EFFECTS OF CONTROLLED BREEDING ON CERVICOVAGINAL MUCUS AND FERTILITY OF THE MERINO EWE Jessie Ward Maddison A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Faculty of Veterinary Science, The University of Sydney © 2017 Declaration Apart from the assistance mentioned in the acknowledgements, the studies contained within this thesis were planned and executed by the author, and have not been previously submitted for any degree to a University or Institution. Jessie Maddison B An Vet Bio Sci (Hon I) Author Attribution Statement Chapter 2 of this thesis is published as Maddison, JW, Rickard, JP, Mooney, E, Bernecic, NC, Soleilhavoup, C, Tsikis, G, Druart, X, Leahy, T & de Graaf, SP 2016, 'Oestrus synchronisation and superovulation alter the production and biochemical constituents of ovine cervicovaginal mucus', Animal Reproduction Science, vol. 172, no., pp. 114-122. I assisted in the design of the study, carried out the experiments, analysed the data and wrote the drafts of the MS. Chapter 3 of this these is published as Maddison, JW, Rickard, JP, Bernecic, NC, Tsikis, G, Soleilhavoup, C, Labas, V, Combes-Soia, L, Harichaux, G, Druart, X, Leahy, T & de Graaf, SP 2017, 'Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe', Journal of Proteomics, vol. 155, no., pp. 1-10. I assisted in the design of the study, carried out the experiments, analysed the data and wrote the drafts of the MS. i Acknowledgements To Simon, Tamara, Xavier and Jess, the supervisor team of the century, thank you all for your support, guidance, patience and encouragement throughout this journey. Simon, thank you, I cannot express how grateful I am for the opportunity you have given me by inviting me into the repro group and setting me on this crazy wonderful experience, I will be forever grateful for the opportunity and I hope that you can be proud of the work we accomplished. Although I feel I may always be, as you put it…a chicken little…this amazing PhD ride has forever changed me, my confidence in myself as well as directed my journey through life, so I must thank you for what it has brought me, and will continue to do, throughout my life. Tamara, your editing skills, stickers, supportive comments and solutions to sticky experimental issues, of which there have been many, have helped me so much. You brought a new life to the group and I am so glad to have had your support. Xavier, thank you for welcoming me into your lab in France, as well as your home, not once but twice, I had such an amazing time with you all at INRA and appreciate all your patience with me and showing me the French way! Coffees, food, comics, it was an absolute blast, even if all our westerns will never see the light of day!! Jess, what can I say, you have been such a big part of this journey, from being the honours boss till now, my supervisor. You have helped me with it all and I really treasure the friendship we have developed over these last 5 years. Thanks for your words of wisdom, encouragement, and your unwavering support and belief in me, all the laughs, and a few crazy sleep deprived tears too, Daft Punk will forever remind me of mucus and you # Team Ram!!. To the other PhD students and now Drs, Danielle and Cassie, thank you all so much for welcoming me into the fold in those early years, you made the transition from honours to PhD ii life much more enjoyable and manageable with all your wise words and anal lab tendencies!! Which I will proudly carry on into my new work adventures. To the new kids in town, Dannielle, Cameron, Taylor and even my own young padawan Naomi, it took a while to adjust to the new office situation but I am so thankful for these last years with you all, our 2:30 pm life chats and animal video distractions will be missed. To my two honours students; Ethan and Naomi, it was a ride and thank you both for all your hard work, we did good kids. Thank you all for being willing to drop it all and help me collect mucus, wrangle sheep and make lamb babies across the countryside, rain, hail or shine, and at all hours of the day too, I couldn’t have done it without you all. To the rest of the repro team, Ros and Angela, thank you for all the wise words from down the hall, they will be missed!! To the Camden boys, past and present, Byron, Keith, Sam and Cameron, thank you for being so accommodating with my requests, you made my life so much easier and I appreciate you all going above and beyond for me time and time again. To the lab veterans, Andrew and Kim, thank you both for all your help and tolerance, of yet another PhD student to come in and take over the lab, your knowledge and skill were crucial over the years for me in many situations. Kim your countless hours running bloods for me, answering all manner of questions and hallway chats have helped me get through these years, so a special thanks to you! To the French boys, Guilluame and Clement, you were both so welcoming and helpful during both of my INRA visits, I have many fond memories of my time there, I dedicate all my westerns to you both! A special thankyou has to go out to my Team Ram buddies….You know who you are. We started as an awesome foursome and have grown to the dominating force it is today, you are a special bunch of people and I am very proud to have been a part of it all with you. iii Last but by no means least, I would like to thank my friends and family for all their support throughout my PhD, especially to Brad and my father Anthony. Bradley James, thank you for supporting and encouraging me throughout this crazy ride, even if it meant me occasionally checking out of our life, not making numerous occasions of climbing and weekend frivolities, your love has helped me so much the last few years, muchos gracias mi amore. Pa, my rock, thank you for the numerous chats, cries, laughs, pep talks and also for just caring about me and all of this so so much, to have you backing me through this whole uni journey of mine has been immeasurably important to me and I am so very grateful that you are my dad. I couldn’t have done it, any of it without you..smashalacken is complete!! You have both lived this mucus-laden journey from the get go and we can celebrate its completion together!! iv Summary This thesis examines the impact of oestrus synchronisation and superovulation on the cervicovaginal (CV) mucus of ewes during their oestrous cycle. The quantity, biochemistry and protein composition of ovine CV mucus produced under these controlled breeding regimes are investigated. Artificial methods of mucus modification (in vitro) and their impact on sperm interaction with this important biological fluid are also examined. The abundance, composition, physiochemical characteristics and proteome of cervicovaginal mucus collected during oestrus and the luteal phase were compared between naturally cycling, progesterone synchronised, superovulated and prostaglandin-F2α synchronised Merino ewes. Oestrus CV mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus. Superovulation resulted in a marked increase in CV mucus production and total protein concentration while synchronisation using Prostandin-F2a significantly reduced mucus volume. Contrary to popular theory, mucus pH (oestrus 6.2-6.5), biochemical profile and penetration by spermatozoa were largely unchanged by superovulation and/or oestrus synchronisation. Quantitative and qualitative proteomic analysis of mucus identified 60 proteins to be more abundant during oestrus, whilst 127 were more abundant during the luteal phase. Furthermore, specific proteins were found to be only present during oestrus (27 proteins; one example being the mucolytic enzyme neuraminidase (NEU1)) or the luteal phase (40 proteins). Superovulation and/or oestrus synchronisation also greatly altered the proteome of CV mucus increasing and decreasing the abundance of a variety of proteins during oestrus when compared to CV mucus obtained during the oestrus of naturally cycling ewes. This study represents the most comprehensive proteomic description of the changes to cervicovaginal v mucus of the ewe both over the oestrous cycle and the first to detail the effects of controlled breeding practices on the CV mucus proteome. The effects of proteomic changes of key structural mucus proteins was investigated in an in vitro setting to ascertain if such changes might influence sperm motility and viability when incubated in a cervicovaginal mucus simulant (CMS). Specifically, variation in the percent composition of mucin, the gel forming protein which provides mucus with its structural characteristics, and variation in the amount of the enzyme neuraminidase (NEU1) which acts to denature mucin proteins and alter the gel structure of mucus, was investigated. Increasing mucin levels of a CMS caused increased mucus viscosity, but interestingly did not affect sperm motility, and resulted in more frozen-thawed spermatozoa migrating through CMS. The inclusion of mucin into CMS resulted in altered sperm velocity, but not always in a dose dependent manner. Spermatozoa had reduced membrane damage and reacted acrosomes when incubated in CMS with 2% or 4% mucins, or CMS pre-treated with NEU1, compared to CMS containing no NEU1 or mucins. These results show that variable mucin and neuraminidase concentrations alters the viscosity of a cervicovaginal mucus simulant, the motility and viability of spermatozoa, and the mucus migrating ability of frozen-thawed spermatozoa.