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This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. A study of an anti-GnRF vaccine as a more welfare friendly method of castration for ram lambs Katarzyna Masłowska A Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Edinburgh 2016 1 To my grandma, parents and husband 1 Declaration I declare that I have undertaken and written the present thesis. This thesis is presenting my own work. Any assistance has been duly acknowledged. The work described has not been submitted for any other degree or professional qualification with exception of the Qualitative Behavioural Assessment study which has been carried out by Fabiana Mizzoni as part of her MSc thesis under supervision of myself and Prof Francoise Wemesfelder. Katarzyna Masłowska 2 Acknowledgements First of all I would like to thank my main supervisors Prof Cathy M Dwyer and Dr Kenny M Rutherford for their ongoing support, expertise, patience and encouragement. I am very grateful that I was given this once in a life time opportunity to share their passion of animal welfare and reduction of unnecessary suffering, distress and pain in farm animals. I am honoured that I could work by their sides during the progression of my PhD. They have provided me with advice and inspiration during the course of my study, helping me to stay on track and achieve set goals. I developed myself professionally and improved my research skills. I was also able to advance my ability to write and speak in public while presenting my achievements. They have accompanied me throughout various scientific and student conferences coaching me in my professional development. There are not enough words to express my gratitude so I will just say simply, thank you! I would like to express my very great appreciation to Prof Eddie Clutton who was my third supervisor. His assistance and involvement in the development and application of anaesthesia protocol was invaluable. He also provided me with continuous support, expertise and advice throughout the course of my PhD in various aspects of my study and development of my research skills and interests. I would like to offer my special thanks to Mrs Eurife Abadin who was my industrial supervisor. She had a very difficult role of being the mediator between the scientific and commercial site of my study which I believe may have been challenging at times. I am very grateful for her patience, advice and expertise, especially in the subject of testosterone analysis. I wish to express my appreciation for organizing the training of testosterone concentration analysis (ELISA) giving me the opportunity to develop lab skills. 3 I would like to extend my gratitude to the staff of Zoetis branch at 45 Poplar Rd., Parkville, Victoria 3052 Melbourne, Australia; who have analysed blood samples for total serum concentration of testosterone. I wish to acknowledge financial support of the Scottish Government and Zoetis. Without their contribution I wouldn’t have been able to perform this study. I would like also to acknowledge Zoetis, The Worshipful Company of Woolman and Birrel-Grey scholarship for their input towards travel funds which allowed me to attend the following scientific conferences: The British Society of Animal Science, Nottingham, United Kingdom, April 2013, The 47th ISAE conference, Florianopolis, Brazil, June 2013 and The 48th ISAE conference, Victoria-Gasteiz, Spain, July 2014. I am particularly grateful to Zoetis for the provision of a novel anti-GnRF vaccine which was the main focus of my study; furthermore I wish to acknowledge Zoetis for the provision of Simcro Autovaccinator which allowed for the quick, sufficient and safe vaccination of lambs during the course of the study. Assistance provided by Prof Francoise Wemelsfelder in QBA preparation, performance and analysis of the results is greatly appreciated. I wish to acknowledge the help provided by MSc student Fabiana Mizzoni who carried out Qualitative Behavioural Analysis (QBA) as part of her dissertation and evaluated the results. Advice and practical training given by Prof Vince Molony has been of a great help in development and application of the best practice during traditional castration and tail docking procedures. I am particularly grateful for the assistance given by Prof Sheryl Ashworth, Charis Hogg and Robert Fleming in the testes tissue collection and analysis. I would like to thank following staff of the Bio Support Unit at the University of Nottingham Sutton Bonington Campus for their assistance with the collection of meat quality and carcass conformation data: Dave Edwards, Deborah Surgay, Richard Plant; Michael Baker, John Corbett and Collin Ward. 4 Mr Alistair Macrae and Prof Natalie Warren as members of R(D)SVS Postgraduate Studies Thesis Committee provided me with very valuable comments, guidance and help to set up appropriate direction and prioritise the work throughout time of my PhD for which I am very grateful. I would like to acknowledge all of my colleagues form SRUC for their help in data collection. My special thanks are going to Jo Donbavand who was the NACWO assigned to my project and provided assistance in all aspects of animal care, handling as well as data collection. I am very grateful to Agnieszka Futro and Lucille Bellegarde for their priceless help during my first lambing, facial expression recordings, castration, tail docking, ear tagging, blood, weights and scrotal measurements collection as well as vaccination and plasma handling. Support and training given by Marianne Farish, and Kirstin McIlvaney during blood sampling, video-recording and solving issues related to behavioural scoring in Observer XT program is gratefully acknowledged. I am also indebted to Mhairi Jack, Eilidh Baker, Susan Richmond and Charlotte George for their help with blood, weights and scrotal measurements collection as well as anxiety behaviours recordings. Thanks also to Neil Clelland, Kirsty McLean and John Skelton who have helped with CT scanning and meat quality data collection. I wish to acknowledge Dr Sarah Hall and Ms Jenny Coe for their assistance and training in ELISA tests for the measure of serum cortisol concentration as well as results transformation and analysis. I wish to express my appreciation to colleagues and friends who have offered their assistance and helped in data collection in times when no other support was available. Firstly I would like to direct my thanks to Jan Woźny for his ongoing assistance in behavioural observations, anxiety and sexual behaviour recordings, lambing, feeding rams and ewes as well as cleaning pens and construction of pens for specific tests, induction of ewes into oestrus, blood, weights and scrotal measurements collection as well as vaccination and plasma handling. My exceptional gratitude goes to Michał Duszyński for his help in handling and feeding rams during sexual behaviour recordings as well as his assistance in management of technical issues related with video-tapes, 5 memory discs, cameras etc. I would like also to thank Urszula Luckiweicz, Carol Thompson, Piotr Mierzejewski and Magdalena Mierzejewska for their help with blood, weights and scrotal measurements collection as well as vaccination and plasma handling. My special thanks are extended to the staff of Woodhouselee and Easter Howgate Farm: Ralph Seaton who has covered all aspects of standard sheep management practices and handling, health checks, gathers, vaccinations, sheering and feeding etc., Scott Grey, Laura Nicoll, and Alex Moir for their help during lambing and lambs allocation to treatment groups. At the end I would like to thank all of my family and friends. Very special thanks are going to my grandma, parents, husband and mother in law. Their unconditional belief in my abilities was inspiring for me, gave me strength and will to achieve my goals on time and to the best standards. To all of you I wish to say thank you! 6 Conference abstracts K. Masłowska, C.M Dwyer. (2012). The Effect of immunization against gonadotropin- releasing factor (GnRF) on circulating testosterone and the development of sexual behaviour in ram lambs. British Society of Animal Science, Nottingham, United Kingdom, April 2012, p113. K. Masłowska, A. Futro, C.M Dwyer. (2013). Ewes direct more maternal attention toward lambs that express the most severe pain-related responses. 47th ISAE conference, Florianopolis, Brazil, June 2013, p97. K. Masłowska, E Abadin, C.M Dwyer. (2014). Influence of castration method and sex on lamb behaviour, the development of ewe-lamb bonding and stress responses. 48th ISAE conference, Victora-Gaistez, Spain, July-August 2014, p125. 7 List of Tables Chapter 1 General Introduction ...............................................................................................