Ovarian hormones AMH and E2 in juvenile gilts as markers of reproductive success Alicia Steel BAnVetBioSci (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sydney Sydney School of Veterinary Science Faculty of Science 2019 Page 1 of 213 Declaration This thesis is submitted to The University of Sydney in fulfilment of the requirements for the Degree of Doctor of Philosophy. The work presented in this thesis is original except as acknowledged in the text. I hereby declare that I have not submitted this material, in either full or in part, for a degree at this or any other university. Signature: Date: 28/02/19 Alicia Steel BAnVetBioSci (Hons) Page 2 of 213 Acknowledgements Chris Grupen –You have made this PhD so enjoyable and easy-going. I really can’t tell you how much I appreciate your support and understanding. Thank you for your guidance and knowledgeable input into this project and thesis. I greatly appreciate every minute you took to help me through this! …. Annnd I’m sorry for all those times I pretended everything was no big deal at the highest pressure-points of my PhD journey. I know this stressed you out, but I couldn’t help myself hahahaha. Thank you for believing me, it means the world! Rebecca Athorn – Thank you for all the generosity you showed me throughout this project. You really made me feel at home in the little town of Corowa! I really appreciate the many times you put me at ease when I thought the sky was falling. I am forever grateful for your calm and collected nature and all of your industry knowledge! Peter Thomson – Thanks for always responding to my (many) emails with fiddly questions…especially the ones where I’d email you an hour later only to tell you “Nevermind! I have figured it out on my own!”. Thank you for always making time for the little guys. Even when you were swamped with your own work, your patience and kindness never wavered. Thank you for always encouraging me to expand my knowledge and get back into coding. I will never ever forget that time you PM’d me code a week before Christmas in your much- deserved holiday period!! You made me feel less alone in this PhD journey. I thoroughly enjoyed every meeting we had and I always left inspired to learn more! The scientific community needs more people like you and it would be an absolute honour to work with you again someday. Jenna Lowe – You made my honours so enjoyable I continued on to do a PhD…. You are to blame :p . Just kidding, but I do owe this largely to you! I am so thankful for your friendship and ongoing support, even from afar. Your passion is infectious. I hope your fire never goes out! Charley, Dom, Alysia, Louise, Jess, Alex, Ha, Harriet, Evan, Danni x2, … and all the rest of the PhD students I met along the way! Can’t forget about Ruby and Bandit also! You helped me keep my sanity. Thank you for your ongoing support, I love you all. Paula –You are so compassionate and bubbly and the breath of fresh air on the other side of a PhD dungeon. Your smile started my day off right every day for four years and I really miss this! You always do your best to lend a hand (even if way outside of your job description) and I admire you for this. I hope we remain great friends! Technical and farm staff – Thank you to Craig Kristo for always going over and above what is required of you. You got me out of strife a few times! Thank you to the staff at Wollondilly Abattoir, Rivalea and Sunpork, I could not have done this without your guidance and willingness to help! Page 3 of 213 To my Mamma Rose, thanks for taking me to science camp and enduring my talks about the life cycles of insects I found when I was little. Without you I wouldn’t have even dared to try this career path, you’re the reason why this whole adventure started. I thank you from the bottom of my heart for giving me the courage to dream big. Your zest, excitement, and appreciation for the small wonders in life is so infectious and has contributed to my passion for the natural world – this is something we tend to lose as adults and you, unknowingly, always remind me how wonderful the world truly is. To my Pappa Scott, thanks for all those times throughout my childhood you cleaned out those caterpillar tanks when I was too scared to touch them when they turned into moths, instead of the butterflies I thought I was growing. Thanks for also supporting my young dreams of becoming a fish breeder. I appreciate, so much, all the time you took to really understand what I was doing in my PhD and your amazing ability to add perspective. You spent endless hours lending me a much-needed ear and this has been invaluable to me. You always ground me and have taught me to think critically and to never take anything at face value. Ever since I was little you cultivated my curiosity, teaching me to build things and showing me how stuff worked and that it's okay to be interested in things outside the realm of typical ‘girl’ activities. Most of all, I want to thank you for giving me my most valued trait, independence. To my dear little sister, Sarah, you are so special and inspiring to me. I hope to be as courageous and confident in my career as you are one day. May our agreement of unconditional support remain unsaid, just to forbid Mum the satisfaction of hearing it…. Oh, and I’m really sorry Mum made you go to science camp. I am truly privileged to be surrounded by such great people, Thank you all, Alicia Page 4 of 213 Abstract Poor sow retention is a common inefficiency in piggeries. In Australia, only around 60% of gilts being retained to parity three (Plush et al., 2016). This is concerning as gilts do not reach optimal reproductive performance until parity three (Engblom et al., 2007). Reproductive inadequacy is the main contributing factor for this premature culling (Plush et al., 2016). Thus, the traditional process for selecting breeding gilts is inadequate. Previously, circulating E2, ovarian follicle populations and hormonal profiles in response to gonadotropin stimulation have been linked with reproductive potential (Laufer et al., 1986; Kondapalli et al., 2012). Anti-Müllerian hormone (AMH) is the best known endocrine marker for antral follicle counts and ovarian reserve in other livestock species (cattle: Batista et al., 2014; sheep: Torres-Rovira et al., 2014; mares: Claes et al., 2014). This thesis aimed to determine the serum E2 and AMH levels in juvenile gilts prior to and after gonadotrophin stimulation and assess their associations with fertility and reproductive performance in order to determine their use as markers for gilts with greater reproductive success. Experiment One examined serum AMH and E2 levels in juvenile gilts 0, 2 and 4 days after gonadotropin administration and compared these measurements with mating, litter and culling information for three parities. Experiment Two assessed whether juvenile levels of E2 and AMH were associated with ovarian and uterine properties at 160 days. Experiment three involved two geographically different farms to validate results of Experiment One. The final experiment was similar to Experiment two but was longitudinal and involved a more detailed ovarian assessment. Page 5 of 213 To our knowledge, the quantification of AMH in juvenile gilts in this thesis was novel. Results showed serum AMH was negatively associated with ovarian follicle numbers, but its association with uterine properties was inconsistent. A negative association between E2 and future litter numbers was also found. Whether serum AMH and E2 levels in juvenile gilts are associated with uterine traits requires further investigation. The results highlight the complexities of endocrinology, emphasising the difficulty of determining hormonal markers for reproductive potential in a production setting. Page 6 of 213 Contents Declaration ................................................................................................................................. 2 Acknowledgements .................................................................................................................... 3 Abstract ...................................................................................................................................... 5 List of peer-reviewed publications........................................................................................... 12 List of conference presentations .............................................................................................. 13 List of grants ............................................................................................................................ 15 Thesis style............................................................................................................................... 16 Disclosure and author contributions ........................................................................................ 17 Chapter Three ................................................................................................................... 18 Chapter Four ..................................................................................................................... 19 Chapter Five ...................................................................................................................... 20 Chapter Six ....................................................................................................................... 21 List of Abbreviations ..............................................................................................................