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INVESTIGATION OF THE SUBCLINICAL TOXICOLOGICAL EFFECTS OF ERGOT ALKALOID MYCOTOXIN (Claviceps purpurea) EXPOSURE IN BEEF COWS AND BULLS A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Toxicology Graduate Program University of Saskatchewan Saskatoon By Vanessa Elizabeth Cowan © Copyright Vanessa Elizabeth Cowan, August 2020. All rights reserved i PERMISSION TO USE In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Chair of the Toxicology Graduate Program 44 Campus Drive University of Saskatchewan Saskatoon, Saskatchewan S7N5B3 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N5C9 Canada i ABSTRACT In my dissertation, I examine the effects of ergot alkaloid mycotoxins on vascular and reproductive systems in beef cows and bulls. Ergot alkaloids are toxic secondary metabolites produced by the pathogenic plant fungus Claviceps purpurea. Ergot alkaloids are commonly occurring adulterating toxins in livestock feed and constitute a great concern for the health of animals that consume such feeds. Consumption of these toxins can cause a broad suite of pathophysiological effects. Relevant and up-to-date scientific information on ergotism in livestock is largely unavailable to address this growing issue. The purpose of this research was to better characterize and understand the effects of ergot alkaloids in Canadian beef cattle and to ascertain concentrations at which these effects may occur. In my first two chapters, beef cows were fed increasing concentrations of ergot alkaloids over a short-term (Chapter 2) and long-term (Chapter 3) basis. As ergot alkaloids have a well- known vasoactive effect, hemodynamics of different arteries were evaluated with ultrasonography (B-mode and Doppler). In both studies, concentration-dependent, subclinical physiological changes in hemodynamics were observed in the caudal artery. These results are significant as a common end-stage manifestation of ergot alkaloid mycotoxicosis is the ischemic necrosis of the tail of exposed cattle. Further, these results indicate that subclinical changes occur at concentrations below current Canadian permissible values. Therefore, vascular changes appear to be the more sensitive indicator of ergot exposure than plasma prolactin changes in cows. Plasma or serum prolactin concentration is an accepted biomarker of ergot alkaloid exposure in livestock. To address the lack of pharmacokinetic information available on ergot alkaloids in cattle, I conducted two oral pharmacokinetics studies and attempted to develop an analytical method to detect ergot alkaloids in bovine plasma (Chapter 4). Although the method was promising for spiked plasma, ergot alkaloids were not detected in plasma samples collected from ergot-exposed cows. Likely, low oral bioavailability explained the lack of detection of ergot alkaloids in plasma from ergot-exposed cattle. An important practical conclusion of this work is that blood samples from suspected poisoning cases will not be clinically useful. In my last research chapter (Chapter 5), adult beef bulls were fed diets containing ergot alkaloids for one spermatogenic cycle (i.e., 61 days) to assess the potential negative effects of ergot exposure on sperm production or function. Results of this study indicated that ergot ii exposure had, at most, a subtle effect on bull sperm endpoints. However, plasma prolactin was affected by treatment. Spermatogenesis is not a sensitive endpoint for ergot exposure in adult bulls. Overall, this work answered questions related to ergot alkaloid exposure that are practically important. This work will enable policy makers to make scientifically-based decisions on guidelines for ergot alkaloids in cattle feed; will bolster the working knowledge of clinicians diagnosing and treating ergot-exposed cattle in the field; and will provide the much sought after information for producers working with ergot-contaminated grain or ergot exposed animals. iii ACKNOWLEDGEMENTS I gratefully acknowledge the guidance and mentorship received from my supervisors throughout my doctorate, Dr. Barry Blakley and Dr. Jaswant Singh. In addition, I acknowledge the assistance and support received from the members of my Advisory Committee: Dr. Mark Wickstrom, Dr. Jane Alcorn, Dr. Muhammed Anzar, and Dr. John McKinnon. I would also like to thank Dr. Lynn Weber, Chair of the Toxicology Graduate Program, and Dr. Gillian Muir, Department Head of Veterinary Biomedical Sciences, for their support. In addition, thank you to Dr. Tim Evans for serving as the external examiner for my dissertation defense. The research described in this dissertation was carried out with the support from the Government of Saskatchewan Agriculture Development Fund (#20130258 and #20160104) and the Saskatchewan Cattlemen’s Association (U of S Fund # 419027). This research was also supported by Dr. Singh’s NSERC Discovery Grant and in-kind contributions from Prairie Diagnostic Services. I was personally supported by the Natural Science of Engineering Research Council Canada Graduate Scholarship (at both the Master and Doctoral levels), the University of Saskatchewan Dean’s Scholarship, the Saskatchewan Innovation and Opportunity Scholarship, and the Toxicology Centre Devolved Scholarship. iv DEDICATION I happily dedicate this dissertation to my family and friends for their unwavering support throughout my degree program. I would especially like to dedicate this thesis to my grandparents, Myrt and (late) Bill Ryhorchuk, my mother, Christina Cowan, and my twin sister, Victoria Cowan. Thank you to my toxicology friends (Jen Briens, Dayna Schultz, Bryanna Eisner, and Shannon Bray) for the fun times and support throughout the grind. Thank you to my Brazilian jiu-jitsu friends and teammates (Shar Cairns and Jane Bryson) for letting me take my school stresses out on them on the mats. Thank you to Charlie Swain, Lianne Price, Chandler Giasson, Erin Matthews, Brad Campbell, Nick Charpentier, Cheryl Cho, and Alicia Unger for being steadfast friends and supports throughout it all. This thesis would not have been possible without the support and guidance of Dr. Barry Blakley. The gratitude I feel for Barry’s support over the years cannot be adequately expressed with words. I first started working for Barry in 2013 as a research assistant in Prairie Diagnostic Services. This is where I first introduced to analysis of ergot alkaloids in livestock feed. Barry and I were able to collaborate on four peer-reviewed manuscripts based on diagnostic case records in toxicology from Prairie Diagnostic Services. These publications undoubtedly helped me win scholarships and recognitions during my degree. In addition to his role my academic career, Barry also has also given me hours of his personal time. In 2014, he spent a day with me helping me purchase a car and set up my car insurance. In 2016, Barry hosted myself and my two friends from Germany at his cabin in Northern Saskatchewan for a day so they could have a new experience while they were in Canada. In 2018, he introduced me to the building manager at his daughter’s condominium complex which enabled me to rent an apartment from that complex. Thank you for everything you have done for me, Barry. I hope that I can make you proud in my career as a toxicologist. v TABLE OF CONTENTS Permission to Use............................................................................................................................i Abstract..........................................................................................................................................ii Acknowledgements.......................................................................................................................iv Dedication.......................................................................................................................................v List of tables................................................................................................................................xiv List of figures............................................................................................................................xviii Abbreviations..............................................................................................................................xxi 1. CHAPTER 1 – GENERAL INTRODUCTION......................................................................1