabbit breeds because of the limited number of their individuals becoming in risk of extinction. In the light of this, conservation and valorization projects of Italian poultry and rabbit breeds have been developing thanks to the financial support of regional and local public institutions. These projects are only based on the in situ conservation strategy. While recognizing that in situ strategy is a priority, the ideal conservation plan should consist in the integration of both strategies, in situ and ex situ (FAO, 2008; FAO, 2012). Regarding the ex situ in vitro technique, the semen cryopreservation represents a valuable tool to safeguard the genetic animal resources by cryobanks. This technology becomes particularly precious in poultry species for the reason that the semen cryopreservation in this species is the only reproductive procedure currently available because of the unique biological features of birds and in rabbit because cryopreserving sperm is easier and cheaper than obtaining embryos (Mocè and Vicente, 2009). In this regard, the present doctoral thesis aims at the identification of reference procedures for the cryopreservation of poultry (turkey and chicken) and rabbit semen according to the “FAO guidelines - Cryoconservation of animal genetic resources ” (2012). The reference procedures will be implemented for the constitution of a sperm cryobank of Italian, turkey, chicken and rabbit breeds, that are not available at national level right now. The following specific aims of the present doctoral thesis are reported in short below: 1. improvement of the cryopreservation success of both turkey (study 1) and chicken (study 2) semen packaged into straws; 2. development of effective protocol for rabbit semen (study 3) in order to reduce cryoinjuries during the freezing procedure and improve the sperm integrity after freezing/thawing II To my family III AKNOWLEDGEMENT The realization of this thesis was only possible due to the several people's collaboration, to which I desire to express my gratefulness. I would like to express my deepest gratitude to my supervisor, Prof. Nicolaia Iaffaldano for her guidance, patience, motivation, and support. I would like to express my gratitude to Dr. Michele Di Iorio for his help, encouragement, teaching and advice. I would like to thank also my Slovak advisor Prof. Peter Chrenek for giving me the possibility to work in his laboratory, at the Animal Production Research Centre (NPPC Nitra) and his collaborators Dr. Barbora Kulíková, Dr. Vaší čekYaromir and Dr. Andrej Baláži. I would like to thank the General Coordinator Prof. Giuseppe Maiorano for his accurate organization of the Ph.D. course. Many thanks to the president Dr. Michele Schiavitto of Italian National Rabbit Breeders (ANCI-AIA) for allowing me to use the Central Breeding Farm. I also thank Drs. Paola Principe for the technical assistance with semen collection and artificial insemination. I would like to thank also Prof. Luca Romagnoli for help with the statistical analysis of data, and Amber Burchell and Francesca Di Nezza for the English revision. Moreover, I would like also to thank all my PhD colleagues. I also thank all my friends, who supported me during these years. Finally, but not the least, I would like to thank my family, for their love, constant support and encouragement. IV EVALUATION REPORT OF DOCTORAL THESIS Title of the thesis Semen cryopreservation as a tool for preserving genetic biodiversity of avian and rabbit species: national cryobank launch Name of the PhD candidate Marsia Miranda Name and affiliation of the thesis supervisor NICOLAIA IAFFALDANO – University of Molise Name and affiliation of the referee PETER MASSANYI – Slovak University of Agriculture in Nitra, Slovak Republic Evaluation: Overall clarity and organization of the thesis Outstanding Very good Good Sufficient Poor Clarity ☒ ☐ ☐ ☐ ☐ Organization ☒ ☐ ☐ ☐ ☐ Comments: Generally the submitted PhD. thesis is very well prepared, all parts are clear and the overall impression is very positive. There are only some minor typo and grammar errors. I has to be state that this PhD. thesis of a very good quality, the review is very well prepared, aim of the study are clear and real, all methods are acceptable and giving significant results. The role of supervisor and coordinator of the course (thesis) should be very highly appreciated. Clarity of the Objectives Outstanding Very good Good Sufficient Poor ☒ ☐ ☐ ☐ ☐ Comments: Objectives of the PhD. thesis are clearly established, very actual and fully in the scope of the scientific section. V Novelty Outstanding Very good Good Sufficient Poor ☒ ☐ ☐ ☐ ☐ Comments: There are some new and very interesting outcomes that should be used in basic as well as applied science related to animal biology and biotechnology. Adequacy of the methods used and methodological rigor Outstanding Very good Good Sufficient Poor Adequacy of methods ☒ ☒ ☐ ☐ ☐ Methodological rigor ☒ ☐ ☐ ☐ ☐ Comments: Methods used in these studies are very actual and ensure significant results and possible clear conclusions. Relevance of Results and Conclusions Out Very good Good Sufficient Poor standing Results ☒ ☒ ☐ ☐ ☐ Conclusion ☒ ☐ ☐ ☒ ☐ Comments: Results are well prepared, clear and finely described. As all results were submitted to scientific journals, underwent serious revision and were accepted for publication and/or were published no additional questions are necessary. Contribution to the advancement of knowledge Outstanding Very good Good Sufficient Poor ☐ ☐ ☐ ☐ ☐ VI Comments: All results are very significant and very useful for the development of knowledge mainly in the field of animal biotechnology/physiology and animal production and reproduction. Potential impact and applicability of the results Outstanding
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