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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

Vanessa Elizabeth Cowan

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

Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N5C9 Canada

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.

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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.

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.

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 1.1 Introduction ...... 1 1.2 The ergot alkaloids...... 2 1.2.1 Physicochemical properties of ergopeptine alkaloids ...... 5 1.2.2 Mechanisms of toxic action of ergopeptine alkaloids...... 8 1.3 Ergotism in livestock ...... 13 1.3.1 Convulsive (nervous) ergotism...... 16

1.3.2 Gangrenous ergotism...... 16 1.3.3 Hyperthermic ergotism...... 18 1.3.4 Reproductive ergotism...... 19

1.4. Comparison of C. purpurea ergotism with fescue toxicosis in livestock...... 21 1.5. Ergot-mediated prolactin suppression in livestock...... 23 1.5.1 Early work...... 25

1.5.2 Administration of ergot alkaloids via injection...... 25 1.5.3 Feeding trials and grazing exposure...... 26 1.6. Ergot-induced peripheral vasoconstriction in livestock...... 27 1.6.1 In vitro and ex vivo studies of contractility...... 27 1.6.2 In vivo studies of vasoconstriction...... 29 1.6.2.1 Cattle...... 30 1.6.2.2 Horses...... 31 1.6.2.3 Small ruminants...... 32

1.7. Pharmacokinetics of ergot alkaloids...... 33 1.7.1 Absorption, Distribution, Metabolism, and Elimination...... 33 1.7.2 Pharmacokinetics versus pharmacodynamics of ergot alkaloids...... 47

1.7.3 Active metabolites...... 48 1.7.4 Livestock pharmacokinetics studies...... 49 1.7.5 Detection of ergot alkaloids in blood...... 50

1.8 Effect of ergot alkaloid exposure on male livestock semen characteristics and fertility 56 1.9 OBJECTIVES & HYPOTHESES FOR RESEARCH CHAPTERS...... 61 2. CHAPTER 2 – ARTERIAL RESPONSES TO SHORT-TERM LOW CONCENTRATION ERGOT ALKALOID EXPOSURE IN HEREFORD COWS...... 63 2.1 ABSTRACT...... 64

2.2 INTRODUCTION...... 65 2.2 MATERIALS AND METHODS ...... 66 2.2.1 Statement of animal ethics...... 66 2.2.2 Ergot alkaloid extraction and quantification procedure with Liquid Chromatography Mass Spectrometry (LC-MS)...... 67 2.2.3 Feed formulation and treatment groups...... 67

2.2.4 Animal husbandry and experimental design...... 68 2.2.5 Plasma samples...... 70 2.2.6 B-mode and Doppler Vascular Ultrasonography...... 70 2.2.7 Hemodynamic variable measurements ...... 71 2.2.8 Enzyme-linked immunosorbent assay (ELISA) for bovine prolactin (PRL) (antigen detection)...... 74 2.2.9 Statistical analysis – repeated measures analysis of variance...... 74 2.3 RESULTS...... 75 2.3.1 General...... 75 2.3.2 Plasma prolactin concentration, weight, and rectal temperature...... 75 2.3.3 Hemodynamic endpoints ...... 75 2.3.3.1 Diameter ...... 77 2.3.3.2 Peak systolic velocity...... 77 2.3.3.3 Blood volume per pulse ...... 77

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2.3.3.4 Blood flow ...... 80 2.3.3.5 Pulsatility index...... 80 2.3.3.6 Pulse rate...... 80 2.4 DISCUSSION...... 80 3. CHAPTER 3 – Arterial responses in periparturient beef cows following a nine-week exposure to ergot (Claviceps purpurea) in feed ...... 87 3.1 ABSTRACT ...... 88 3.2 INTRODUCTION...... 89 3. MATERIALS AND METHODS ...... 90 3.3.1 Statement of animal ethics ...... 90 3.3.2 Ration formulation and ergot alkaloid quantification in feed...... 90 3.3.3 Animal husbandry and experimental design...... 92 3.3.4 Ultrasonography of the caudal and internal iliac arteries...... 93 3.3.5 Blood collection ...... 93 3.3.6 Enzyme-linked immunosorbent assay (ELISA) for bovine plasma prolactin 94 3.3.7 Statistical analysis – SAS mixed procedure ...... 94 3.4. RESULTS...... 95 3.4.1. General...... 95 3.4.2. Hemodynamic endpoints...... 95 3.4.3. Caudal artery hemodynamics ...... 95 3.4.3.1. Caudal artery diameter...... 95 3.4.3.2. Caudal artery blood flow and volume...... 95 3.4.3.3. Caudal artery blood velocities...... 102 3.4.3.4. Caudal artery pulse rate, pulsatility index, and resistivity index...... 102 3.4.4. Internal iliac artery hemodynamics ...... 102 3.4.4.1. Internal iliac artery diameter...... 102 3.4.4.2. Internal iliac artery blood flow and volume...... 103 3.4.4.3. Internal iliac artery blood velocities ...... 103 3.4.4.4. Internal iliac artery pulse rate, pulsatility index, and resistivity index...... 103 3.4.5. Plasma prolactin concentration and rectal temperature...... 104

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3.5. DISCUSSION ...... 104 4. CHAPTER 4 – Development and partial validation of a method to detect and quantify ergopeptine alkaloids in bovine plasma with Liquid Chromatography Tandem Mass Spectrometry ...... 110 4.1. ABSTRACT ...... 111 4.2 INTRODUCTION...... 112 4.3. MATERIALS AND METHODS ...... 113 4.3.1. Chemicals and reagents...... 113 4.3.2. Ergot alkaloid standards ...... 114 4.3.3. Spiked plasma preparation...... 114 4.3.4. Quality control sample preparation ...... 115 4.3.5. Sample extraction by protein precipitation...... 115 4.3.6. Instrument and liquid chromatography-mass spectrometry (LC-MS) conditions...... 115 4.3.7. Acceptance criteria for calibration curve and quality control samples ...... 118 4.3.8. Practical applications – oral pharmacokinetics studies in beef cows ...... 118 4.3.8.1. Animal ethics statement...... 118 4.3.8.2. Animal husbandry ...... 120 4.3.8.3. Ergotized pellet preparation and ergot alkaloid quantification with liquid chromatography mass spectrometry (LC-MS)...... 120 4.3.8.4. Jugular catheterization ...... 120 4.3.8.4.1. Replicate 1...... 120 4.3.8.4.2. Replicate 2...... 122 4.3.8.4.3. Dose regimen and experimental protocol...... 122 4.3.8.4.3.1. Replicate 1 ...... 122 4.3.8.4.3.2. Replicate 2...... 123 4.3.9. Method development procedures ...... 124 4.3.9.1. Detection limit test of ergot alkaloids in spiked plasma based on Prairie Diagnostic Services method for ergot alkaloids in feedstuffs...... 124 4.3.9.2. Linearity test based on detection limits using six calibration points ...... 124 4.3.9.3. Extended calibration curve to expand upper quantitation range ... 125

4.3.9.4.Additional calibration curve test and preliminary low, medium, and high quality control sample analysis ...... 125 4.3.9.5. Full QC test including first LLOQ set and analysis of experimental cow samples (replicate 1)...... 126 4.3.9.6. Partial method validation with refined QCs...... 126

4.3.9.7. Autosampler stability ...... 127 4.3.9.8. Statistical analysis – linear regression for comparison of slopes and Y-intercepts from three-day partial validation ...... 127 4.3.9.9. Experimental cow samples from 2017 pharmacokinetics study in cows...... 127 4.4. RESULTS...... 128 4.4.1. Detection limit test of ergot alkaloids in spiked plasma based on Prairie Diagnostic Services method for ergot alkaloids in feedstuffs ...... 128 4.4.2. Linearity test based on detection limits using six calibration points...... 132 4.4.3. Extended standard curve to extend upper range of quantitation ...... 132 4.4.4. Accuracy and precision for preliminary low, medium, and high quality control (QC) sample analysis ...... 135 4.4.5 Full QC test including first LLOQ set and analysis of cow samples (replicate 1)...... 140

4.4.6. Partial method validation...... 140 4.4.6.1. General ...... 140 4.4.6.2. Linearity ...... 145 4.4.6.3. Slope...... 145 4.4.6.4 Y-intercepts...... 145 4.4.6.5 Precision and accuracy...... 145

4.4.6.6. Autosampler stability...... 149 4.4.7. Experimental samples (replicate 2 - 2017 pharmacokinetics study) ...... 149 4.5. DISCUSSION...... 149 4.6. FUTURE DIRECTIONS...... 157 5. CHAPTER 5 – Plasma prolactin, scrotal circumference, and semen quality following long-term exposure to ergot alkaloids (Claviceps purpurea) in adult Angus bulls...... 159 5.1. ABSTRACT ...... 160

5.2. INTRODUCTION...... 161 5.3.1. Animals...... 163 5.3.1.1. Animal Ethics Statement ...... 163 5.3.1.2. Animal Husbandry...... 163 5.3.2. Experiment 1: Pilot ergot alkaloid feeding study...... 164 5.3.2.1. Experimental design ...... 164 5.3.3. Experiment 2: Long term ergot alkaloid feeding study...... 167 5.3.3.1. Experimental design...... 167 5.3.3.2 Sample collection and animal handling...... 168 5.3.3.3 Rectal temperature, body weight, and scrotal circumference.....168 5.3.3.4 Semen collection...... 171 5.3.3.5 Blood collection and plasma separation...... 171 5.3.3.6 Semen assays...... 172

5.3.3.6.1. Computer assisted sperm analyzer (CASA)...... 172 5.3.3.6.2 Flow cytometer analysis (PI, FITC-PNA, MT Deep Red)...... 172 5.3.3.6.3. Sperm morphology (eosin-nigrosin vital staining). ... 176 5.3.3.7. Plasma prolactin quantification by radioimmunoassay...... 176 5.3.4. Statistical analysis...... 177 5.3.4.1. Experiment 1 – pilot ergot alkaloid feeding study ...... 177 5.3.4.2. Experiment 2 – long-term ergot alkaloid feeding study...... 177 5.4. RESULTS...... 178 5.4.1. Experiment 1 – Pilot ergot alkaloid feeding study ...... 178 5.4.2. Experiment 2 – Long term ergot alkaloid feeding study ...... 178 5.4.2.1. Prolactin, body weight, rectal temperature, and scrotal circumference...... 180 5.4.2.2. Sperm volume, concentration, and motility parameters (CASA)...... 180 5.4.2.3. Mitochondrial membrane potential and acrosome intactness (flow cytometry) 185 5.4.2.4. Sperm morphology (eosin-nigrosin stains)...... 188

5.5. DISCUSSION ...... 188 6. CHAPTER 6 – GENERAL DISCUSSION ...... 198 6.1 Vascular changes...... 198 6.2 Diagnostic considerations...... 201 6.3 Effect of season...... 202 6.4 Prolactin...... 202 6.5 Semen characteristics...... 203 6.6 Detection of ergot alkaloids in bovine plasma...... 204 6.7 Comparison with fescue toxicosis...... 205 6.8 Establishing a no-effect concentration and impact on regulations...... 206 6.9 Food safety...... 206 6.10 Future directions...... 207 6.11 Conclusion...... 207 7. REFERENCES...... 208

8. APPENDICES ...... 242 Appendix A. Syntax for statistical analysis in SAS Proc mixed in Chapter 2...... 242 Appendix B. Tabular presentation of measured and calcualted hemodynamic variables from Chapter 2...... 243 Appendix C. Tabular presentation of measured and calculated hemodynamic variables from Chapter 3...... 249 Appendix D. Syntax for statistical analysis used in Chapter 3...... 253 Appendix E. Preparation of calibration curve and quality control samples (4.1) and quality control sample concentrations (4.2) for Chapter 4...... 254 Appendix F. Pharmacokinetic studies (Chapter 4) – additional information ...... 255 Appendix G. Preliminary matrix effects analysis for Chapter 4...... 257 Appendix H. Solid phase extraction attempts for Chapter 4...... 258 Appendix I. Breeding soundness evaluations for bulls used in ergot feeding trials...... 260 Appendix J. Alternate feed analysis for ergot alkaloid concentration. Analysis of wheat screenings and pellet samples was conducted by the Missouri University Veterinary Medical Diagnostic Laboratory and Romer Labs...... 261

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Appendix K. Flow cytometric rationale and endpoints...... 263 Appendix L. Syntax used for statistical analysis for Chapter 5...... 265 Appendix M. Tabular results of experimental data from Chapter 5...... 266

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LIST OF TABLES TABLE 1.1. Chemistry of the major toxicologically relevant ergopeptine alkaloids found in C. purpurea sclerotia...... 7

TABLE 1.2. Broad physiologic functions of prolactin endocrinology in mammals...... 12

TABLE 1.3. Clinical signs and symptoms of ergot alkaloid exposure and ergotism observed in domestic livestock species...... 14

TABLE 1.4. Comparison of C. purpurea ergotism and E. coenephialum fescue toxicosis in cattle...... 24

TABLE 1.5. Summary of ergot alkaloid and ergot alkaloid derivative pharmacokinetic data from human studies...... 34

TABLE 1.6. Summary of pharmacokinetic parameters in livestock for intravenous administration of ergovaline tartrate...... 43

TABLE 1.7. Summary of liquid chromatographic-mass spectrometric methods for detecting and quantifying ergot alkaloids or ergot alkaloid derivatives in blood, plasma, and/or serum...... 52

TABLE 2.1. Treatment ration composition and corresponding total ergot alkaloid concentrations of diets fed to lactating Hereford cows...... 69

TABLE 3.1. Ergot alkaloid concentration (µg/kg; mean ± SEM) and percent of total (in parentheses) in the three pellet formulations...... 91

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TABLE 4.1. Definition of quality control samples and associated passing criteria for method validation...... 117

TABLE 4.2. Q1 to Q3 Transitions of ergot alkaloids analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) by Multiple Reaction Monitoring (MRM)...... 119

TABLE 4.9. Calibration curve information for ergopeptine alkaloids spiked in bovine plasma. Spiked plasma samples were extracted by protein precipitation and analyzed by liquid

xv chromatography tandem mass spectrometry (LC-MS/MS). Ergocristine was twice as concentrated as the other alkaloids...... 141

TABLE 4.12. Intra-day precision and accuracy following a three-day partial validation to quantify ergot alkaloids in bovine plasma. Plasma samples with ergopeptine alkaloids were quantified consecutively over a three-day period with liquid chromatography tandem mass spectrometry (LC-MS/MS)...... 148

TABLE 4.13. Inter-day accuracy and precision of four ergopeptine alkaloids spiked in bovine plasma by liquid-chromatography tandem mass spectrometry (LC-MS/MS)...... 150

TABLE 5.1. Ergot alkaloid concentration of pellets (µg/kg) used in Experiment 1 (pilot ergot alkaloid feeding study) in adult Angus bulls as determined by liquid chromatography mass spectrometry (LC-MS) analysis...... 165

TABLE 5.2 Ration of adult Angus bulls used in Experiment 1 (pilot ergot alkaloid feeding study)...... 166

TABLE 5.3. Concentration (mean ± standard deviation; µg/kg) of six ergot alkaloids in pelleted rations as determined by LC-MS following solvent extraction (Prairie Diagnostic Services, Saskatoon SK Canada)...... 169

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TABLE 5.4. Total mixed ration offered to adult Angus bulls (n=14) in Experiment 2 (long term ergot alkaloid feeding study)...... 170

TABLE 6.1. Major indicators and effects of ergot alkaloid exposure in beef cattle...... 199

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LIST OF FIGURES FIGURE 1.1. Representative structures of each class of ergot alkaloid...... 4

FIGURE 1.2. Structures of toxicologically relevant ergot alkaloids produced by Claviceps purpurea...... 6

FIGURE 1.3. Comparison of ergopeptine alkaloid structure with biogenic amines...... 9

FIGURE 1.4. Structure of the ergopeptine alkaloid ergovaline that produced by the fungal endophyte N. coenophialum in endophyte-infected tall fescue...... 22

FIGURE 2.1. Measurement of the hemodynamic endpoints using B-mode and color Doppler ultrasonography of the caudal, median sacral, and internal iliac arteries in cows...... 72

FIGURE 2.2 Diameter of the a) caudal artery, b) median sacral artery, and c) internal iliac artery of lactating Hereford cows (n=4 per treatment group) before (4 days), during (7 days), and after (4 days) feeding increasing concentrations of ergot alkaloids in Control (<15 µg/kg), Low (132 µg/kg), Medium (529 µg/kg) and High (2115 µg/kg) ergot groups...... 78

FIGURE 3.1. Diameter (mm), Blood flow (mL/min), and Blood volume per pulse (mL) of the caudal artery and internal iliac artery of periparturient Hereford cows (n=32) before (2 weeks), during (8 weeks), and after (3 weeks) feeding increasing concentrations of ergot alkaloids in Control (<15 μg/kg dry matter intake), Low (48 μg/kg), Medium (201 μg/kg), and High (822 μg/kg) ergot groups...... 96

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FIGURE 3.3. Pulse rate (bpm), pulsatility index, and resistivity index of the caudal artery and internal iliac artery of periparturient Hereford cows (n=32) before (2 weeks), during (8 weeks), and after (3 weeks) feeding increasing concentrations of ergot alkaloids in Control (<15 μg/kg dry matter intake), Low (48 μg/kg), Medium (201 μg/kg), and High (822 μg/kg) ergot groups100

FIGURE 4.5. Representative chromatograms for experimental cow plasma samples analyzed for ergot alkaloids by liquid chromatography tandem mass spectrometry (LC-MS/MS) after a one- time high concentration exposure in feed. No peaks were observed for any of the cow samples analyzed...... 143

FIGURE 4.6. Representative chromatograms of four ergot alkaloids over a three-day partial method validation. Sample were quantified with liquid chromatography tandem mass spectrometry (LC-MS/MS). Clear peaks for all alkaloids were observed on each day of the validation...... 144

FIGURE 4.7. Representative chromatograms for experimental cow plasma samples analyzed for ergot alkaloids by liquid chromatography tandem mass spectrometry (LC-MS/MS) after a one- time high concentration exposure in feed. No peaks were observed for any of the cow samples analyzed...... 153

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FIGURE 5.1. Representative histograms of triple-stain flow cytometric analysis of fresh adult bull sperm exposed to ergot alkaloids...... 174

FIGURE 5.2. Feed consumption (percentage of pellets offered that was consumed; mean ± standard error) in adult Angus bulls (n=14)...... 179

FIGURE 5.3. Plasma prolactin concentration, body weight, rectal temperature, and scrotal circumference of adult Angus bulls in low ergot (n=8; 1113 µg/kg DMI) and high ergot (n=6; 2227 µg/kg DMI) groups before (12 weeks/84 days), during (9 weeks/61 days), and after (10 weeks/68 days) daily ergot alkaloid exposure in their feed...... 181

FIGURE 5.4. Semen characteristics of fresh adult Angus bull semen before (twelve weeks/84 days), during (9 weeks/61 days), and after (10 weeks/68 days) ergot treatment...... 183

FIGURE 5.5. Sperm populations with intact plasma membrane and different levels of mitochondrial membrane potentials and acrosome integrity of fresh adult Angus bull semen before (six weeks; 36 days), during (9 weeks; 61 days), and after (10 weeks; 68 days) ergot treatment...... 186

FIGURE 5.6. Sperm morphology determined by eosin-nigrosin staining and differential microscopic counting of fresh adult Angus bull semen before (six weeks/36 days), during (9 weeks/61 days), and after (10 weeks/68 days) ergot treatment...... 189

LIST OF ABBREVIATIONS 5HT 5-hydroxytryptamine (a.k.a. serotonin) ACD acid citrate dextrose AICc Akaike information criterion AUC area under the plasma concentration versus time curve BLF blood flow bPRL bovine prolactin BW body weight C0 concentration of drug at time 0 CASA computer assisted sperm analyzer Cl systemic or total clearance ClR renal clearance Cmax maximum concentration of analyte in plasma/serum CV coefficient of variation CYP cytochrome p450 monooxygenase DA dopamine DM dry matter DMI dry matter intake EA ergot alkaloid EDV end diastolic velocity ELISA enzyme linked immunosorbent assay ESI electrospray ionization E+ endophyte infected tall fescue (i.e., ergot alkaloid containing) E- endophyte negative tall fescue F bioavailability fe,u fraction of drug excreted in urine FI feed intake FITC-PNA fluorescein-isothiocyanate conjugated peanut agglutinin HPLC high performance liquid chromatography HQC high quality control HR heart rate I.M. intramuscular I.U. international units I.V. intravenous LC-MS(-MS) liquid chromatography (tandem) mass spectrometry LLOQ lowest limit of quantitation LOD limit of detection Log Kow octanol:water partition coefficient LSD lysergic acid diethylamide LQC low quality control MMP mitochondrial membrane potential MnV mean blood flow velocity MQC medium/middle quality control MRM multiple reaction monitoring MRT mean residence time MtDR MitoTracker® deep red

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NE norepinephrine PI pulsatility index (chapters 2 and 3) PI propidium iodide (chapter 5) pKa acid dissociation constant PRL prolactin PRM parallel reaction monitoring PSV peak systolic velocity R correlation coefficient R2 coefficient of determination RI resistivity index RIA radioimmunoassay RR respiration rate TCA tris-citric-acid (buffer) TMR total mixed ration t1/2 half-life tmax time at which Cmax occurred S.C. subcutaneous SEM standard error of the mean SRM selected reaction monitoring STDEV standard deviation S:N signal-to-noise ratio QC quality control Vd volume of distribution XIC chromatogram

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1. CHAPTER 1 – GENERAL INTRODUCTION 1.1 Introduction Ergot alkaloids produced by the parasitic fungus Claviceps purpurea have long been known as poisonous to humans and animals (Barceloux 2008; Lee 2009; Belser-Ehrlich et al. 2013). Today, ergot poisoning in humans is uncommon due to stringent grain grading standards, but still poses a considerable risk to livestock. Livestock may become exposed to ergot while grazing contaminated pastures or consuming rations with contaminated grain ingredients. Ergotism has been observed in most livestock species, including cattle, pigs, horses, goats, and poultry. Ergot is a fungal pathogen that infects grasses and cultivated grains across Canada. Ergot contamination has become especially problematic for Canadian farmers and livestock producers in the past five to ten years. Outbreaks of C. purpurea infestation in western Canada were reported in 2005 (Manitoba), 2008, and 2011 (Alberta, Saskatchewan, Manitoba) (Menzies and Turkington 2015). In red spring wheat samples evaluated at grain elevators, ergot contamination rose from 12 to 15% in 2008 to 15 to 29% in 2011. In durum wheat samples, contamination was 15% in 2008 and 14% in 2011. Yield loss associated with ergot contamination is approximately 5 to 10% (Miedaner and Geiger 2015). With less grain suitable for human consumption, more grain has been reduced to livestock feed grade or salvage. Ergot infestation follows a predictable life cycle. The C. purpurea fungus targets the flowering stage of plants in the early summer months. A mature ergot body is formed approximately five weeks after initial infection (Schiff 2006; Wegulo and Carlson 2011; Belser- Ehrlich et al. 2013; Mai and Li 2013). Ascospore deposition during the plant’s flowering period enables C. purpurea mycelia to parasitize the ovary. As this stage progresses, a sugar secretion called “honeydew” is visible. This is the first apparent indication of ergot infection (Tudzynski and Scheffer 2004). Honeydew attracts insects to the infected plant to enable spread of conidia. The honeydew eventually hardens into the characteristic kernel-shaped sclerotia and replaces the entire seed head of the grass or grain approximately two-weeks post-infection (Tudzynski and Scheffer 2004; Miedaner and Geiger 2015). The ergot sclerotia fall to the soil below the plant substrate and remain stable until the following spring (Lorenz and Hoseney 1979). Due to the stability of the sclerotia, repeated contamination issues can arise annually. Rye is the predominant substrate of C. purpurea, as these grains have a prolonged flowering stage and

1 reliance on cross-fertilization by wind (Lee 2009), leading to a longer period of susceptibility for infection. Other economically important grain substrates include wheat, oats, barley, and sorghum (Lorenz and Hoseney1979; EFSA 2012; Tittlemeier et al. 2015). Ergot also affects most natural and introduced grasses found in Canada, including smooth brome grass, blue grass, quack grass, sedge grass, and orchard grass. Cool, wet conditions during the spring and summer months enhance both the ascospore production and sclerotia formation of ergot (Burfening 1973; Barceloux 2008; Lee 2009). Modern farming techniques may enhance the spread of ergot infection. Low- or no-till farming tends to increase the spread of ergot infection because mature ergot bodies can form and germinate in-field when favourable conditions arise. Fungicide application can prevent the spread of ergot contamination (Shelby 1999); however, treatment is largely ineffective once the sclerotia are mature. In addition, applying fungicides to crops may enhance the opportunity for C. purpurea infection due to mechanical damage of the plant stalk, leaving the plant more vulnerable to ascospore deposition. Considering recent ergot contamination issues in Canada, innovative crop management strategies are becoming the new industry standard. The advent of spectrophotometric sorting methods to clean grain and crop-switching with non-ergotized substrates between years have been suggested; however, the economic and time costs associated with these methods may deter farmers and producers from routine use of these methods.

1.2 The ergot alkaloids Ergot alkaloids are bioactive secondary metabolites produced by C. purpurea and other fungal species. Secondary metabolites are generally bioactive, low molecular weight compounds that are not required for growth of the producing organism (Keller, Turner, Bennett 2005). Numerous studies have indicated that the range of total ergot alkaloid concentration in C. purpurea sclerotia is extremely variable; in rye samples, concentrations can range from 1,000 to 6,003,000 µg/kg (Mainka et al. 2007; Appelt and Ellner 2009; Franzmann et al. 2010; Mulder et al. 2012; Orlando, Maumené, and Piraux 2017). In addition, ergot alkaloid content and abundance in different grain types also varies. Grusie et al. (2018a) indicated that ergocristine was the predominant ergopeptine alkaloids in western Canadian grain samples. The same study also found that ergot alkaloids could vary between grain types (e.g., rye, wheat, triticale, and barley).

Ergot alkaloids, which are indolic derivatives of the amino acid tryptophan, are all comprised of a common tetracyclic ergoline ring moiety (Mai and Li 2013; Bräse et al. 2013; Crews 2015). Ergot alkaloids are produced by prenylation of tryptophan by dimethylallyl tryptophan synthetase, followed by N-methylation and numerous oxidation reactions to produce lysergic acid and the ergoline ring (Keller, Turner, Bennett 2005). The ergoline ring is methylated on the N6 nitrogen. The different alkaloids are produced by various substitutions at carbon C8 and differences in structure on one of the four rings of the ergoline moiety (Arroyo- Manzanares et al. 2017). Over 70 ergot alkaloids have been identified from Claviceps spp. (Bräse et al. 2009; Arroyo-Manzanares et al. 2017). The ergot alkaloids can be broadly classified into four categories based on their structure (Figure 1.1). Each group differs in their physiological properties and potencies. The first group, the clavine alkaloids, are only comprised of the ergoline ring and includes elymoclavine and agroclavine (Schiff 2006; Arroyo-Manzanares et al. 2017). The second group of ergot alkaloids are the ergoamide alkaloids, i.e., lysergic acid amides, which are water soluble amino alcohol derivatives. The lysergic acid amides comprise approximately 20% of the alkaloids found in the sclerotia (Schiff 2006; Mai and Li 2013; Crews 2015). The major ergoamide alkaloid is ergometrine, which has a long history of use in human obstetrics to prevent and treat postpartum hemorrhage (van Dongen and de Groot 1995; de Costa 2002). The third and fourth groups – ergopeptams and ergopeptines – are similar as both groups have tripeptide groups in addition to the ergoline ring. The difference between the ergopeptams and ergopeptines is substitution of L- proline for D-proline on the tripeptide group, leading the ergopeptams to have a chain (i.e., non- cyclol lactam) conformation as opposed to a ring structure, as seen with the ergopeptines (Arroyo-Manzanares et al. 2017). An example of an ergopeptam alkaloid is ergocristam. Ergopeptine (or ergopeptide) alkaloids contain a tricyclic peptide group and lysergic acid. All ergopeptides contain the amino acid L-proline. Further differentiation of the ergopeptines occurs based on different amino acid substitution on the two of the peptide moieties of the tripeptide group (Arroyo-Manzanares et al. 2017). In addition, the ergopeptines (R-conformation) undergo epimerization at the C8 carbon to produce their corresponding (S)-inine epimers (i.e., ergotamine ⇌ ergotaminine) (Krska and Crews 2008). Epimerization of the ergopeptine alkaloids occurs spontaneously and variably in alkaline or protic solutions, during heating, or by UV light exposure (Krska and Crews 2008; Schummer et al. 2020). Although previously thought to be

Clavine type – agroclavine Ergoamides – ergometrine

Ergopeptines – ergotamine Ergopeptams – ergocristam

FIGURE 1.1. Representative structures of each class of ergot alkaloid. There are four classes of ergot alkaloids – clavine alkaloids, ergoamides, ergopeptines, and ergopeptams. The ergopeptine class of alkaloids are toxicologically important. Structures retrieved from Wikimedia Commons. Ergocristam structure retrieved from PubChem.

4 marginally active or inactive compared to the parent compounds (Berde and Sturmer 1978; Pierri et al. 1982; Komarova and Tolkachev 2001; Krska and Crews 2008), a recent in vitro study suggests that ergopeptinines are vasoactive (Cherewyk et al. 2020). This finding could be significant for the future of ergot alkaloid research. The ergopeptines comprise 80% of the total alkaloids in the sclerotia (Schiff 2006). In C. purpurea sclerotia, the most common ergopeptine alkaloids are ergocristine, ergotamine, ergocryptine, ergocornine, and ergosine (Figure 1.2) (Barceloux 2008; EFSA 2012; Crews 2015; Grusie et al. 2018a). The R1 and R2 substitution of each of the major ergopeptine alkaloids is described in Table 1.1. Ergot alkaloids vary in potency and toxicity. However, the most toxicologically important class of ergot alkaloids in relation to livestock health are the ergopeptine alkaloids. The European Food Safety Administration has identified ergotamine, ergosine, ergocristine, ergocryptine, ergocornine, their corresponding -inine epimers, and ergometrine (a lysergic acid amide) as priority alkaloids for monitoring in food and feed (EFSA 2012).

1.2.1 Physicochemical properties of ergopeptine alkaloids. The ergopeptine alkaloids are weak bases with reported pKa values ranging from 5.5 (for ergocristine) to 6 (ergometrine, a lysergic acid amide) (Maulding and Zoglio 1970; Krska and Crews 2008; Arroyo-Manzanares et al. 2017). The basic nature of the ergopeptine alkaloids is associated with the N6 nitrogen on the ergoline ring (Mai and Li 2003; Arroyo-Manzanares et al. 2017). As weak bases, the ergopeptine alkaloids have a positive charge in acidic solutions and are neutrally charged in alkaline solutions (Crews 2015). The basic nature of the ergot alkaloids decreases from ergotamine > ergocryptine > ergocristine (Komarova and Tolkachev 2001). In terms of dissolution properties, the ergopeptine alkaloids are slightly insoluble in water and are soluble in organic solvents like acetonitrile and methanol (Kumar and Bhansali 2007; Crews 2015). Most high performance liquid chromatography (HPLC) methods to extract and quantify ergot alkaloids use methanol- water or acetonitrile-water solutions adjusted to alkaline conditions to elute the alkaloids

(Jegorov 1999). The reported octanol:water partition coefficients (log Kow) for the ergot alkaloids include 2.53 for ergotamine (Dänicke and Flachowsky 2017; NCBI), 3.44 for ergocristine (NCBI), and 0.52 for ergometrine (Dänicke and Flachowsky 2017; NCBI). Apart from ergometrine, the ergopeptine alkaloids appear to be moderately lipid soluble and would be expected to cross biological membranes with ease.

Ergotamine Ergosine Ergocristine R 2

R 1

Ergocornine Ergocryptine Ergometrine

ergoline ring system

FIGURE 1.2. Structures of toxicologically relevant ergot alkaloids produced by Claviceps purpurea. Ergotamine, ergosine, ergocristine, ergocornine, and ergocryptine are ergopeptine alkaloids. Ergometrine (also known as ergonovine) is a lysergic acid amide. Ergot alkaloids all contain a tetracyclic ergoline ring system (indicated on the structure of ergometrine). Ergopeptine alkaloids vary by the their R1 and R2 moieties (R1 and R2 carbons indicated on ergotamine). Structures retrieved from Wikimedia Commons. purpurea.

TABLE 1.1. Chemistry of the major toxicologically relevant ergopeptine alkaloids found in C. purpurea sclerotia. Table adapted from information described in Arroyo-Manzanares et al. (2017) Ergot alkaloid R1 R2

Ergotamine -CH3 -CH2-Phe

Ergocristine -CH(CH3)2 -CH2-Phe

-Ergocryptine -CH(CH3)2 -CH2CH(CH3)2

-Ergocryptine -CH(CH3)2 -CH(CH3)CH2CH3

Ergocornine -CH(CH3)2 -CH(CH3)2

-Ergosine -CH3 -CH2CH(CH3)2

-Ergosine -CH3 -CH(CH3)CH2CH3 Phe = phenyl

1.2.2 Mechanisms of toxic action of ergopeptine alkaloids. The toxic basis of ergot is the ability of the ergot alkaloids to act as receptor-binding agonists or partial-agonists of dopaminergic, serotonergic, and -adrenergic receptors (Figure 1.3; Aellig and Berder 1969; Berde and Stürmer 1978; Pertz and Eich 1999). Dopamine, serotonin (i.e., 5-hydroxytryptamine; 5HT), and norepinephrine are derived from aromatic amino acids, including tryptophan, by decarboxylation and hydroxylation (Schardl 2006). Production of the ergot alkaloids, as mentioned previously, follows a similar synthetic pathway. This leads to structural similarity between the ergoline ring and the neurotransmitters. The potential for physiological alterations with this mechanism is substantial; however, the major pharmacological effects exerted by ergot alkaloids appear to be stimulation of smooth muscle, central sympatholytic activity, and peripheral adrenergic blockade (Merhoff and Porter 1974). The most well-described pathophysiological changes related to ergot exposure are peripheral vasoconstriction and decreased circulating concentration of the hormone prolactin. Ergot-induced vasoconstriction results from alkaloid binding to peripheral serotonergic and adrenergic receptors on smooth muscle cells as agonists (Merhoff and Porter 1974; Silberstein 1997; Pertz and Eich 1999; Eadie 2003; Silberstein and McCrory 2003). Ergot alkaloids are antagonistic at vasodilatory dopamine D1 receptors (Pertz and Eich 1999; Schiff 2006). The ergopeptine alkaloids act on the vascular smooth muscle cells of the arterial tunica media (Boor 2003). Prolonged vasoconstriction induced by ergot can produce thrombosis, blood flow stasis, and, eventually, capillary endothelial damage (Eadie 2003; Silberstein and McCrory 2003). Restriction of blood flow to peripheral tissues through constriction of the smooth muscle cells in blood vessels reduces oxygen delivery and may result in ischemic damage (Eadie 2003; Boor 2003; Rostoff et al. 2010). What is unique about ergot-induced vasoconstriction is that the effect gradually increases over time in a fashion that is not predicted by the plasma ergot alkaloid concentrations (Bigal and Tepper 2003). The exact biochemical mechanism for this prolonged constriction is undetermined, but it has been suggested that delayed dissociation of the alkaloids from the bioamine receptor interface may be involved (Silberstein 1997; Schöning et al. 2001; Klotz et al. 2007; Pesquiera et al. 2014) – this will be discussed further in section 1.7.2. The serotonergic activity of ergot alkaloids has been well-known for decades, largely due to the use of ergotamine and semi-synthetic ergot alkaloid derivatives (e.g., bromocriptine and

FIGURE 1.3. Comparison of ergopeptine alkaloid structure with biogenic amines. The tetracyclic ergoline skeleton – common to all ergot alkaloids – shares structural similarities with dopamine, serotonin, and norepinephrine and imparts their pathophysiological activity. Structures retrieved from Wikimedia Commons.

9 methysergide) in the treatment of migraine headaches and other psychoactive human disorders (Silberstein 1997; Tfelt-Hansen et al. 2000; Dawson and Moffatt 2012). The potency of vasoactivity varies by location and the associated serotonin receptor subtypes present in the tissue. Overall, however, ergot alkaloids bind non-specifically to serotonin receptors, including the 5HT1, 5HT2, and 5HT4 subtypes (Pertz and Eich 1999; Eadie 2003). Of the 5HT1 receptors, ergotamine binds with high affinity to the 1A, 1B, 1D, and 1F isoforms. Of the 5HT2 receptors, ergotamine binds the 2A, 2B, and 2C isoforms. The anti-migraine action of ergotamine results from binding to 5HT1B/1D receptors in the carotid vascular bed (Silberstein 1997; Silberstein and McCrory 2003). In the peripheral vascular beds, however, a higher concentration of the vasoconstrictive 5HT2A receptors and leads to smooth muscle contraction and platelet aggregation. This produces the undesirable side effect of peripheral vasoconstriction in migraine patients taking ergotamine on a long-term basis (Silberstein and McCrory 2003). Binding to serotonin receptors has been described as the major mechanism of vasoconstriction and induction of smooth muscle proliferation induced by ergot alkaloids. Prolonged binding to 5HT2B receptors in tissues, e.g., human heart valves or peripheral blood vessels, results in sustained endothelial contraction leading to tissue fibrosis and arterial stiffening (i.e., reduced elasticity) (Baron and Tepper 2010). Human clinical cases regarding chronic ergotamine use have highlighted the adverse vascular events (Redfield et al. 1992; Garcia et al. 2000; Dawson and Moffatt 2012). The structural similarity of the ergopeptine alkaloids to norepinephrine also induces peripheral vasoconstriction (Schöning et al. 2001; Barceloux 2008). Norepinephrine is the principle compound that mediates blood flow resistance through constriction of vessels to individual organs. Ergot alkaloids may act as agonists, partial agonists, or antagonists at adrenergic receptors, depending on the receptor subtype and the chemical composition of the alkaloid mixture (Roquebert and Grenie 1986; Pertz and Eich 1999). The pharmacological and biochemical information on binding to adrenergic receptors is comparatively less than that of ergot alkaloid binding to serotonin and dopamine receptors. This is largely due to the therapeutic applications associated with these receptors (e.g., Parkinson’s Disease (Berde and Stürmer 1978)). Ergotamine binds to -adrenergic receptors, but not -adrenergic receptors, with a high affinity (Tfelt-Hansen et al. 2000; Silberstein and Hargreaves 2000). The alkaloids bind to both the vasoconstrictive 1 and 2 receptor subtypes. Under normal physiological conditions, norepinephrine binding to peripheral  receptors on smooth muscle cells in arterioles dictates

10 peripheral vascular resistance. An early study found that dihydroergotoxine increased blood pressure in anaesthetized rats by vasoconstriction via the 2 receptors (Roquebert and Demichel 1985). Roquebert and Grenie (1986) found that, in pithed rats, vasoconstriction was caused by ergotamine and dihydroergotamine acting as partial agonists of peripheral 2 receptors and competitive antagonists at peripheral 1 receptors. In addition, vasoconstriction in canine external carotid arteries was reported following intracarotid infusions of methysergide, ergotamine, and dihydroergotamine (Villalón et al. 2009). This effect was mediated by binding to 5HT1B/1D receptors and 2-adrenergic receptors. It is not known whether the net effect of binding to serotonin and adrenergic receptors is additive or synergistic on peripheral vasoconstriction. In addition to peripheral vasoconstriction, reduced circulating prolactin represents one of the most well-established indicators of ergot alkaloid exposure in livestock. Prolactin is a polypeptide hormone released from anterior pituitary lactotrophs. The name given to the hormone reveals its most well-known role in milk production (i.e., lactogenesis). The endocrinology of prolactin is unique. Firstly, pituitary lactotrophs are specialized endocrine cells that have basal secretory activity, unlike other cell types. Secondly, the actions of prolactin are pleiotropic: identified biological functions of prolactin in vertebrates are over 300 in number (Table 1.2) (Bole-Feysot et al. 1998; Freeman et al. 2000; Goffin et al. 2002). The vast suite of biological functions effected by prolactin is greater than that of all other pituitary hormones combined (Goffin et al. 2002). Lastly, prolactin is one of the only pituitary hormones that acts to stimulate its own inhibition. This is accomplished through negative feedback to the hypothalamus, referred to as “short-loop feedback”, where high circulating concentrations of prolactin stimulate the release of dopamine. Dopamine, a hypothalamic hormone, is the predominant inhibitor of prolactin secretion (Ben-Jonathan and Hnasko 2001). Its action is to prevent exocytosis of prolactin from the pituitary lactrotrophs (Ben-Jonathan and Hnasko 2001).

The pathway occurs via the D2 subtype of dopamine receptors lactotrophic cell membranes. Dopamine may also inhibit synthesis of prolactin (Maurer 1980; Auchtung et al. 2003).

Ergot alkaloids have been shown to bind to dopamine D2 and D1 receptors with high affinity (Nasr and Pearson 1975; Anlezark, Pycock, and Meldrum 1976; Sibley and Creese 1983;

Eadie 2003; Barceloux 2008). In particular, binding to D2 receptors inhibits prolactin secretion,

TABLE 1.2. Broad physiologic functions of prolactin endocrinology in mammals (reviews by Bole- Feysot et al. 1998; Freeman et al. 2000; Ben-Jonathan and Hnasko 2001; Goffin et al 2002; Grattan and Kokay 2008; Ignacak et al. 2012). Categories of biological functions divided based as per Bole- Feysot et al. 1998.

Water and electrolyte balance (kidney, intestines, sweat glands, uterus, and placenta)

Growth and development (liver, fetal lungs, smooth muscle, gonads, germ cells, accessory sex glands, hair growth)

Endocrinology and energy metabolism (lipid and carbohydrate metabolism; steroid metabolism; signal transduction)

Brain and behaviour (neurogenesis; maternal and grooming behaviour; sleep-wake cycles; maturation of the neonatal neuroendocrine system; activation of dopamine neurons; appetite and body weight regulation)

Reproduction (mammary gland growth and development; ovarian maturation, including effects on granulosa cells; corpus luteum modulation; uterus effects; oxytocin regulation; suppression of fertility; increased testicular activities in general, including effects on Leydig, Sertoli, and germ cells/spermatozoa)

Immunoregulation (thymic and splenic growth; increased cell mediated and humoral immunity; increased natural killer cell and macrophage activity)

12 thereby reducing circulating prolactin concentrations (Hökfelt and Fuxe 1972; Floss, Cassady, and Robbers 1973; Nasr and Pearson 1975; Anlezark, Pycock, and Meldrum 1976; Holohean et al. 1982; Sibley and Creese 1983; Šoškić et al. 1986; Ben-Jonathan and Hnasko 2001). Under normal physiological conditions, dopamine inhibits the secretion of prolactin from the anterior pituitary through the arcuate nuclei of the hypothalamus (Ben-Jonathan and Hnasko 2003). A major target of the endocrine action of prolactin is the mammary gland with its high concentration of prolactin receptors. The role of prolactin in mammary gland growth and development has been described (Freeman et al. 2000). Its action appears to be involved in the lobular alveolar gland development. During lactation, prolactin and the prolactin receptor is localized within mammary epithelial cells (Freeman et al. 2000). Ergot alkaloids disrupt lactogenesis via the inhibition of mammary gland maturation, manifested as decreased milk production or complete agalactia (Mantle 1969; Blaney et al. 2000b; Copetti et al. 2002; Cross 2003). Another less well-described mechanism of ergopeptine alkaloids is dysfunction of glutaminergic neurotransmission. Glutamate is the predominant excitatory neurotransmitter in the vertebrate nervous system. A study in isolated bovine cerebral synaptic vesicles found that various ergot alkaloids (ergovaline, ergotamine, ergocornine, and bromocriptine) inhibited Glutamate uptake by acting on vesicular Glutamate receptors (Xue et al. 2011). Ergovaline appeared to inhibit the vesicular glutamate receptors by a non-competitive mechanism. The authors of this study concluded that ergot alkaloids (from endophyte-infected tall fescue) could decrease glutaminergic neurotransmission in livestock consuming these alkaloids.

1.3 Ergotism in livestock. The term ‘ergotism’ can describe multiple syndromes in livestock. In addition, the manifestation of ergotism varies greatly by species (Table 1.3). The two classic manifestations of ergot alkaloid mycotoxicosis are convulsive ergotism and gangrenous ergotism (Robbins, Porter, and Bacon 1986; Shelby 1999). However, two other disease states – hyperthermic ergotism and reproductive ergotism – have also been described. Differences in exposure, i.e., grazing on contaminated pasture and consumption of contaminated rations, from other livestock species may be involved. Cattle and sheep, both ruminants, may be more resistant to ergot alkaloid exposure due to the increased metabolic and

TABLE 1.3. Clinical signs and symptoms of ergot alkaloid exposure and ergotism observed in domestic livestock species. Cattle - ↓ circulating prolactin concentration Woods, Jones, and Mantle 1966; Dinnusson et al. 1971; - Hindlimb lameness McKeon and Egan 1971; Schams, Karg, Rheinhart - Gangrene (sloughing of tail tip, ear trips, and/or hooves) 1972; Skarland and Thomas 1972; Hurley et al. 1980; - Hyperthermia/↑ rectal temperatures Schmidt et al. 1982; Appleyard 1986; Robbins, Porter, - Feed refusal/↓ feed intake and Bacon 1986; Jessep et al. 1987; Stuedemann and - ↓ average daily gain/weight gain Hoveland 1988; Ross et al. 1989; Burfening 1994; Hill - ↓ milk yield et al. 1994; Browning et al. 1998; Browning 2000, - Failure to shed winter coat/rough hair coat 2004; Al-Tamimi et al. 2003; Bourke et al. 2000; - ↑ blood pressure Bourke 2003; Botha et al. 2004; Watson et al. 2004; - Abortion Schuenemann et al. 2005a,b; Aiken et al. 2007, 2009, - ↓ birth weight 2011a; Blaney et al. 2011; Craig, Klotz, and Duringer 2015; Miskimins et al. 2015

1 4

Horses - ↓ circulating prolactin concentration Poppenga et al., 1984; Taylor et al., 1985; Monroe et - Agalactia al., 1988; Riet-Correa et al. 1988; Ireland et al. 1991; - Prolonged gestation McCann et al. 1992; Porter and Thompson 1992; - Dystocia Cross, Redmond, and Strickland 1995; Green and - Thickened placentas Raisbeck 1997; Blodgett 2001; Evans, Rottinghaus, and - Neonate mortality Casteel 2004a,b; Copetti et al. 2002; Evans 2011 - Fetal dysmaturity

Pigs - ↓ circulating prolactin concentration Shone et al. 1959; Whittemore et al. 1976; Whittemore, - ↓ feed intake, feed conversion, and/or growth rate Miller, and Mantle 1977; Whitacre and Threlfall 1981; - Agalactia Coitinho, Feippe and Riet 1984; Schneider et al. 1986; - ↓ litter weight gain Blaney and Kopinski 1998; Blaney et al. 1997; Blaney - Neonate mortality et al. 2000a,b; Oresanya et al. 2003; Kopinski et al. - Intestinal lesions 2007,2008; Kanora and Maes 2009; Waret-Szkuta et al. - Hindlimb lameness 2019 - Necrosis/gangrene of tails, ears, hooves

Small - ↓ circulating prolactin concentration Burfening 1973; Niswender 1974; Greatorex and

ruminants - ↓ lambing Mantle 1974; Stilham et al. 1982; Robbins, Porter, and

1 5 (sheep, - ↓ milk production Bacon 1986; Elsasser and Bolt 1987; Bond et al. 1988; goats) - Abortion Burfening 1994; Engeland et al. 1998; Emile, Bony, and - Depression Ghesquiere 2000; Parish et al. 2003; Duckett, Andrae, - ↓ feed intake and Pratt 2014; Zbib et al. 2014 - ↓ live birth weights - ↓ fetal growth - ↑ rectal temperature - Gangrene (ear tips) - Tongue lesions, gastrointestinal lesions

15 detoxifying capacity of their rumen microflora seen with other plant toxins (Kiessling et al. 1984; Stuedemann et al. 1998; Fink-Gremmels 2008). Lactating sows and mares appear to be the most vulnerable livestock species to ergopeptine alkaloids due to the severity of reproductive effects, including complete agalactia, observed in these species. Manifestations in these species may include full reproductive failure following ergot exposure in feed (Blaney et al. 2000; Evans 2002; Kopinski et al. 2007; Evans 2011). A partial explanation for this may be attributed to differences in prolactin physiology in milk production. Prolactin appears to be the primary lactotrophic hormone involved in lactogenesis in horses and swine. This is in contrast the cattle, where growth hormone, placental lactogen, and prolactin both contribute to lactogenesis (Flint and Knight 1997; Vitala et al. 2006; Takahashi 2006). Consequently, loss of prolactin function in horses and swine relating to milk production may be more critical than for cattle. Further research in species differences of prolactin and role in development of ergotism is required.

1.3.1 Convulsive (nervous) ergotism. Nervous or convulsive ergotism is a syndrome manifested as vertigo, hyperexcitability, intermittent convulsions, belligerence, drowsiness, temporary posterior paralysis, and potentially death (Burfening 1973; Shelby 1999). This syndrome, however, is rare in livestock; cases have been reported in horses and sheep. Sheep tend to display less severe clinical signs of gangrenous ergot poisoning than cattle but may present with convulsions indicative of nervous ergotism (Robbins, Porter, and Bacon 1986). The concentrations of ergot alkaloids in feed that produce convulsive ergotism have not been reported. It has been suggested that convulsive ergotism results from overstimulation of the dopaminergic and serotonergic pathways (Eadie 2003). The mortality rate of convulsive ergotism is not known, but death appears to be a rare occurrence. Very few case reports describing convulsive ergotism in livestock are available.

1.3.2 Gangrenous ergotism. Gangrenous ergotism of livestock was previously thought to be an ‘old’ disease, being described in the scientific literature of the late 1950s and 1960s (Stocker 1959; Mantle 1969). Due to recent contamination issues, however, there appears to be a resurgence of disease occurrence in cattle. Ergot sclerotia content of 0.2% (w/w), approximately 2400 µg/kg, in feed has been associated with gangrene development (Burfening 1994; Shelby 1999). Sustained peripheral vasoconstriction induced by ergot alkaloids progresses to ischemic necrosis due to lack of blood flow and oxygen to the vascular beds. A major contributing factor

16 in its etiology is the profound interaction with cold ambient temperatures encountered during Canadian winters. The vasoconstrictive effect of ergot is exacerbated by cold ambient temperatures. In addition, cows are often pregnant during the winter season in Canada and consume more feed to meet their energetic requirements. This leads to greater exposure to ergot alkaloids in the winter. To maintain a normal core body temperature during cold weather, peripheral vasoconstriction acts as a normal physiological mechanism to divert blood flow to critical organs and prevent peripheral heat dissipation. When combined with ergot alkaloid exposure, the vasoconstriction is predominantly pathophysiologic rather than physiologic. Livestock become more vulnerable to frostbite and full limb freezing when exposed to ergot. Removal of contaminated feed and good livestock management practices are necessary to prevent the development of gangrene. Once sloughing of the hooves occurs, however, the damage is irreversible. Gangrenous ergotism of domestic animals is most frequently observed in cattle, and thus the etiology of the disease in these animals is well described (Robbins, Porter, and Bacon 1986; Belser-Ehrlich et al. 2013). Severe gangrenous ergotism in cattle is marked by complete sloughing of hooves. In sheep, lesions of the tongue and gastrointestinal inflammation occur, without sloughing of appendages (Robbins, Porter, and Bacon 1986). The first signs of intoxication are ataxia and hind limb lameness and may manifest as an animal being unable to bear weight on one of its hind limbs. Following prolonged exposure to ergot, i.e., long-term consumption of contaminated feed, cattle eventually develop swelling and erythema in the distal portions of their limbs and tail tips. Necrosis is indicated by a visible line of demarcation between viable and non-viable tissue. Sloughing of the necrotic skin or the entire limb may occur. In affected hooves, gangrene is found at the fetlock and pastern regions. Outbreaks of gangrenous ergotism have been reported worldwide in the last half-century, including Canada, the United States, Australia, the United Kingdom, and South Africa (Schneider et al. 1986; Botha et al. 2004; Millar, Smith, and May 2010; Belser-Ehrlich et al. 2013; Craig, Klotz, and Duringer 2015). Case reports of gangrenous ergotism with ergot alkaloid concentration information feed provide insight into the etiology of vascular toxicity. Craig, Klotz and Duringer (2015) summarized numerous cases of cattle exhibiting gangrene of appendages. In a Canadian case during February (-20ºC), ergotamine concentration of 473 µg/kg feed was associated with tail tip

17 necrosis. Notably, this concentration is well within the current Canadian standards for ergot alkaloids in cattle feed of 2000 to 3000 µg/kg. Another Canadian case, this time occurring in April (-4ºC), saw full hoof loss in steers. As a result, nearly 40% of the herd was culled. The concentration of ergot alkaloids in the diet was found to be 11 538 µg/kg. The authors also reported this concentration in terms of “ergotamine equivalence” as ergotamine is the most potent C. purpurea ergopeptine alkaloid – this concentration was 1161 µg/kg. The final case reported by the authors occurred in Idaho during January (-1ºC) was associated with a feed concentration of 62 245 µg/kg (ergotamine equivalence = 10 124 µg/kg) with 75% of cattle in the herd being terminated due to hoof gangrene. A report from southern Africa described mid- winter tail tip and hoof necrosis in cattle grazing endophyte infected tall fescue pasture (Botha et al. 2004). The concentration of ergovaline in the fescue was determined to be 1700 to 8170 µg/kg, whereas the ergopeptine alkaloids (presumably) were present at 1000 µg/kg. Clearly, high concentrations of ergot alkaloids in feed do cause gangrenous ergotism. In addition, clinical symptoms of gangrenous ergotism occur at concentrations current allowed by the Canadian Feed Inspection Agency. What is largely unknown, however, is a no effect concentration in feed for gangrene development. There is also uncertainty surrounding the interaction of dose and duration of exposure.

1.3.3 Hyperthermic ergotism. The dynamics between peripheral vasodilation and vasoconstriction mediate evaporative heat loss for many mammalian species as an adaptive mechanism for elevated ambient temperature and humidity. Clinical signs of hyperthermic ergotism have been observed starting at concentrations of approximately 1000 µg/kg of feed (Bourke 2003; Blaney, Molloy, and Brock 2009). The vasoconstrictive effect of ergot alkaloids leads to increased core body temperatures, as peripheral convective cooling mechanisms are insufficient (Aldrich et al. 1993). Hyperthermia in livestock results in decreased feed intake to reduce the ability of the metabolic heat production. Other consequences of hyperthermia include rectal temperatures greater than 40ºC, depressed milk production, poor animal performance and weight gains, and increased vulnerability to death from heat exhaustion. The vasoconstrictive activity of ergot alkaloids led to further research into the interaction of ambient temperature with ergot exposure in livestock. The first recognition of this thermoregulatory disruption was in 1987, in which Jessep et al. (1987) reported case studies in cattle displaying hyperthermia, respiratory distress, and decreased production. It was later

18 determined that ergot sclerotia were present in the feed at concentrations of 0.02 to 0.08% (w/w). Cattle have now been widely studied in this area due to the identification of hyperthermic ergotism. Typical clinical signs associated with ergot-induced bovine hyperthermia include high rectal temperatures (≥39.4ºC), decreased feed intake, poor weight gains and performance, increased water intake and urination, preference for shade, salivation, panting, open-mouth breathing, protruding tongues, increased respiratory and heart rates, and decreased milk production. Multiple studies worldwide describe hyperthermic ergotism in cattle. In Hereford steers fed diets containing 0.5% ergot (w/w), hyperthermia developed within three days of ingestion (Ross et al. 1989). Hyperthermia was accompanied by decreased feed intake of up to 50%, weight loss, and depressed serum prolactin concentrations. It was observed that these steers returned to a normal state overnight, indicative of recovery once the ambient temperature decreased. A case study from Australia reported a severe outbreak of hyperthermic ergotism in a feedlot (Bourke 2000). A total of 126 steers and heifers out of 1565 died following 33 days of consumption of ergotized ryegrass seed in a 60% barley ration (no ergot alkaloid concentration given). Observations during the outbreak including that the animals were anorexic, failed to gain weight, and displayed symptoms of hyperthermia (lethargy, excessive salivation, and panting). Rectal temperatures of the affected cattle were between 41ºC to 43ºC. Ergotism was diagnosed after the outbreak, however no ergot alkaloid concentration in feed was given. In another study, steers fed a capsule of 180 mg/kg of ground ergot bodies developed hyperthermia within one week of ergot exposure (Bourke 2003). These steers only returned to normal after three weeks of consuming ergot-free feed. This study concluded that not only do ambient temperature and humidity influence hyperthermic ergotism, but so does exposure to high solar radiation. Lastly, hyperthermia was induced in dairy cows that were fed ergotized barley diets at a concentration of 10 µg ergot alkaloids/kg BW per day for 10 days (Al-Tamimi et al. 2003). This dosage corresponds to approximately 400 µg/kg per day based on 680 kg BW and 2% DMI.

1.3.4 Reproductive ergotism. Ergot has a long history of use in human obstetrics due to its modulating effect on uterine tonicity. Ergometrine has been used extensively to induce uterine contractions (i.e., its oxytocic action) and prevent excessive uterine bleeding following childbirth (de Groot et al. 1998). In animals, however, there is a spectrum of reproductive effects that have been observed. These adverse effects appear to be related to the ergopeptine alkaloids and not

19 necessarily ergometrine. The impact of ergot on female reproduction is better understood than male reproduction. The reproductive effects of ergot alkaloids (specifically fescue toxicosis) on female livestock have been recently reviewed by Poole and Poole (2019). Dysfunction in secretion of prolactin is problematic for lactating livestock. Insufficient prolactin, clinically defined as hypoprolactinemia, leads to underdevelopment of the mammary glands, which may progress to poor milk secretion or agalactia, the complete cessation of milk production (Diekman and Green 1992). In addition, prolactin is necessary for the maintenance of pregnancy and corpus luteum function through its interactions with progesterone (Evans 2002, 2011), thus making prolactin suppression problematic for pregnant mammals in general. Agalactia is commonly observed in ergot-exposed lactating livestock (Robbins, Porter, and Bacon 1986; Bacon et al. 1986; Diekman and Green 1992), although the severity of agalactia varies by species. Abortion has been observed in livestock exposed to ergot. Pigs and horses appear to be the most sensitive species to the reproductive effects of ergot. Numerous studies of swine demonstrate reduced reproductive performance following ergot exposure in feed. Abortion in livestock was reported in the scientific literature as early as 1945 (Nordskog and Clark 1945). A study of sows fed 1% ergot in their diet resulted in 100% agalactia and reduced live birth weights and survival of piglets (Schneider et al. 1986). Sows fed 1.5% ergot- contaminated sorghum (Claviceps africana; 7 mg ergot alkaloids/kg) experienced complete agalactia after farrowing (Kopinski et al. 2007). However, a study in which sows were fed 1.1 mg dihydroergosine/kg prior to farrowing and until piglet weaning did not find significant changes in lactation, piglet mortality, or prolactin (Kopinski, Blaney, and Downing 2008). Case studies of piggeries in Australia showed severe agalactia and high incidence of litter mortality following consumption of 5 to 40 mg ergot alkaloids per kg feed over varying periods of time (Blaney et al. 2000b). The adverse effects of ergopeptine alkaloids on equine reproduction have been reviewed previously (Evans 2002; Evans 2011; Riet-Correa et al. 2013); therefore, detail in the following section will be limited. Briefly, clinical signs of reproductive ergotism in horses include agalactia, prolonged gestation, dystocia, placental thickening and edema, neonate weakness and death, and premature placental separation (Riet-Correa et al. 1988; Ireland et al. 1991). In a case report of periparturient mares, C. purpurea sclerotia that comprised 0.22% of the tested ration

20 were implicated in poor mammary gland development and agalactia with subsequent foal mortality (Copetti et al. 2002). Ruminants appear to be more resistant to reproductive ergotism compared to swine and horses, as the associated symptoms are less severe overall. In cows, a suite of effects has been reported, including abortion (which appears to be rare), reduced calf birth weights, reduced calf weight gains, and impaired fertility from prolonged exposure to ergot from various Claviceps species and endophyte infected tall fescue. A study of cows that consumed C. purpurea ergot sclerotia in late pregnancy described abortion in 11/34 cows (Appleyard 1986). In ewes, exposure to 800 µg ergovaline and ergovalinine per kg dry matter from day 35 of gestation to parturition resulted in a decreased gestational length and concurrent reduction in lamb birth weight by 37% compared to control ewes (Duckett, Andrae, and Pratt 2014). Despite cases of ergot-induced abortion, the predominant adverse reproductive effects experienced by ruminants appear to be decreased milk production and reduced offspring performance.

1.4 Comparison of C. purpurea ergotism with fescue toxicosis in livestock. Ergot alkaloids have long been a cause for concern in the United States, but not necessarily due to C. purpurea. Of greater relevance to livestock health in the United States is a syndrome called fescue toxicosis. Tall fescue (Lolium arundinaceum) is a predominant forage for grazing animals and comprises over 15 million hectares of pasture in certain regions of the States (Hoveland 1993). The fungal endophytic species Neotyphodium coenephialum exists in a symbiotic relationship with tall fescue grasses. Neotyphodium coenophialum is a phylogenetic relative of the Claviceps genus and, consequently, produces ergot alkaloids (Schiff 2006; Mai and Li 2013; Tudzynski and Neubauer 2014). Ergovaline and ergometrine, the former of which is not found in C. purpurea sclerotia, are produced in the greatest quantities in endophyte-infected tall fescue (Figure 1.4). Ergovaline has been identified as the major toxic component of tall fescue (Robbins, Porter, and Bacon 1986; Bacon et al. 1986; Klotz et al. 2009; Klotz et al. 2007; Foote et al. 2012; Pesquiera et al. 2014). Interestingly, ergovaline has comparable in vitro potency to ergotamine (Klotz, Bush, and Strickland 2011). Further, ergocristine is the most abundant C.

Ergovaline

FIGURE 1.4. Structure of the ergopeptine alkaloid ergovaline that produced by the fungal endophyte E. coenophialum in endophyte-infected tall fescue. Ergovaline is considered the most toxic component of endophyte-infected tall fescue. Structure retrieved from Wikimedia Commons.

22 purpurea ergot alkaloid in cereal grains (Grusie et al. 2018a). Concentrations of 200 to 800 µg ergovaline per kg feed are associated with clinical signs of fescue toxicosis (Cornell et al. 1990; Hovermale and Craig 2001). Fescue toxicosis includes a suite of different syndromes: summer slump, fescue foot, and fat necrosis. Summer slump is the most common syndrome of fescue toxicosis observed. As the name suggests, it is most pronounced during months with high ambient temperatures (≥31 °C). Summer slump is characterized by impaired thermoregulation, leading to decreased productivity (i.e., decreased feed intake, weight gain, and average daily gain), hyperthermia, lethargy, and shade-seeking behaviour (Evans, Rottinghaus, and Casteel 2004). Other symptoms of summer slump include retained winter coats (further exacerbating the hyperthermia) and decreased reproductive efficiency. Fescue foot is mechanistically identical to classical gangrenous ergotism. As in gangrenous ergotism, mild hindlimb lameness progresses to severe hoof swelling and lameness, eventually terminating in ischemic necrosis and sloughing of appendages (Evans, Rottinghaus, and Casteel 2004). Lastly, fat necrosis (also known as lipomatosis) is unique to ruminants, in which abdominal and pelvic fat becomes necrotic. Decreased circulating prolactin is considered a biomarker of fescue toxicosis. There are clear similarities between ergot-of-rye (C. purpurea) poisoning and fescue toxicosis; severe fescue toxicosis is similar to classic gangrenous ergotism (Bacon et al. 1986; Henry and Bosch 2001). These diseases of livestock, however, should be considered distinct. A comparison of the two syndromes is summarized in Table 1.4. Research on fescue toxicosis and the involvement of the ergot alkaloid-producing fungal endophyte have provided a wealth of information of pathophysiological effects in livestock. Although the disease states are not identical, information from fescue toxicosis is highly useful in informing potential effects of C. purpurea exposure and at what concentrations effects may occur. The notation used to describe tall fescue with the fungal endophyte, i.e., toxic/ergot alkaloid producing tall fescue, is E+. Conversely, E- is commonly used to describe non-toxic fescue.

1.5 Ergot-mediated prolactin suppression in livestock Studies evaluating the effects of ergot alkaloid exposure in livestock, reduced prolactin is commonly reported to indicate the presence of ergot exposure. The following studies, while multiple endpoints being measured, have been included only to highlight the effect of ergot on prolactin. Numerous studies have established that ergot alkaloids are prolactin inhibitors due to the dopaminergic mechanism described earlier. However, what is lacking in current research, are feeding trials reporting the total mixture of

TABLE 1.4. Comparison of C. purpurea ergotism and E. coenephialum fescue toxicosis in cattle. Comparison based on information from Robbins, Porter, and Bacon 1986 and Evans, Rottinghaus, and Casteel 2004a,b. C. purpurea ergotism Fescue toxicosis Source of ergot alkaloids Claviceps purpurea (fungus) Epichloë coenophiala (fungal endophyte) Major substrate Rye, triticale, wheat, barley, most Tall fescue (Lolium arundinaceum) grasses Class of ergot alkaloids Ergopeptine Ergopeptine Principle causative ergotamine, ergocornine, ergovaline, ergocornine, agent(s) ergocryptine, ergocristine, ergosine ergocryptine, ergocristine, ergosine Mechanism of action Biogenic amine receptor agonists Biogenic amine receptor agonists Clinical symptoms ↓ feed intake and body weight gain Rough hair coat ↓ milk production Lethargy ↓ reproductive efficiency ↓ feed intake and body weight gain Tissue necrosis ↓ milk production Inability to dissipate body heat ↓ reproductive efficiency Sloughing of tail tips, hooves Tissue necrosis Inability to dissipate body heat Rough hair coat

Physiologic alterations ↑ blood pressure ↑ blood pressure ↓ prolactin concentration ↓ prolactin concentration ↑ respiratory rate, heart rate ↑ respiratory rate, heart rate Peripheral vasoconstriction Peripheral vasoconstriction Interaction with ambient temperature Yes Yes

Season of disease Summer – hyperthermic ergotism Summer (summer slump syndrome) emergence (in temperate regions; not Canada) Winter (fescue foot) Winter – gangrenous ergotism

24 ergot alkaloids and the minimum concentration in feed that reduces prolactin in pregnant and lactating livestock.

1.5.1 Early work. Ergot alkaloids were first described as prolactin inhibitors in the late 1960s. The inhibitory effect of ergot alkaloids on early pregnancy in rodents was known prior to that time (Shelesnyak 1958), but the mechanism of such activity was not described. A study in rats with mammary tumours found that ergocornine administration decreased both serum and pituitary prolactin, in addition to suppressing tumour growth and subsequent tumour development (Nagasawa and Meites 1970). A rat study from 1971 demonstrated that ergocornine suppressed prolactin release from an anterior pituitary graft in hypophysectomised and ovariectomized rats (Lu, Koch, and Meites 1971). Another early study revealed that subcutaneous administration of each ergocornine, ergonovine, ergotamine, ergocryptine depressed both prolactin secretion and lactation in postpartum rats (Shaar and Clemens 1972).

1.5.2 Administration of ergot alkaloids via injection. Some of the first work documenting prolactin suppression by ergot alkaloids in cattle was conducted in the early 1970s. Karg, Schams, and Reinhardt (1972) demonstrated that 2-Br--ergocryptine-methane-sulfonate (3 mL I.M. or S.C.) reduced plasma prolactin concentration in lactating cows. In a subsequent study, Schams, Reinhardt, and Karg (1972) found that 2-Br--ergocryptine-methane-sulfonate substantially reduced plasma prolactin prior to parturition. The authors concluded that the ergot alkaloid derivative interfered with the prolactin surge that is normally seen prior to parturition. Steers (n=7) administered a bolus intravenous dose of either ergotamine tartrate or ergonovine maleate (7 mg) had reduced serum prolactin as little as 30 minutes following dosing (Browning et al. 1997). Pregnant pony mares administered intramuscular injections of 800 µg/kg BW bromocriptine (per day) from gestational day 295 until parturition resulted in clinical signs similar to fescue toxicosis. Bromocriptine-exposed mares had prolactin concentrations of 12.35 ± 3.64 ng/mL compared to 29.04 ± 4.12 ng/mL in saline-control mares (Ireland et al. 1991). In addition, these bromocriptine-exposed mares had a prolonged gestation and displayed agalactia at foaling. Lactating Holstein cows (n=10) administered 80 mg ergocryptine (0.01 to 10 µg/mL vehicle) had prolactin concentrations of 1.3 to 1.4 ng/mL compared to 20 to 35 ng/mL in the vehicle control group, which corresponded to a 93.5% to 96% reduction of prolactin between these groups (Smith et al. 1974).

1.5.3 Feeding trials and grazing exposure. Numerous animal studies have described prolactin endocrinology following exposure to ergot alkaloids. The consensus is that prolactin is reduced in all livestock species of both sexes. A small selection of these studies will be described below. Toxic tall fescue studies of prolactin are numerous. Late gestational mares (n=30) in a 3- year tall fescue endophyte grazing trial were assessed for changes in serum prolactin (McCann et al. 1992). In two out of three years, mares that grazed the 100% infected tall fescue pasture had lower circulating prolactin compared to the mares that grazed the 0% infected pasture. Holstein calves fed toxic tall fescue had a mean prolactin concentration of 1.8 ± 0.1 ng/mL compared to 6.0 ± 1.2 ng/mL in the ‘less toxic’ fescue fed-calves, corresponding to a 70% reduction (Hurley et al. 1980). Unfortunately, alkaloid concentration was not stated in these studies, thus limiting the practical use of the information generated. Feeding trials in swine have provided robust information regarding ergot alkaloid concentration and prolactin depression. Following a 28-day feeding trial of 35 mg/kg (2.5% sclerotia) and 70 mg/kg (5% sclerotia) ergot alkaloids, growing pigs of both sexes had prolactin reduced by >80% (Blaney et al. 2000a). In a study of sows 6 to 10 days before farrowing, inclusion of 1.5% sorghum ergot (C. africana; 7 mg alkaloids/kg) in the diet resulted in a decreased prolactin compared to the control animals (Kopinski et al. 2007). This decline corresponded to no pre-farrow prolactin surge and complete agalactia in the sows. Consequently, entire litters from certain sows died. Kopinski et al. (2008) fed diets to mid-lactational sows containing 3% sorghum ergot (16 mg ergot alkaloids/kg feed) for 14 days, corresponding to 14 days post-farrow to weaning. Plasma prolactin was reduced in the ergot-exposed sows both at 7 days and 14 days of feeding compared to the control animals. Prolactin secretion is affected by multiple environmental and physical factors. These include stress, ambient temperature, photoperiod, mechanical stimulation (i.e., calf suckling, handling), and physiological status (i.e., pregnant or lactating, anestrus or diestrus, etc.) (Webb and Lamming 1981; Ben-Jonathan and Hnasko 2003, Auchtung et al. 2003). In an experimental setting, the dose and duration of exposure are important determinants. There is great individual variation among animals. Species differences among animals cannot be ignored; for example, horses are seasonal breeders whereas cattle can breed year-round. In horses and pigs, prolactin is the primary hormone associated with milk production, thus chronic ergot exposure often leads to complete and severe agalactia (Blaney et al. 2000; Bony et al. 2001; Cross 2003; Abdelrahim,

Richardson, and Gueye 2012). In cattle, however, growth hormone and prolactin are involved in milk production (Flint and Knight 1997; Vitala et al. 2006). Ergot alkaloids do not appear to affect growth hormone to the same degree as prolactin (Browning et al. 1997), therefore decreased milk production is seen in modest exposure situations compared to complete agalactia. Based on the aforementioned studies, it is clear that ergot alkaloids suppress circulating prolactin. What is not characterized, however, is the minimum concentration of ergot alkaloids that reduces prolactin, and at which point this reduction becomes clinically significant. One Canadian study in piglets attempted to find the minimum concentration of ergot alkaloids in feed that decreased prolactin. Weaned pigs (n=192) fed diets containing 1.04, 2.07, 5.21, 10.41, and 20.82 mg/kg ergopeptine alkaloids for 28 days all had decreased serum prolactin (Oresanya et al. 2003). Feeding of these diets were not associated with development of ergotism. Collectively, these results demonstrated that ergot alkaloids affect prolactin secretion at very low concentrations in the feed, i.e., 100 µg/kg, long before the development of clinical signs. Consequently, decreased circulating prolactin in livestock represents both a biomarker of exposure and physiological impact from ergot alkaloids in feed.

1.6 Ergot-induced peripheral vasoconstriction in livestock 1.6.1 In vitro and ex vivo studies of contractility. The development of in vitro bioassays using isolated arteries and veins of livestock has enabled the characterization of contractile potency of individual ergot alkaloids. In vitro bioassays are valuable for determining mechanisms of action of compounds, but lack applicability in a whole body system that has processes of metabolism and excretion. Nonetheless, a wealth of information on ergot alkaloids has been generated from in vitro vessel assays. The first in vitro vascular bioassay was conducted using isolated bovine dorsal pedal veins to determine the contractile potential of the ergot alkaloids ergotamine, ergosine, and agroclavine. The use of isolated bovine lateral saphenous veins became a widespread method to test for vasoconstrictive activity of ergot alkaloids. Oliver et al. (1998) used alpha-adrenergic receptor agonists phenylephrine (1) and BHT-90 (2) to elucidate mechanisms of vessel reactivity from cattle fed either E+ or E- experimental diets. Cattle fed E+ diets prior to slaughter had vessels that were more reactive to the 2-adrenoceptor agonist whereas treatment with the 1-adrenoceptor agonist produced similar responses between E+ and

E- animals. This led to the conclusion that vasoconstriction was mediated by binding to the 2

27 subtype of -adrenergic receptors. The authors also postulated that the vasoconstriction induced by E+ consumption likely affects tissue perfusion and heat dissipation in cattle and could likely account for the poor growth rates observed in animals experiencing fescue toxicosis. Research from the last decade using these in vitro bioassays has further characterized the potency of ergot alkaloids and has led to the establishment of a “bioaccumulation” hypothesis of effects in bovine lateral saphenous veins. Klotz et al. (2006) ruled out lysergic acid as the principle component of fescue toxicosis symptomatology when they discovered that it did not produce appreciable vasoconstrictive effects in bovine lateral saphenous veins. Later, ergotamine and ergovaline were compared using the same assay, which led to the determination of both compounds as potent vasoconstrictors that appeared to induce a gradual contractile response (Klotz et al. 2007). This gradual response was thought to be due to a slow dissociation of the ergot alkaloids from biogenic amine receptors (i.e., 5HT) that mediate vasoconstriction (originally postulated by Schöning et al. 2001; cited by Klotz et al. 2009, 2011). In theory, the gradual induction of vasoconstriction coupled with continual exposure to ergot alkaloids would result in a sustained peripheral vasoconstrictive effect, resulting in classic symptoms of fescue toxicosis (and ergot poisoning, for that matter) marked by altered thermoregulation (Klotz et al. 2009). Further research into this hypothesis found that repeated additions of ergovaline induced a dose-response effect on contractility (Klotz, Bush, and Strickland 2011). The bovine lateral saphenous vein bioassay was employed by Pesqueira et al. (2014) and determined the relative potency of the alkaloids ergometrine, -ergocryptine, ergocristine, and ergocornine. The authors found that the alkaloids each individually induced a contractile response at 1x10-7 M during the incubation period. However, ergometrine appeared to produce a more profound response as indicated by a much greater contractile intensity. The other three ergopeptines appeared to be similar in terms of vasospastic potency. Kudupoje et al. (2018) demonstrated the ex vivo vasoactivity of ergotamine in bovine lateral saphenous veins. In addition, the authors reported that adsorbents attenuated ergotamine- induced contractility in the vein preparations. The addition of mycotoxin binding agents, including adsorbents, remains an active area of research for mycotoxin mitigation strategies (Galvano et al. 2001). In an in vitro contractility study of bovine dorsal metatarsal arteries, the (S)-epimers ergotaminine, ergocorninine, ergocristinine, and ergocryptinine produced a contractile response

28 at 1x10-7 M (Cherewyk et al. 2020). In this study, contractile response followed the following pattern: ergotaminine > ergocorninine > ergocristinine > ergocryptinine. The results of these studies have not only illustrated the vasoconstrictive potency of ergot alkaloids in bovine in vitro models, but also illustrated the similarity in toxicity between C. purpurea alkaloids and E+ tall fescue alkaloids. However, few studies have studied mixture effects of ergot alkaloids in vitro as mixtures are most relevant to field exposures. In addition, in vitro studies have not been corroborated and validated with in vivo tests and thus results should be carefully considered.

1.6.2 In vivo studies of vasoconstriction. Doppler ultrasonography has been employed for studies of changes in peripheral blood flow due to consumption of E+ tall fescue, particularly in the American states of Kentucky and Tennessee. The Doppler Effect in ultrasonography has important diagnostic and clinical uses in both human and veterinary medicine. Doppler ultrasound uses the frequency shifts of sound waves reflecting off blood vessels to determine blood flow direction and velocity in an artery (Aiken and Strickland 2012). A signal transducer sends ultrasound waves into tissue where it interacts with red blood cells. This interaction results in the ultrasound wave being reflected and scattered back towards the transducer, where it is detected. Detection of blood flow velocity is achieved through the shift to a higher or lower frequency in the Doppler Effect. In colour flow Doppler, the average blood flow velocity is displayed in each pixel of the area of measurement. In general, red colour indicates flow towards the transducer and blue colour represents flow away from the transducer. Colour brightness is indicative of flow velocity. Within the last decade, Doppler ultrasonography has been applied as a research tool for assessing livestock exposure to ergot alkaloid producing tall fescue. This research has been generated almost exclusively in the United States of America, particularly in the states within the “fescue belt”. These states include Kentucky, Indiana, and Montana, in which tall fescue is a common livestock forage and over 90% of which appears to be infected with the ergovaline-producing endophyte Neotyphodium coenophiala (Porter and Thompson 1992; Browning 2004; Fayrer-Hosken et al. 2012). A recent review article details the significance of Doppler ultrasound in relation to livestock exposed to endophyte-infected tall fescue (Aiken and Strickland 2014). As described in the review, advantages of the method include real-time detection of changes in blood flow related to alkaloid exposure, it being a non-

29 invasive technique, reproducible, and an objective measure. Disadvantages are related to the skill required in both the execution of technique and the interpretation of results.

1.6.2.1 Cattle. Aiken et al. (2007) used colour Doppler ultrasound scanning to characterize the effects of E+ on blood flow in the caudal artery of beef heifers. The E+ diets contained 850 µg ergovaline and 360 µg ergovalinine per kg dry matter. Blood samples were also taken to assess the circulating concentrations of serum prolactin, which have been consistently measured as an indicator of fescue toxicosis (Karg, Schams, and Reinhardt 1972; Smith et al. 1974). Feeding E+ contaminated diets resulted in decreased caudal artery area in as little as four hours following initial feeding compared to baseline measurements. The luminal area remained decreased for the remainder of the study period. In addition, blood flow was found to be lower at 4 hours and 172 hours following initial feeding, indicating a sustained vascular effect occurred. Heart rate was decreased at 100 hours post-initial feed. Heifers on the E- diet did not have decreased caudal artery areas throughout the measurement period. An increase in caudal artery area was observed at 100 hours after initial feeding in these heifers. Heart rate and feed intake rate were not different from baseline for heifers consuming E- diets. In both groups, serum prolactin concentrations decreased at 4 hours following initial feeding, but was distinct in the E+ group. Prolactin appeared to be affected by ambient temperature. Consumption of the E+ diet resulted in a 10-fold decrease in serum prolactin concentration. The authors concluded that symptoms of fescue toxicosis were present within 4 hours of initial exposure and were related to peripheral vasoconstriction and decreased heart rate. A similar study by Aiken et al. (2009) found that an intermediate response in changes in peripheral hemodynamics occurred when beef heifers were fed a 1:1 mixture of E+ and E- compared to E+ and E- in isolation. The E+ diet contained 790 µg ergovaline per kg DM and the E+E- diet contained 390 µg ergovaline per kg DM. Decreased caudal artery luminal area was detected by colour Doppler ultrasound scanning at 27 hours for E+ and 51 hour for E+E- following initial feeding, respectively. In addition, blood flow was decreased for the treatments at 51 hours after feeding and persisted for 96 hours for the E+ and 48 hours for the E+E- heifers. Serum prolactin was also decreased in both groups containing ergovaline, but was observed at 27 hours after initial feeding for E+ compared to 51 hours after feeding for the mixed diet. These results confirm the role of ergovaline as the principle vasoconstrictive component of toxic tall fescue.

In a pen study (126 days) and grazing study (155 days), Aiken et al. (2015) detected vasoconstriction in both the testicular and caudal artery of yearling bulls consuming E+. The authors attributed the effect in the caudal artery as a sign of effectiveness of E+ treatment. Caudal artery and testicular artery luminal areas decreased by 42 and 40%, respectively, compared to bulls consuming nontoxic feed in the pen study. In the grazing study, caudal and testicular artery luminal diameters were 46% and 41% less than those of control arteries.

1.6.2.2 Horses. Douthit et al. (2012) assessed the effect of endophyte-infected fescue consumption on hoof perfusion and lameness in the horses. American Quarter Horses (n=12) were assigned to high and low endophyte treatment groups and assessed on days 0, 30, 60, and 90 of the 90-day feeding study. Animals were fed the low endophyte diet from days 0 to 30, then the treatment hay from days 30 to 60, and were then fed ground treatment feed until day 90. Estimated daily ergovaline consumption was 280 µg/kg in the high endophyte group and 18 µg/kg in the low endophyte group. The medial palmar artery (left forelimb) was imaged using Doppler ultrasonography to determine the diameter and blood flow. The authors found no consistently measured differences between treatment groups in serum prolactin, blood flow velocity, arterial diameter, or hoof temperature. Horses in the high endophyte group tended to show more lameness and hoof sensitivity compared to the low endophyte groups on day 60, although this effect was mostly attenuated by day 90. Overall, there was no observed changes in distal palmar arterial diameter or perfusion in horses receiving the E+ treatment. A study conducted by McDowell et al. (2013) examined vasoconstriction in horses (n=11) following exposure to E+ seed that was either whole or ground compared to E-. The E+ seed contained 4.93 mg/kg. The fescue seed was offered in the diet starting at 0.02% of body weight (76 µg/kg) on the first day of the study and up to 0.22% body weight (i.e., 713 µg/kg) on study days 11 through 14. Doppler ultrasonography was used to image the transverse view of the palmar artery of the left distal palmar artery was observed in both E+ exposed groups. The effect was greater in the ground seed E+ group, presumably due to enhanced bioavailability of the alkaloids in the gastrointestinal tract due to greater particle surface area. Overall, the authors detected a decrease in artery luminal diameter. Tendencies toward decreased area and circumference in the E+ group receiving ground fescue seed were also reported. Other hemodynamic parameters, including peak systolic velocity, end diastolic velocity, were not different among treatment groups. In addition, the authors did not detect any differences in

31 circulating prolactin concentrations among the groups. This result was attributed to non-pregnant mares generally having low serum prolactin concentrations and that alterations are expected in pregnant animals.

1.6.2.3 Small ruminants. In a crossover design from Aiken and Flythe (2014), fistulated goats (n=7) given diets containing either E+ or E- seed were assessed for vasoconstrictive responses at the carotid and auricular arteries with Doppler ultrasound. During the first exposure period, the mean luminal area of the carotid artery in the E+ group was approximately 38% smaller than that of the E- group. The vasoconstrictive response in the auricular artery was present but less pronounced than in the carotid artery. Decreased auricular artery diameter in the E+ group was observed on study days 2, 4, and 12. In the second exposure period (in which the diets were switched for each group), goats that were changed from the E- to the E+ group had decreased carotid luminal area compared to the E- group on days 2, 3,6, and 7 of the study. Vasoconstriction in the auricular artery of this group was observed on day 3 of the study and the study days thereafter. In general, constriction of these arteries due to E+ infusion in the rumen was observed within 2-3 days of the initial exposure. A recent study in fistulated goats from Aiken et al. (2016) assessed if the use of isoflavones could attenuate ergot-induced vasoconstriction of the carotid and interosseous arteries. In the context of the present review, the results of the isoflavone treatments will not be discussed. In the first portion of the study, goats received approximately 800 µg/kg DM ergovaline and ergovalinine. In the second portion, animals received 1100 µg/kg DM ergovaline and ergovalinine hay. In the absence of isoflavone infusion, luminal diameter of both arteries was decreased. These results indicate that the concentrations of ergovaline in the diets were sufficient to produce vasoconstriction in the goats. Lambs grazing endophyte-infected fescue pastures for 18 days were evaluated for constriction in their carotid and auricular arteries (Aiken, Sutherland, and Fletcher 2011). The concentrations of ergovaline in the AR6 pasture and wild-type endophyte ryegrass pasture were 550 and 100 µg/kg, respectively, at the beginning of the study. A linear decrease in luminal area of both arteries was detected in both E+ groups compared to the control. Following crossover of the exposed animals to the endophyte-free pasture (and vice versa), luminal area of the auricular artery increased over 9 days in previously exposed lambs whereas it decreased in lambs switched to the infected pasture. The increase in luminal area of the arteries suggests relaxation of the

32 endophyte-induced constriction. Carotid artery pulsatility index was greater in lambs on both endophyte-infected pastures compared to the lambs on endophyte-free pasture, indicative of increased resistance to flow. Other characteristics of blood flow, including peak systolic velocity, end diastolic velocity, and mean velocity, were not affected by treatment. The above hemodynamics data suggests that ergot alkaloids are vasoconstrictive in numerous peripheral vessels in ruminants at low-to-moderate concentrations (>100 µg/kg) as short as two days after feeding ergot-contaminated feed. Vasoconstriction in peripheral vasculature is consistent with the patterns of gangrene observed in gangrenous ergotism. In terms of other mammalian livestock and studies of vasoactivity, there are limited studies available on horses and no studies available in swine. 1.7 Pharmacokinetics of ergot alkaloids. Pharmacokinetic information on the ergot alkaloids in livestock is generally lacking. Further, understanding of pharmacokinetic profiles of ergot alkaloids following oral ingestion (i.e., the mode of potential livestock poisoning) is absent in the scientific literature. However, due to the use of ergotamine and dihydroergotamine as treatments for migraine headache in human patients, the pharmacokinetics of these compounds have been fairly well-described over the past 5 decades. In addition, interest in the illicit hallucinatory drug LSD, a derivative of lysergic acid, has led to pharmacokinetic characterization of this compound and its metabolites as well.

1.7.1 Absorption, Distribution, Metabolism, and Elimination. Pharmacokinetic data from human and livestock studies are summarized in Tables 1.5 and 1.6, respectively. As the ergopeptine alkaloids are moderately lipophilic compounds, they permeate across biological membranes with ease. For use of ergotamine as an anti-migraine treatment, Tfelt-Hansen et al. (2012) reported that oral absorption of ergotamine to be 60 to 70%. The degree of absorption of ergot alkaloids from livestock diets is unknown, however diet formulation appears to be an important contributor. In a study that assessed vasoconstriction in equine distal palmar arteries,

TABLE 1.5. Summary of ergot alkaloid and ergot alkaloid derivative pharmacokinetic data from human studies. The nomenclature and estimates of variance (if available) of pharmacokinetic parameters is taken from each individual study. Unit conversions have been made for consistency where possible. Abbreviations and terms are defined at the bottom of the table.

Ergot alkaloid or Route of administration, dose, and pharmacokinetic parameters Reference (analytical derivative method) Ergot alkaloids Ergotamine tartrate Oral (2 mg) Rectal (2 mg) I.M. (0.5 mg) Ala-Hurula et al. 1979a

-1 Cmax = 0.36 ± 0.1 ngmL Cmax = 0.42 ± 0.09 ng Cmax = 1.94 ± 0.34 ng (RIA) (SEM) mL-1 mL-1

tmax = 2 h tmax = 1 h tmax = 0.5 h Second peak: Second peak: 0.37 ± 0.09 ng mL-1 (48 0.77 ± 0.62 ng mL-1 (96

3 h) h) 4 Ergotamine tartrate Oral (2 mg – every 24 hr for 3 consecutive days) Ala-Hurula et al. 1979b

-1 Cmax = 0.35 ± 0.05 (SEM) ng mL (first dose) (RIA)

tmax = 1-2 h Accumulation hypothesized

th Cmax = 0.70 ± 0.10 ng/mL at 6 day post-administration Ergotamine tartrate Oral (1-4 mg): Ekbom, Paalzow, and Waldenlind 1981 ND in any plasma samples (detection limit 0.1 ng/mL) (HPLC) Estimated F <1% Ergotamine tartrate I.V. (0.25 mg) I.M. (0.25 mg) Oral (2 mg) Orton and Richardson 1982

-1 -1 ND at 240 min Cmax 1.5 ng mL Cmax = 0.5-0.6 ng mL (RIA)

t1/2, = 2.43 ± 1.03 (SD) tmax = 15 min tmax = 45-60 min min

t1/2, = 2.45 ± 1.0 h Ergotamine tartrate I.V. (0.5 mg) I.M. (0.5 mg) Ibraheem, Paalzow, and Tfelt-Hansen 1982 t1/2, = 3 min tmax= 10 min (HPLC) t1/2, = 1.86 h F = 46.6% (28.3-60.8%)

-1 -1 Cltotal,plasma = 0.6 L h kg

-1 Vd = 1.85 L kg Ergotamine tartrate Oral (2 mg) Rectal suppos. (2 mg) Rectal solution (2 mg) Ibraheem, Paalzow, Tfelt- Fmax~6% Hansen 1983 ND in plasma (10 min-54 Fmax = 4.7% h) AUC: solution > (HPLC) suppository

Fmax~2%

3 5 Ergotamine tartrate I.V. (0.5 mg) I.V. (0.25 mg) I.V. (0.125 mg) Ibraheem, Paalzow, and Tfelt-Hansen 1985 A = 27.61 ± 12.89 (SD) A = 21.04 ± 12.73 A = 16.57 ± 11.6 ng-1mL ng mL-1 (HPLC)  = 28.37 ± 8.30 h-1  = 32.57 ± 11.02 h-1  = 28.67 ± 4.10 h-1 B = 0.44 ± 0.08 ng mL-1 B = 0.29 ± 0.08 ng mL-1 B = 1.12 ± 0.42 ng mL-1  = 0.328 ± 0.031 h-1  = 0.354 ± 0.092 h-1  = 0.331 ± 0.056 h-1 t1/2, = 1.62 ± 0.48 min t1/2, = 1.44 ± 0.66 min t1/2, = 1.5 ± 0.18 min t1/2, = 2.13 ± 0.20 h t1/2, = 2.08 ± 0.55 h t = 2.15 ± 0.35 h 1/2, AUC = 2.04 ± 0.41 ng h AUC = 1.36 ± 0.41 mL-1 nghmL-1

AUC = 4.38 ± 1.40 Cl = 981.1 ± 189.3 Lh- Cl = 809.2 ± 276 L h-1kg- nghmL-1 1kg-1 1

-1 -1 -1 -1 Cl = 0.96 ± 0.26 L h kg Vd,b =2.99 ± 0.44 L kg Vd,b = 2.34 ± 0.74 L kg

-1 -1 -1 Vd, = 2.9 ± 0.78 L kg Vc = 0.13 ± 0.09 L kg Vc = 0.09 ± 0.07 L kg

-1 Vc = 0.17 ± 0.06 L kg BL:plasma=0.41-0.67 Ergotamine Oral (2 mg) Rectal (2 mg) Sanders et al. 1986 AUC = 61 ± 10 pg hmL-1 AUC = 1216 ± 198 pg h mL-1 (MS with direct probe

-1 -1 insertion) Cmax = 21.4 ± 2.5 pg mL Cmax = 454 ± 85 pg mL

tmax = 69 ± 39 min tmax = 50 ± 9 min

VZ/F = 6447 ± 1976 L t1/2,α = 10.5 ± 4.5 min 36

Frelative = 5% t1/2,β = 3.35 ± 0.53 h Cl/F = 2810 ± 677 L h-1

VZ/F = 550.1 ± 11.7 L Ergometrine maleate I.V. (0.075 mg) Oral (0.2 mg) de Groot et al. 1994 Two compartment model One compartment model (method not indicated)

t1/2, = 0.18 ± 0.20 h Abs. t1/2 = 0.19 ± 0.22 h

t1/2, = 2.0 ± 0.90 h t1/2, = 1.90 ± 0.16 h

Cl = 35.9 ± 13.41 L hr-1 Foral = 34-117%

Vss = 73.4 ± 22.1 L Hydrogenated ergot alkaloids

Dihydroergotamine Route not indicated (0.5 mg) Hilke et al. 1978

t1/2, = 1.35 min (RIA)

t1/2, = 23.2 min

-1 Vd = 0.25 L kg

-1 Cltotal,plasma = 93= 1562.8 L h

Dihydroergotamine I.V. (10 g/kg) Oral (10 mg) Little et al. 1982

-1 t1/2, = 13.8 ± 3.6 (SEM) AUC = 0.11-1.19 ng h mL (RIA) h Foral = 0.30-0.64% t1/2, = 2.37 ± 0.29 h Oral (20 mg) AUC = 13.4 ± 3.3 ng h AUC = 1.33-2.46 ng h mL-1 -1

3 mL 7

Foral = 0.20-0.86% Cltotal,plasma= 60.1 ± 10.1 L h-1 Oral (30 mg)

-1 -1 Vd = 14.5 ± 3.1 L kg AUC = 1.32-4.06 ng h mL

fe,u = 11.1 ± 1.0 % Foral = 0.16-0.94% Total urinary excretion: 1.1-7.3%

Dihydroergotoxine Oral solution (4.5 mg) Oral tablet (4.5 mg) Oral delayed release (4.5 Woodcock et al. 1985 mg) tmax 0.52 ± 0.24 h tmax 1.52 ± 1.73 h (RIA)

-1 -1 tmax 3.27 ± 1.7 h Cmax 527 ± 253 pg mL Cmax 378 ± 136 pg mL -1 Cmax 273 ± 116 pg mL t1/2,α 14.4 ± 14.4 min t1/2,α 16.8 ± 21.6 min

t1/2,β 2.02 ± 1.55 h t1/2,β 2.63 ± 1.64 h t1/2,α 54 ± 19.8 min

t1/2,γ 13.23 ± 5.04 h t1/2,γ 14.48 ± 6.15 h t1/2,β 2.46 ± 1.57 h

AUC 3594 ± 906 pg AUC 2570 ± 629 pg h t1/2,γ 12.25 ± 4.29 h hmL-1 mL-1 AUC 3044 ± 657 pg h -1 Frelative = 0.88 ± 0.21% Frelative = 0.78 ± 0.35% mL Dihydroergotamine Intravenous: Oral: Wyss et al. 1991 I.V. (0.5 mg) Oral (5 mg) (RIA) AUC = 9.25 ng h mL-1 AUC = 9.20 ng h mL-1

Vz = 2284 L Vz/F = 2586 L

-1 -1 Clplasma = 180 L h Clplasma/F = 2.04 L h Plasma protein binding = Oral (10 mg) 98.8%

AUC = 7.27 ng-h/mL 3

8 I.V. (0.1 mg) Vz/F = 2604 L -1 AUC = 7.31 ng h mL -1 Clplasma/F = 2.05 L h V = 2895 L z Oral (20 mg) Cl = 136.8 L h-1 plasma AUC = 6.48 ng-h/mL Plasma protein binding = 94.8% Vz/F = 2604 L -1 -1 Clplasma/F = 2.05 L h ClR = 9.6 L h -1 ClR = 6 L h t1/2, = 54 min

t1/2, = 60 min t1/2, = 12.1 h Blood:plasma = 0.41-0.89 t1/2, = 11.8 h

Plasma protein binding = 88.9-94.5%

Fabsolute=1% Dihydroergotoxine Oral tablet (9 mg) Oral solution (9 mg) Setnikar et al. 2001

mesylate -1 Cmax = 124 ± 16 (SEM) Cmax = 176 ± 16 pg mL (RIA) pg mL-1 tmax = 0.50 ± 0.04 h t = 1.15 ± 0.21 h max AUC = 779 ± 94 pg mL-1 AUC = 790 ± 93 pg h mL-1 t1/2, = 6.13 ± 0.76 h

t1/2, = 7.54 ± 1.23 h Dihydroergocristine Dihydroergocristine oral 8’-OH-dihydroergocristine Bicalho et al. 2005 (18 mg)

39 -1 Cmax = 5.63 ± 3.34 μg L (LC-MS/MS)

-1 Cmax = 0.22 ± 0.22 μg L tmax = 1.04 ± 0.66 h

tmax = 0.46 ± 0.26 h -1 AUC(last) = 13.36 ± 5.82 μg h L AUC(last) = 0.39 ± 0.41 μg h L-1 t1/2, = 3.90 ± 1.07 h

t1/2, = 3.50 ± 2.27 h Dihydroergotoxine Dihydroergocornine Bicalho et al. 2008 (dihydroergocornine, Dihydroergocryptine (LC-MS/MS) -ergocryptine, and - ergocristine) Dihydroergocristine (oral 27 mg)

-1 Cmax for all ~0.04 μg L Hydroxy-dihydroergocornine

-1 Cmax = 0.98 μg L

Hydroxy-dihydroergocryptine

-1 Cmax = 0.53 μg L Hydroxy-dihydroergocristine

-1 Cmax = 0.30 μg L Dihydroergotamine S.C. (0.5 mg) de Hoon et al. 2014

t1/2,abs = 0.11 ± 0.03 h (RIA)

-1 Cmax = 1419 ± 88 pg mL

tmax = 20 min

t1/2,λ1 = 55.2 ± 9.6 min

4 t (elim.) = 5.63 ± 1.15 h

0 1/2,λ2

Cl = 95 ± 9 L h-1

Vd = 739 ± 114 L AUC = 5825 ± 667 pg mL-1 Note: estimate of variance not indicated Dihydroergotamine, I.V. and Oral (dose not indicated) Aellig, Nüesch 1977 Dihydroergotoxine, C ~1.52 ng mL-1 max (Tritium labelling) Dihydroergostine, tmax~2 hrs

Dihydroergocornine, Invasion t1/2= 0.5 h

Dihydroergovaline, Elimination t1/2= 1.4-6.2 hr (α); 13-50 hr (β) Dihydroergonine, Quotient of absorption: ergotamine, 1- 25-30% for dehydrogenated EAs (except dihydroergovaline, dihydrogergonine)

methyl-ergotamine, 66% ergotamine and 1-methyl-ergotamine bromocriptine ~100% bromocriptine

Lysergic acid diethylamide and metabolites LSD Oral (200 μg) Dolder et al. 2016

-1 Cmax = 4.5 ± 1.4 (SD) ng mL (LC-MS/MS)

tmax = 1.5 h First order kinetics

t1/2, = 3.6 ± 0.9 h (up to 12 h)

t1/2, = 8.9 ± 5.9 h

4 -1

1 AUC0-24= 26 ng h mL

-1 AUC∞= 28 ng h mL

-1 ClR= 4.7 ± 2.2 L h

fe,u LSD = 1%

fe,u O-H-LSD = 13% LSD Oral (100 μg) Oral (200 μg) Dolder et al. 2017

-1 -1 Cmax = 1.3 ng mL Cmax = 3.1 ng mL (LC-MS/MS)

tmax = 1.4 h tmax = 1.5 h

t1/2 = 2.6 h LSD + O-H-LSD Oral (100 μg) O-H-LSD Holze et al. 2019

-1 First order kinetics Cmax = 0.11 ng mL (LC-MS/MS)

-1 Cmax 1.7 ng mL tmax = 5 h

tmax = 1.7 h t1/2 = 5.2 h

-1 t1/2 = 3.6 h AUC∞ = 1.7 ng h mL

-1 AUC∞ = 13 ng h/mL

-t -t -t - Abbreviations defined: A, pre-exponential constant (Cp=Ae + Be ); , exponential constant (Cp=Ae + Be ); AUC, area under the

plasma concentration versus time curve; AUC∞, area under the plasma concentration versus time curve extrapolated to infinity; AUC0-

24h, area under the plasma concentration versus time curve from 0 to 24 hours; AUClast, area under the plasma concentration versus

-t -t -t - time curve extrapolated to the last time point; B, pre-exponential constant (Cp=Ae + Be ); b, exponential constant (Cp=Ae + Be );

Cl, systemic clearance; Clplasma, plasma clearance; ClR, renal clearance; Cmax, maximum plasma concentration of drug; F,

bioavailability; Foral, oral bioavailability; Frelative, relative bioavailability; fe,u, fraction of drug excreted in urine; HPLC, high

performance liquid chromatography; LC-MS/MS; liquid-chromatography (tandem) mass spectrometry; LSD, lysergic acid 4

2 diethylamide; ND, non-detectable; O-H-LSD, 2-oxo-3-hydroxy-lysergic acid diethylamide; RIA, radioimmunoassay; tmax, time at

which Cmax occurred; t1/2abs, absorption half-life; t1/2, (or t1/2,λ1), distribution half-life; t1/2, (or t1/2,λ2), elimination half-life; VC, volume

of the central compartment; Vd (or Vz or Vd,), volume of distribution; Vss, volume at steady state concentration

TABLE 1.6. Summary of pharmacokinetic parameters in livestock for intravenous administration of ergovaline tartrate. Detection of ergovaline in blood in each study was by high performance liquid chromatography. Pharmacokinetic Jaussaud et al. 1998* Bony et al. (2001)** parameters n=4 sheep n=4 gelding horses Ergovaline (17 µg/kg BW) Ergovaline (15 µg/kg BW)

Exponential  0.751 ± 0.389 λ1 1.690 ± 0.354 constants (min-1)  0.029 ± 0.003 λ2 0.237 ± 0.118

λ3 0.012 ± 0.002

Pre-exponential constants A 122.87 ± 45.76 A1 251.99 ± 105.12 (ng mL-1) B 18.59 ± 4.48 A2 14.32 ± 7.79

A3 7.60 ± 0.83 t1/2 (min) 23.60 ± 2.81 56.83 ± 13.48 Cl (L h-1kg-1) 1.2 ± 0.06 1.2 ± 0.24 AUC (ng min mL-1) 820.9 ± 74.76 828.78 ± 139.61 MRT (min) 26.48 ± 3.88 62.65 ± 20.86

-1 Vss (L kg ) Vss 0.56 ± 0.10 N/A

-1 Vc (L kg ) N/A Vc 0.062 ± 0.024

-1 C0 (ng mL ) N/A 273.92 ± 115.52

AUC, area under the plasma concentration time curve; A, y-intercept(s); B, y-intercept;  or λ, absorption rate constant;  or λ, elimination rate constant (= 0.693/t1/2); C0, concentration of drug at time

= 0 (A1+A2+A3); MRT, mean residence time; Vss, volume at steady state concentration; Vc, volume of the central compartment (Vc = dose/C0)

* kinetic model used by Jaussaud et al. 1998: C = Ae-at + Be-bt

–λ1t –λ2t -λ3t ** kinetic model used by Bony et al. 2001: C = A1e + A2e + A3e

43 dosing the animals with ground fescue seed produced a more pronounced effect compared to whole seed (McDowell et al. 2013). Ground seed had a greater surface area for absorption in the equine gastrointestinal tract. Hill et al. (2001) described transport of the water-soluble ergoline alkaloids across ruminant forestomach epithelia but could not demonstrate this for the lipophilic ergopeptine alkaloids. In fact, it appears that ergopeptine alkaloids reduce ruminal blood flow, thereby decreasing nutrient absorption (Foote et al. 2013). In addition, there is some evidence to suggest that ergot alkaloids cross the blood-brain-barrier (Mulac et al. 2012). Further research is required to characterize the absorption of ergot alkaloids.

Pharmacokinetic parameters that describe the absorption of drugs are Cmax, tmax, and area under the plasma concentration versus time curve (i.e., area under the curve; AUC). As most of the pharmacokinetic data on ergot alkaloids is from human trials, human data will be relied upon heavily in the following discussion. In general, ergot alkaloids appear to be rapidly absorbed, with tmax values for ergotamine tartrate (2 mg) occurring within 1 to 2 hours following oral administration (Ala-Hurula et al. 1979a,b; Orton and Richardson 1982; Sanders et al. 1986). The reported Cmax values of ergotamine tartrate (2 mg) in humans following oral exposure is within 0.35 ng/mL to 0.6 ng/mL. The AUC, which estimates the total body exposure to a compound, for ergotamine in humans is 1.36 to 4.38 ng-hr/mL for I.V. (0.125, 0.25, and 0.5 mg doses; Ibraheem, Paalzow, and Tfelt-Hansen 1985) and 0.061 ng-hr/mL for oral (2 mg dose; Sanders et al. 1986) administration, respectively. For dehydrogenated ergot alkaloids, oral Cmax and tmax are 40 to 527 ng/mL and 0.46 to 3.27 hr, respectively, for doses between 4.5 and 27 mg (Woodcock et al. 1985; Setnikar et al. 2001; Bicalho et al. 2005, 2008). Similarly, LSD reaches its Cmax (1.7 to 4.5 ng/mL) within approximately 1.5 hours after oral administration (100-200 µg doses; Dolder et al. 2016, 2017; Holze et al. 2019). As the onset of pharmacological effects of ergot alkaloids, whether therapeutic (in human medicine) or unintended side-effects (e.g., peripheral vasoconstriction in humans and domestic animals), occurs within hours to days of administration, the compounds appear to be well- distributed to their target sites of action. This is corroborated by the large volume of distribution

(Vd or Vz) values available in the literature. Reported Vd values for humans range from 1.85 L/kg to 2.9 L/kg for ergotamine administered I.V. (corrected for body weight; Ibraheem et al. 1983, 1985), 14.5 L/kg to 2284 L for I.V. dihydroergotamine (Little et al. 1982; Wyss et al. 1991). Ergot alkaloids appear to distribute to blood cells to a certain degree, as reported values for

44 blood:plasma ratio are 0.41-0.89 (Ibraheem, Paalzow, and Tfelt-Hansen 1985; Wyss et al. 1991). Ergot alkaloids may also bind to plasma proteins, which would contribute to a longer biological half-life (discussed below). Wyss et al. 1991 reported that dihydroergotamine administered I.V. and orally resulted in plasma protein binding between 88.9 to 99.8%. Oral bioavailability in the ergot alkaloids is reportedly low (<2%) due to first-pass metabolism in the intestine and liver (Aellig and Nuesch 1977; Eckert et al. 1978; Ekbom, Paalzow, and Waldenlind 1981; Perrin 1985; Tfelt-Hansen et al. 2000, 2012). Bioavailability, the fraction of the parent compound that reaches the systemic circulating unchanged following pre-systemic metabolism, estimates the amount of drug available to elicit pharmacological effect (assuming all pharmacologic effect comes from the parent compound). Pre-systemic and systemic metabolic transformation of ergot alkaloids occurs in intestinal epithelial cells and predominantly within the liver through the cytochrome p450 enzyme (CYP) system and phase II conjugation reactions (Moubarak and Rosenkrans 2000; Tfelt-Hansen et al. 2000). In particular, the CYP3A4 enzyme system is principally responsible for metabolism of ergot alkaloids (Tfelt- Hansen et al. 2000) and is responsible for the metabolism of more than 50% of drugs (Guengerich 1999). Human studies have indicated that CYP3A4 inhibitors administered concomitantly with ergotamine for migraine therapy lead to development of peripheral vasoconstrictive symptoms consistent with ergotism (Hayton 1969; Horowitz, Dar, and Gomez 1996; Caballero-Granado et al. 1997; Liaudet 1999; Dresser et al. 2000; Rostoff et al. 2010). Hepatic microsome studies have further elucidated the role of CYP3A metabolism of ergot alkaloids in humans and animals. Peyronneau et al. (1994) indicated that ergotamine, ergocryptine, ergocristine, bromocriptine, as well as derivatives (dihydro-ergotamine, - ergocryptine, and – ergocristine) strongly interacted with rat CYP3A and human CYP3A4 enzymes. Specifically, the tripeptide ring system of the ergopeptines was important for recognition by CYP3A isozyme (Peyronneau et al. 1994). Moubarak et al. (2000) reported that ergotamine had a high affinity for bovine hepatic microsomal CYP3A and its metabolism produced hydroxy and dihydroxy metabolites. A gene expression study by Settivari et al. (2008) noted increased hepatic expression of numerous CYP isoforms (CYP1A1, CYP2C9, CYP2E1, and CYP3A1) in rats following dietary ergovaline exposure. Hydroxylation, the P450-catalyzed addition of a hydroxyl group to the parent compound, of ergot alkaloids has been demonstrated by in vitro studies in rat (dihydroergotamine - Bauermeister et al. 2016), mouse (ergotamine -

Duringer et al. 2005), and bovine (ergotamine - Moubarak et al. 2000; dihydroergocristine/dihydroergotoxine - Bicalho et al. 2005, 2008) hepatic microsomes. Hydroxylation of ergot alkaloids has also been demonstrated by cell culture studies, including immortalized HT29 colon cancer cells (Mulac et al. 2011) and human hepatocytes (Althaus et al. 2000). Hydroxylation appears to occur at the C9 position of the ergoline ring moiety for ergopeptine alkaloids (Rudolph et al. 2018). In contrast, some evidence suggests that ergot alkaloids inhibit CYP450 activity (Moubarak et al 2003a,b; Rosenkrans and Ezell 2015). Therefore, further investigation on the metabolism of ergot alkaloids is necessary. Ergot alkaloids and their metabolites are excreted via urine or bile, depending on polarity and size of the compound. Stuedemann et al. (1998) reported that 96% of ergot alkaloids consumed by steers were eliminated in the urine following a 126-day E+ grazing trial. In horses that grazed E+ infected pastures, Schultz et al. (2006) noted that lysergic acid was the predominant alkaloid excreted in urine, although some fecal excretion of ergovaline also occurred. In both studies, the authors postulated that ergot alkaloids were metabolized to lysergic acid prior to urinary excretion. Clearance, the volume of blood cleared of a drug per unit time, estimates elimination of a drug from the body. Total plasma clearance for ergotamine following I.V. administration in humans ranges from 0.66 L/hr-kg to 0.955 L/hr-kg (corrected for body weight; Ibraheem, Paalzow, and Tfelt-Hansen 1982, 1985). Renal clearance of dihydroergotamine in humans is 9.6 L/hr and 6 L/hr for I.V. and oral administration (assuming

Foral=1%), respectively (Wyss et al. 1991), indicating the importance of the kidneys in excretion of ergot alkaloids. The renal clearance of LSD is 47.4 L/hr, with 1% of LSD and 13% of its main metabolite (O-H-LSD) being excreted in the urine (Dolder et al. 2016). Hepatic and biliary excretion plays a role as well – the rapid disappearance of the ergot alkaloids from the blood is due to extraction by liver (Cheeke 1998). Molecular weight of the ergot alkaloids may influence their route of excretion. Eckert et al. 1978 suggested that the larger ergopeptine alkaloids undergo biliary excretion whereas smaller ergoline alkaloids are excreted in urine. Milk does not appear to be a route of excretion for ergot alkaloids (Cheeke 1998; Durix et al. 1999; Schumann et al. 2009), suggesting limited food safety concerns for humans. It should be noted that first pass metabolism may produce bioactive metabolites (Müller-Schweinitzer 1984), however this will be discussed later (see section 1.7.2).

Half-life is used to estimate the time required for irreversible elimination of the drug from the body. Most ergot alkaloid pharmacokinetics studies report two phases of plasma decline for ergot alkaloids – an alpha () phase and a beta () phase. The  phase, or distribution half-life, is the time required for plasma concentrations of a drug to decrease due to distribution into peripheral tissues (Teboul and Chouinard 1990). Distribution half-life for ergotamine ranges from 1.4 to 3 minutes (Orton and Richardson 1982; Ibraheem, Paalzow, and Tfelt-Hansen 1982, 1985) for intravenous administration (Sanders et al. 1986). For dihydrogenated ergot alkaloids, the  phase ranges from 1.4 to 2.6 minutes (Hilke et al. 1978; Woodcock et al. 1985). The second half-life, the  phase or terminal/elimination half-life, is decreasing plasma concentrations of the drug due to elimination processes (Teboul and Chouinard 1990). Reported elimination half-life ( phase) values of ergotamine tartrate in humans ranges from 2 to 3 hours (Orton and Richardson 1982; Ibraheem, Paalzow, and Tfelt-Hansen 1982, 1983; Sanders et al. 1986). For dihydrogenated ergot alkaloids, elimination half-life values in humans ranges from 2 to 12 hours (Little et al. 1982; Woodcock et al. 1985; Wyss et al. 1991; Setnikar et al. 2001; Bicalho et al. 2005; de Hoon et al. 2014). One study reported a third phase of elimination for dihydroergotoxine (i.e., a gamma phase of 12 to 15 hours) (Wyss et al. 1991). The gamma phase may be related to slow dissociation from receptors and resdistribution from tissues into the systemic circulation. Based on this information, ergot alkaloids do not remain detectable in plasma for very long. However, as will discussed below (section 1.7.2), the pharmacologic effect of ergot alkaloids remains despite apparent undetectable levels of the alkaloids in plasma.

1.7.2. Pharmacokinetics versus pharmacodynamics of ergot alkaloids. A discrepancy between plasma concentrations and pharmacological effect of ergot alkaloids has been noted (Müller-Schweinitzer and Rosenthaler 1987; Bigal and Tepper 2003). Studies often cite that the vasoconstrictive activity of the ergot alkaloids long persists after the compounds are low or undetectable in blood (Tfelt-Hansen, Eickhoff, Olesen 1980; Tfelt-Hansen 1988; Bigal and Tepper 2003). In isolated human coronary arteries, ergotamine-induced contractility persisted despite numerous washes (Maassen Van Den Brink et al. 1998). In beagles administered dihydroergotamine, Müller-Schweinitzter and Rosenthaler (1987) described a greater duration of venoconstrictor effect that expected based on the half-life of dihydroergotamine. An in vitro study of canine femoral and saphenous veins demonstrated that dihydroergotamine caused

47 increased vessel tension that remained for hours after the drug was removed from the incubating solution (Müller-Schweinitzer 1979). As described in the in vitro contractility section earlier (section 1.6.1), in vitro studies with animal tissues also demonstrated that contractility of various ergopeptine alkaloids is prolonged. de Hoon et al. (2014) performed a pharmacodynamics study following S.C. administration of dihydroergotamine to human volunteers and described decreased brachial artery diameter and compliance long after the maximum plasma concentration was reached (tmax ~20 minutes post-administration). It has been speculated that slow diffusion from receptors (Bülow et al. 1986; Müller-Schweinitzer 1987; Tfelt-Hansen and Johnson 1993) and/or the formation of active metabolites contribute to the prolonged constrictor effect (Müller- Schweinitzer 1987; Müller-Schweinitzer and Rosenthaler 1987; Wyss et al. 1991). Muller- Schweinitzer (1984) also suggested accumulation of active compound at target receptors in smooth muscle cells.

1.7.3. Active metabolites. As an important but brief aside, the potential pharmacological activity of ergot alkaloid metabolites should be acknowledged. Pharmacological and pharmacokinetic activity of metabolites of the parent ergot alkaloids are even less described than those of the parent compounds. Perrin (1985) attributed the apparent contraction between plasma concentration and pharmacodynamic effect of ergotamine in the treatment of migraine headache to either high potency of ergotamine or active metabolites. One study suggested venoconstriction of canine saphenous veins was due to incubation in ergosine or its epimer ergosinine (Müller- Schweinitzer, Ellis, and Ziegler 1992); however, the pharmacologic effect of ergosinine was attributed to reconversion to ergosine. A study of conscious beagles administered DHE found that local administration of some DHE metabolites constricted the saphenous vein (Müller- Schwienitzer 1987). Some studies have also hypothesized accumulation of parent compound at the site of action (Ala-Hurula et al. 1979b) but could not discount the possibility of formation of active metabolites. Hydroxylated metabolites of ergot alkaloids have been described kinetically more recently (Bicalho et al. 2005, 2008); however, pharmacodynamic studies have not been conducted on these metabolites. More recently, ergot alkaloid epimers have received considerable interest due to the vasoactive potential. As such, the European Food Safety Authority has indicated that the ergopeptine alkaloids epimers (ergotaminine, ergosinine, ergocryptinine, ergocorninine, and ergocristinine) as also alkaloids of interest for monitoring in food and feed (EFSA 2012).

1.7.4 Livestock pharmacokinetics studies. A limited number of pharmacokinetic studies have been conducted in livestock using intravenous injection of ergovaline (Table 1.6). Jaussaud et al. (1998) characterized the kinetics of ergovaline in four sheep following a bolus I.V. injection of 17 µg/kg body weight. Ergovaline was quantified in serum using HPLC, the methodology of which was the focus of their publication (limit of quantitation: 3.5 ng/mL; limit of detection: 1.2 ng/mL). The authors found that ergovaline was rapid cleared from the blood, with an elimination half-life of 23.6 minutes. The kinetics followed a two-compartment model. Total clearance of ergovaline was 0.020 L/min-kg. Durix et al. (1999) developed an HPLC method for quantification of ergovaline in goat milk; however, they also described the kinetics of ergovaline in plasma of goats following administration of 32 µg/kg BW. The kinetics of ergovaline in lactating goats (n=4) followed a two-compartment model with a biphasic elimination. Excretion of ergovaline was rapid, as indicated by plasma concentrations reaching the limit of quantitation (3.5 ng/mL) within 1-2 hours of dosing. The elimination half-life of ergovaline and total clearance were found to be 32.41 minutes and 2.0 L/h-kg, respectively. The authors concluded that these parameters were in the same order of those found by Jaussaud et al. (1998) and were comparable between the species. The details of these kinetic parameters, however, were not published. Bony et al. (2001) also used HPLC to quantify ergovaline in the blood of gelding horses (n=4) following a bolus I.V. administration of 15 µg/kg BW. In contrast to the results of Jaussaud et al., ergovaline kinetics appeared to follow a three-compartment model. The elimination half-life of ergovaline from plasma was 56.83 minutes. The total clearance of ergovaline was 1.2 L/hr-kg. Initial comparison of the results described in these three studies suggests that total ergovaline clearance is comparable among sheep, goats, and horses. Interestingly, horses appear to have a longer half- life of elimination of ergovaline compared to the other two species. This may partially explain the increased susceptibility of horses to ergot exposure in addition to their prolactin physiology. One study is available in cattle that assesses a broader variety of ergot alkaloids with high performance liquid chromatography detection. Moubarak et al. (1996) individually administered ergosine, ergotamine, and ergine were intravenously to Holstein calves. Ergosine was given at doses of 1.8 and 7 µg/kg BW. Ergotamine and ergine were each given at 14 µg/kg BW (n=4/alkaloid). Classical kinetic parameters were not described in this study; however, useful information regarding the kinetic profile of these alkaloids was available. The authors reported

49 that all alkaloids reached Cmax in serum within 30 minutes of injection. Specifically, ergosine was at its highest in serum at five minutes post-injection and rapidly declined 10 to 15 minutes thereafter. The higher dose of ergosine appeared to elicit a slower elimination. Serum ergotamine concentration peaked at five minutes post-injection and declined to a plateau for the following 20 minutes. Ergotamine concentration was nearly zero for the rest of the time period of collection. Ergine concentration also peaked within five minutes of dosing. These absorption observations are contrary to commonly-held pharmacokinetic principle that absorption is 100% following intravenous administration of a drug, meaning a drug is at Cmax at the time of I.V. administration. It is unclear why the authors reported intravenous pharmacokinetic information in this manner. The authors postulated that these ergot alkaloids appeared to follow a three-phase pharmacokinetic model. Collectively, these results indicate that ergot alkaloids are rapidly excreted by calves when dosed intravenously. This agrees with the aforementioned studies in horses, sheep, and goats. It should be noted that ruminant livestock are considered more resistant to the effects of mycotoxins than monogastric species due to ruminal detoxification (Kurmanov 1977; Fink- Gremmels 2008); however, few dedicated studies on ruminal detoxification of ergot alkaloids are currently available. One study in sheep described ruminal metabolism of ergovaline to lysergic acid (Duringer et al. 2007), suggesting that ergopeptine alkaloids are preliminarily degraded in the rumen, from which they cross the intestinal epithelia and distribute to the liver for further degradation.

1.7.5 Detection of ergot alkaloids in blood. To conduct pharmacokinetic studies and analysis, methods for detection and quantification for ergot alkaloids in biological fluids is necessary. Many review papers are available that discuss analytical methods to detect ergot alkaloids in feed and biological fluids (Flieger, Wurst, and Shelby 1997; Jegorov 1999; Komarova and Tolkachev 2001; Scott 2007; Krska and Crews 2008; Crews 2015; Arroyo-Manzanares et al. 2017). However, a short description of this topic will be provided below with emphasis on detection in blood (including plasma and/or serum) as this matrix is the scope of this dissertation. A review on analytical techniques for all mycotoxins has been published recently (Agriopoulou, Stamatelopoulou, and Varzakas 2020).

Numerous technologies that have been used to detect ergot alkaloids and their various derivatives in blood, plasma, and serum. These include radio-labelling (Meier and Schrier 1976; Aellig and Nüesch 1977), radioimmunoassay (RIA) (Kleimola 1978; Ala-Hurula et al. 1979a,b; Schran et al. 1979; Orton and Richardson 1982; Collignon and Pradelles 1984), enzyme-linked immunosorbent assay (ELISA) (Scott et al. 1994); HPLC (Edlund 1981; Žorž et al. 1985; de Groot et al. 1994; Moubarak et al. 1996; Jaussaud et al. 1998; Bony et al. 2001), and, most recently, (HP)LC-MS(/MS) (See Table 1.7). Mass spectrometry (MS) is useful for mycotoxin analysis due to its high specificity and sensitivity (Eckers and Henion 1985). Mass spectrometry produces characteristic fragmentation patterns for a given analyte that allows for its identification. In contrast to gas chromatography, liquid chromatography (LC), particularly high-performance liquid chromatography (HPLC), is useful for bioactive compounds such as toxins, drugs, and peptides (Eckers and Henion 1985). Coupling (HP)LC to MS allows for very specific and sensitive characterization of compounds, making this method the current state-of-the-art detection for mycotoxins (Krska et al. 2008a; Agriopoulou, Stamatelopoulou, and Varzakas 2020). In addition, (HP)LC-MS(/MS) is considered the current ‘gold standard’ for simultaneous determination of mycotoxins in complex martrices (Krska et al. 2008; Leite et al. 2020; Agriopoulou, Stamatelopoulou, and Varzakas 2020). A summary of LC-MS and LC-MS/MS methods for detecting ergot alkaloids and various ergot alkaloid derivatives in blood, plasma, and/or serum are provided in Table 1.7. In general, the limits of detection and quantification of ergot alkaloids are generally within the pg/mL to ng/mL range. For the ergopeptine alkaloids, reported LODs and LOQs in plasma range from 0.05 to 5 ng/mL and 0.1 to 10 ng/mL, respectively (human - Favretto et al. 2007b; equine - Rudolph et al. 2018). The LOQs dihydrogenated ergot alkaloids range from 0.04 to 25 pg/mL (Chen et al. 2002; Friedrich et al. 2004; Bicalho et al. 2005; El-Din et al. 2016). The data summarized in Table 1.7 indicate that detection of ergot alkaloids and derivatives are quantifiable reliably in blood, plasma, and serum. However, there are no studies available that quantify ergot alkaloids in livestock blood samples with following oral exposure. The low oral bioavailability of ergot alkaloids and the uncertainty surrounding the contribution of active metabolites to toxicity makes analytical detection and pharmacokinetic characterization of these compounds difficult. However, there would be great diagnostic utility to detect ergot alkaloids in blood of putatively exposed or intoxicated animals. Should a reliable and accurate analytical

TABLE 1.7. Summary of liquid chromatographic-mass spectrometric methods for detecting and quantifying ergot alkaloids or ergot alkaloid derivatives in blood, plasma, and/or serum. All samples are from human subjects unless indicated otherwise. Abbreviations are defined at bottom of the table. Ergot alkaloid or Method of extraction, detection, Method validation parameters (linear range, limit Reference ergot alkaloid and specimen used of detection, etc.) derivative Ergopeptine alkaloids Ergotamine Liquid-liquid extraction Linearity: 10 – 10 000 pg/mL Favretto et al. LC-MS/MS (+ESI) LOD: 5 pg/mL 2007b Blood LLOQ: 10 pg/mL

Recovery: 82-110%

Ergocristine, UHPLC-HRMS/MS LODs: 0.05-0.5 ng/mL Rudolph et al. ergocryptine, (+ESI and PRM mode) LOQs: 0.1-1.0 ng/mL 2018 ergotamine, Equine serum Linearity: 0.1-50 ng/mL ergovaline Recovery: >73.6% Semi-synthetic analogs Bromocriptine Solid phase extraction Linearity: 2-500 pg/mL Salvador et al. LC-MS/MS (MRM, +ESI) LLOQ: 2 pg/mL 2005 Plasma

Bromocriptine Liquid-liquid extraction Linearity: 0.1-10 ng/mL Rudy and Dixon LC-MS (+ESI) 2008 Equine plasma

Cabergoline Protein precipitation Linearity: 5-250 ng/mL Igarashi et al. LC-MS/MS (MRM, +ESI) LOD: 2 pg/mL 2003 Hydrogenated and methylated ergot alkaloid derivatives Dihydroergotamine Liquid-liquid extraction Linearity: 10-1000 pg/mL Chen et al. 2002 LC-MS/MS (+ESI) LOQ: 10 pg/mL

Plasma Recovery: 58% 5

3 Dihydroergocristine LC-MS/MS LOQ: 10 pg/mL Bicalho et al.

Plasma 2005

Dihydroergocryptine LC-MS/MS (SRM, +ESI) Linearity: 25-1000 pg/mL Friedrich et al. Plasma LOQ: 25 pg/mL 2004 Recovery: 99%

Dihydroergotoxine Liquid-liquid extraction Linearity: 0.05-40 ng/mL El-Din et al. 2016 HPLC-MS/MS (+ESI, MRM) LLOQ: 0.04-1 ng/mL Plasma Recovery: >86.16%

Methylergonovine Liquid-liquid extraction Linearity: 0.025-10 ng/mL Gao et al. 2016 LC-MS/MS (+MRM) LLOQ: 0.025 ng/mL Plasma Lysergic acid diethylamide and metabolites LSD, iso-LSD LC-MS (+ESI) LLOQ: 0.02 mg/L Canezin et al. 2001 LSD, iso-LSD Liquid-liquid extraction LOQ: 0.01 mg/kg Johansen and LC-MS (MRM, +ESI) Linearity: 0.01-50 mg/kg Jensen 2005

LSD, iso-LSD, nor- LC-MS/MS (+ESI) Linearity: 20 pg/mL-10 ng/mL Favretto et al. LSD, 2-oxo-3- Blood LOQ: 20 pg/mL (LSD, nor-LSD) 2007

hydroxy-LSD Recovery: 60-107%

5 4

LSD, iso-LSD, nor- UHPLC-MS/MS (MRM, +ESI) LOD: 5 pg/mL (LSD, iso-LSD), 10 pg/mL (nor- Chung, Hudson, LSD, O-H-LSD Blood LSD, O-H-LSD) and McKay 2009 LOQ: 20 ng/mL (LSD, iso-LSD), 50 pg/mL (nor- LSD, O-H-LSD) Linearity: 10-2000 pg/mL (LSD, iso-LSD), 20- 2000 pg/mL (nor-LSD, O-H-LSD)

LSD, iso-LSD, nor- Solid phase extraction Linearity: 20-800 pg/mL (40-1600 pg/mL O-H- Martin et al. 2013 LSD, nor-iso-LSD, LC-MS/MS (+ESI) LSD) O-H-LSD Plasma and serum LOD (serum): 10-30 pg/mL LOD (plasma): 15-30 pg/mL LOQ (serum): 12-34 pg/mL LOQ (plasma): 15-30 pg/mL Recovery: >86%

LSD, 2-oxo-3- Protein precipitation LLOQ: 0.1 ng/mL Dolder, Liechti, hydroxy-LSD LC-MS/MS Linearity: 0.1-10 ng/mL Rentsch 2014

Serum Recovery: 64%

5 5

LSD, iso-LSD, O-H- Solid phase extraction LOQ: 0.01 ng/mL Steuer et al. 2017 LSD MFLC-MS (MRM, +ESI) Linearity: 0.01-20 ng/mL Plasma

+ ESI, positive electrospray ionization; HPLC, high performance liquid chromatography; LC-MS(/MS), liquid chromatography (tandem) mass spectrometry; LOD, limit of detection; LOQ, limit of quantification; LSD, lysergic acid diethylamide; MFLC, microflow liquid chromatography; MFLC; microflow liquid chromatography; MRM, multiple reaction monitoring (mode); PRM, parallel reaction monitoring (mode); SRM, selective reaction monitoring (mode); UHPLC, ultra-high performance liquid chromatography

55 method be developed, information of withdrawal times could be derived from elimination half- life data from pharmacokinetics studies.

1.8 Effect of ergot alkaloid exposure on male livestock semen characteristics and fertility As discussed previously, ergot is known to impair female reproductive efficiency. The reproductive effects of ergot alkaloids in female mammalian livestock have been examined by Grusie et al. 2018b and reviewed by Poole and Poole (2019). In contrast, the impact of ergot alkaloid exposure on male reproduction is unclear. Due to the vasoconstrictive activity of ergot, there exists a strong mechanistic basis for the putative toxicity of ergot alkaloids to spermatogenesis. These are impaired thermoregulation and/or testicular tissue hypoxia with resulting oxidative stress. Vasoconstriction from ergot exposure may affect the ability of the testes to dissipate excess heat. Alternatively, prolonged vasoconstriction may deplete the testicular tissue of oxygen and lead to the generation of reactive oxygen species. Prolactin- dependent mechanisms have also been proposed (Pratt and Andrae 2015) as prolactin has been detected in in bull seminal plasma (Pratt et al. 2015b) and the prolactin receptor is present in the testis and sperm cells, including the epididymis and differentiating spermatids (Pratt et al. 2015a). Normal thermoregulation of the testes is crucial for proper spermatogenesis in bulls. In animals with external testes, the scrotum is maintained 2 to 6 ºC below the core body temperature (Cook, Coulter, and Kastelic 1994; Kastelic, Cook, and Coulter 1996; Brito et al. 2012; Romano and Brinsko 2020). This temperature gradient is required for production of viable spermatozoa with normal morphology. In particular, the cauda epididymis stores most sperm and thus heavily relies on a low scrotal temperature (Cook, Coulter, and Kastelic 1994; Kastelic, Cook, and Coulter 1997). In a study of bulls with scrotal insulation (to mimic high environmental temperatures), Rahman et al. (2011) found that insulation generally reduced motility and increased the incidence of defects in sperm morphology. Specifically, the authors concluded that the stages of spermatogenesis most sensitive to increased ambient temperature are the spermiogenic and meiotic stages of development. Additionally, spermatocytes and spermatids appear to be particularly sensitive to heat stress (Brito et al. 2004; Setchell 2006). Thermoregulation of external testes has a complex physiology. This information is described by Romano and Brinsko (2020). Briefly, this temperature difference is maintained by a countercurrent heat exchange mechanism between the arterial and venous testicular vasculature.

The testis is highly vascularized with a venous pampiniform plexus network that surrounds the coiled testicular artery. The artery traverses the length of the testis before branching dorsally and laterally to reach the testicular parenchyma. The blood entering the testes via the testicular artery loses its heat to the nearby venous system. This vascular system allows for significant radiative heat loss in high ambient temperature environments. Highly metabolically active tissue such as the testes are more susceptible to oxidative stress and generation of reactive oxygen species. Lipid peroxidation, a consequence of reduced tissue perfusion and oxygen, greatly reduces sperm function. Sperm are uniquely sensitive to lipid peroxidation due to a high polyunsaturated fatty acid content (Aitken and Baker 2004; Aitken and Baker 2006; Tuncer et al. 2010). Semen contains a certain degree of antioxidant ability, with compounds such as ascorbic acid, tocopherol, catalase, glutathione peroxidase, superoxide dismutase (Aurich et al. 1997; Baumber et al. 2000; Bansal and Bilaspuri 2011). As reactive oxygen species generation is a normal by-product of metabolism in spermatozoa, the presence of antioxidants keeps balance between pro-oxidant and antioxidant conditions (Baumber et al. 2000; Bansal and Bilaspuri 2010). Reactive oxygen produced in semen as a by- product of metabolism and signalling include hydrogen peroxide, superoxide anion, the hydroxyl radical, and the hypochlorite radical (Aitken and Clarkson, 1987; Bilodeau et al. 2001; Bansal and Bilaspuri 2010). In an in vitro study of equine semen, the reactive oxygen radical hydrogen peroxide was found to be abundant and sufficient to reduce motility of the spermatozoa via lipid peroxidation (Baumber et al. 2000). Lipid peroxidation of the sperm plasma membrane is associated with impaired motility, increased apoptosis, and impaired binding of the sperm cell with the zona pellucida to achieve fertilization (Aitken, Clarkson, and Fishel 1989; Aitken and Baker 2006; Bansal and Bilaspuri 2010). Collectively, fertility of the semen donor becomes compromised. Whether or not ergot alkaloids induce these pathophysiological changes during spermatogenesis and/or in the sperm cells remains to be demonstrated. The results of in vitro studies have provided a partial explanation of mechanisms of ergot that may impair male fertility. Wang et al. (2009) demonstrated that ergotamine and dihydroergotamine decreased the proportion of motile bovine spermatozoa via -adrenergic receptors. Page et al. (2013) also found that ergotamine and dihydroergotamine decreased overall sperm motility and increased the percentage of nonmotile sperm in a dose- and time-dependent fashion. At concentrations of

57 ergotamine and dihydroergotamine >66 µM, the percentage of rapid and progressive spermatozoa decreased. Gallagher and Senger (1989) tested the effect of ergonovine (i.e., ergometrine) in semen extender on post-thaw motility and percentage of intact acrosomes of cryopreserved bull sperm. The authors found that 20 mg/mL ergonovine reduced motility and percentage of intact acrosomes at both 0 and 4-hours post-thaw. Collectively, these results suggest a direct effect of ergot alkaloids on sperm motility, thereby potentially reducing fertility, however the relevance is limited due to the complex physiology of testicular function. A large body of in vivo evidence exists that suggests that exposure to fescue ergot alkaloids decreases semen quality and fertility of bulls. However, a consistently altered sperm characteristic has not been identified. This may be due to differences in experimental concentrations and length of the exposure periods used. A study in bulls (n=6) from Jones et al. (2004) using experimental diets containing 1005 µg ergovaline per kg feed found that, following one month of exposure, ergot exposed bulls had higher scrotal temperatures, smaller scrotal circumferences, and higher spermatozoa concentrations without any adverse effects on sperm morphology or motility. The elevations in scrotal temperature for these bulls were evident without elevated rectal temperatures. The reduced scrotal circumference observed was thought to be due to scrotal sweating or a dopaminergic mechanism that would reduce testicular fluid volume and induce hypertrophy in Leydig cells (Dirami, Teerds, and Cooks 1996; Jones et al. 2004). The authors attributed increased spermatozoa concentration to the dopaminergic activity of ergot alkaloids having a direct effect on accessory glands or the pituitary gland. In a two-year grazing study, beef bulls on the E+ pasture (containing 270 to 340 µg ergovaline per kg feed) exhibited signs of fescue toxicosis, including reduced body weight, increased rectal temperature, and decreased serum prolactin compared to bulls on E- pasture (Schuenemann et al. 2005a). There were no observed differences in sperm morphology and motility or in vitro development of embryos from the blastocyst stage. Only a decrease in in vitro cleavage of semen from E+ exposed bulls was observed. A grazing study from Looper et al. (2009) demonstrated subtle changes on semen characteristics from yearling Brahman influenced bulls consuming 600 µg ergovaline per kg on a dry matter basis. In bulls grazing the E+ pasture, no change in scrotal circumference was observed. However, serum prolactin was decreased in these bulls. In addition, bulls on the E+ pasture had decreased motile sperm, decreased proportion of each progressive and rapid sperm, and reduced sperm velocity compared to bulls grazing E- pasture. The authors

58 concluded that sperm motility and morphology were reduced in bulls exposed to toxic tall fescue. In a study from Stowe et al. (2013), when Angus and Hereford yearling bulls were fed an E+ diet containing 800 µg ergovaline and ergovalinine per kg DM for 126 days, no difference compared to E- bulls was observed for any semen characteristics (measured by computer assisted sperm analyzer, i.e., CASA) or the proportion of bulls passing a breeding soundness evaluation (Stowe et al. 2013). However, serum prolactin and scrotal circumference (day 0 to day 126) decreased in bulls grazing E+. Angus bulls grazing E+ tall fescue pasture (with a 98% infection rate; no ergot alkaloid concentration given) displayed decreased serum prolactin, decreased total gain, average daily gain, and body weight by day 140, reduced sperm concentration, decreased proportion of morphologically normal sperm, and decreased post-thaw motility compared to bulls grazing E- pasture (Pratt et al. 2015b). In a 112-day E+ feeding study in yearling Angus bulls, cryopreserved ejaculates from ergot alkaloid exposed bulls had lower post-thaw progressive motile concentration (day 28, 84), lower sperm concentration (day 84), decreased percent motility (days 28, 84, and 168), decreased motile concentration (days 28, 84, 168) compared to cryopreserved ejaculates from non-toxic fescue exposed bulls (Burnett, Bridges, and Pratt 2017). In contrast, studies of yearling and adult Angus bulls allowed to graze endophyte infected tall fescue pasture (no ergot alkaloid concentration given) for 56 days did not find adverse impacts on semen quality (Burnett et al. 2018). This study reported contradictory results on pregnancy rates in cows inseminated by E+ exposed bulls. The potential for vasoconstriction in the testicular artery as a mechanism of reproductive toxicity in male livestock has been studied using ultrasonography. A study from Aiken et al. (2015) found that the testicular artery cross sectional area was reduced in bulls fed 800 µg ergovaline per kg DM for 126 days; this result was consistent with ergot-induced vasoconstriction of the caudal artery in the same study and previous studies (Aiken et al. 2007; Aiken et al. 2009; Aiken et al. 2015). The second part of the study, a 155-day grazing study (no ergot alkaloid concentration given), also reported reduced area in both the caudal artery and testicular artery in bulls grazing E+ pasture. The constriction in the testicular artery was detected for 70 days after the bulls removed from the E+ pasture. Some studies of fescue ergot alkaloids and spermatogenesis have also been conducted in stallions. Stallions fed tall fescue seed (no ergot alkaloid concentration given) for 70 days had spermiograms that were not different from control animals; there were no differences in sperm

59 concentration, motility, total number of cells, total number of artificial insemination doses, or percentage of abnormal sperm (Fayrer-Hosken et al. 2013). The only effect detected was a lower gel-free ejaculate volume, which the authors speculated could be prolactin-mediated. However, prolactin concentration was not different between treated and control animals. In a separate study, it was reported that sperm from E+ exposed stallions were less likely to undergo a Ca2+- ionophore induced acrosome reaction after storage at 5 ºC for 48 hours (Fayrer-Hosken et al. 2012). The role of ergot alkaloids in male reproduction is not completely clear, however some compelling evidence exists to suggest that endophyte infected tall fescue ergot alkaloids adversely affected sperm production, motility, and morphology. Currently, there are no published studies that investigate the effect of C. purpurea derived ergot alkaloids on male livestock reproduction.

1.9 OBJECTIVES & HYPOTHESES FOR RESEARCH CHAPTERS Chapter 2:

Summary and objective. Ergot alkaloids are known vasoconstrictive agents that are commonly encountered in livestock feed. Current Canadian tolerance concentrations for ergot alkaloids in cattle feed are 2000 to 3000 µg/kg. The purpose of this study was to test if hemodynamic changes were present in a survey of arteries of beef cows fed ergot alkaloids near the current guidance values over a one-week period.

Hypothesis. Concentration-dependent changes in hemodynamic parameters in the caudal, median sacral, and internal iliac arteries were hypothesized following a one-week exposure to ergot alkaloids in feed near the current Canadian tolerance concentrations.

Chapter 3:

Summary and objective. This study was conducted to address if hemodynamic changes occurred in cows following long-term exposure to ergot alkaloids in feed at lower concentrations than the first study. A long-term, chronic-type exposure is more relevant to farm conditions.

Hypothesis. Concentration-dependent changes in hemodynamic parameters in the caudal and internal iliac arteries following long-term (i.e., nine-week) exposure to ergot alkaloids in feed were hypothesized.

Chapter 4:

Summary. Pharmacokinetic information on ergot alkaloids is largely under characterized in livestock, especially following on-farm relevant oral exposure. In order to characterize the oral pharmacokinetic parameters of ergot alkaloids in cattle, development of a sensitive analytical method was necessary. Liquid chromatography tandem mass spectrometry was chosen because it is routinely used for quantification of ergot alkaloids in feed samples and would provide the sensitivity needed for detecting low concentrations of ergot alkaloids anticipated to be found in plasma.

Objectives. Pharmacokinetic studies were undertaken to collect plasma samples from cows exposed to a one-time high concentration (27 000 to 29 000 µg/kg) exposure to ergot alkaloid in feed. In turn, quantification of plasma concentrations would allow for the construction of a

61 plasma concentration versus time curve and the subsequent determination of oral pharmacokinetic parameters for ergot alkaloids. The other objective of this work was to develop a sensitive method to quantify ergopeptine alkaloids in bovine plasma in order to generate pharmacokinetic information on these compounds following oral exposure in cows.

Chapter 5:

Summary. Ergot alkaloids in livestock feed are known to impair female reproductive performance. However, male reproductive performance following exposure to ergot is not well characterized. This study will assess reproductive endpoints of beef bulls that have been exposed to low and permissible concentrations (i.e., below the CFIA standard for cattle) of ergot in their feed. Endpoints include semen concentration, sperm motility, sperm morphology, and flow cytometric staining of sperm subcellular organelles. The objective of this work was to determine if the fertility of adult bulls, an economically important livestock group, would be negatively affected by ergot exposure.

Hypothesis. Ergot alkaloids will decrease bull fertility as indicated by reduced motility, increased morphological defects, increased prematurely acrosome reacted sperm, and increased sperm with low mitochondrial membrane potential.

2. CHAPTER 2 – ARTERIAL RESPONSES TO SHORT-TERM LOW CONCENTRATION ERGOT ALKALOID EXPOSURE IN HEREFORD COWS PREFACE The material described within this chapter was published as a manuscript in Frontiers in Veterinary Science. This chapter has been reformatted from the original published version for inclusion in the thesis. Cowan, V., Neumann, A., McKinnon, J., Blakley, B.R., Grusie, T.G., Singh, J. (2018). Arterial Responses to Acute Low-Level Ergot Exposure in Hereford Cows. Front. Vet. Sci. 16 (12 pages). doi: 10.3389/fvets.2018.00240 VC was responsible for conducting the research, data collection and analysis, and manuscript preparation. AN was involved in animal feeding, experimental procedures, and caudal artery data analysis. JM contributed to ration formulation, nutritional considerations, and feeding considerations. TG contributed to prolactin data analysis. BB was involved in ergot alkaloid concentration selection, experimental design, and consultation. JS was the principle investigator of the grant and contributed toward concept development, hypotheses formulation, experimental design, oversight of the study, statistical analyses and manuscript preparation/revisions. This study was the first conducted in a series of ergot alkaloid investigations. Results of this study provided ergot alkaloid concentration-response information and time course of action of vascular effects. Importantly, results of this study indicated that current Canadian guidelines for ergot alkaloids in livestock feed required re-examination.

2.1 ABSTRACT

Ergot alkaloids are toxic secondary metabolites produced by the fungus Claviceps purpurea that contaminate cereal grains. Current Canadian standards allow 2 to 3 parts per million of ergot alkaloids in animal feed. The purpose of this study was to determine whether hemodynamic parameters were altered when beef cows were fed permissible concentrations of ergot alkaloids (i.e., <3 ppm) on a short-term basis. A dose-response relationship between ergot alkaloid concentration and hemodynamic changes in caudal (coccygeal), median sacral, and internal iliac arteries was hypothesized. Beef cows were randomly allocated to: Control (<15 µg total ergot alkaloids/kg dry matter), Low (132 µg/kg), Medium (529 µg/kg), and High (2115 µg/kg) groups (n=4 per group). Animals were fed 8.8 kg of dry matter daily for 4 days (pre-treatment), 7 days (treatment), and 4 days (post-treatment). The caudal, median sacral, and internal iliac arteries were examined daily using ultrasonography in B-mode and Doppler (colour and spectral) mode and hemodynamics endpoints were analyzed by repeated measures mixed model analyses. Caudal artery diameter decreased in the Medium (p=0.004) and High (p<0.001) groups compared to pre-treatment values and the pulsatility index increased (P≤0.033) in all ergot treatments during the post-exposure period compared to the Control group. Blood volume per pulse (mL) and blood flow (mL/min) through the caudal artery during the treatment period were reduced in the Medium (-1.0 mL reduction; p≤0.004) and High (-1.1 mL p≤0.006) groups compared to pre-treatment values. The median sacral artery diameter decreased in the Medium (p=0.006) and High (p=0.017) treatments compared to the Control group. No differences were detected in any hemodynamic endpoints for the internal iliac artery except changes in pulse rate (p=0.011). There was no treatment (p>0.554) or Treatment*Time interaction (p>0.471) for plasma prolactin concentration or body temperature. In conclusion, alterations in caudal artery hemodynamics were detected when cows were fed 529 and 2115 µg ergot alkaloids per kg dry matter per day for one week. The caudal artery was more sensitive to ergot alkaloids than the median sacral and internal iliac arteries. Our results partially support the hypothesis of a dose- response effect of ergot alkaloids in feed on hemodynamics.

2.2 INTRODUCTION

Ergot alkaloids are biologically active secondary metabolites produced by the pathogenic plant fungus Claviceps purpurea. The name “ergot” refers to the dark “sclerotia” produced by the fungus that replace the kernels in the ripe ear of the cereals such as rye, triticale, barley, and wheat. C. purpurea infection has become widespread across Western Canada within the past decade (Menzies and Turkington 2015) and ergot alkaloids are commonly encountered in Canadian and global livestock feeds (EFSA 2012). Exposure to livestock may occur through consumption of contaminated native pasture during grazing or result from feed formulated with contaminated cereal grains and forage. Ergot toxicity in livestock can manifest in many forms, the most commonly observed of which include the gangrenous, reproductive, and hyperthermic forms. The symptoms result due to the structural similarity of the ergopeptine alkaloids (including ergotamine, ergosine, ergocornine, -ergocryptine and ergocristine) to endogenous biogenic amines (i.e., dopamine, serotonin, and norepinephrine) (Pertz and Eich 1999, Tudzynski, Correia, and Keller 2001). Ergot alkaloids may act as agonists and partial-agonists (Roquebert and Grenie 1986; Tudzynski, Correia, and Keller 2001) at these bioamine receptors. A major mechanism of ergotism results from enhanced peripheral vascular constriction by adrenergic receptor blockade and agonism at peripheral serotonin (5-hydroxytryptamine) receptors (Aellig and Berde 1969; Berde and Stürmer 1978; Hofmann 1978; Roquebert et al., 1984; Villalon et al., 1999; Gröcer and Floss 1998; Tudzynski, Correia, and Keller 2001; Schardl et al., 2006). This is implicated in sustained constriction of peripheral arteries that progress to the development of necrosis and gangrene (Aellig and Berde 1969; Klotz 2015). Clinical manifestations of gangrenous ergotism in livestock first manifest as hindlimb lameness and swelling of the hooves at the pastern and fetlock regions that progress to necrotic lesions and appendage loss (Mantle 1969; Burfening 1973; Klotz 2015; Craig et al., 2015). Development of gangrenous lesions of the tail tips of cattle has been observed at concentrations as low as 473 µg total ergot alkaloids per kg total ration, while complete hoof loss has been reported at concentrations of 12 000 µg/kg (Craig, Klotz, and Duringer 2015), suggestive of a dose-response relationship between ergot alkaloids and gangrenous ergotism.

Fescue toxicosis is caused by the consumption of endophyte (Eplichoë coenophialum)- infected tall fescue (Solomans et al., 1989; Strickland et al. 1996; Aiken et al. 2007; Klotz et al. 2006, 2007, 2008, 2009, 2010; Aiken et al. 2009; Foote et al. 2011; Egert et al. 2014; Pesquiera et al. 2014). Animals experiencing fescue toxicosis also exhibit gangrenous signs similar to ergot poisoning (Porter and Thompson 1992; Thompson and Stuedemann 1993). Although related phylogenetically to classic ‘ergot of rye’ (i.e., C. purpurea) (Glenn et al. 1996; Schardl 2001), the ergot alkaloid profiles produced by the endophyte are different than that of C. purpurea and, thus, direct comparisons have limited relevance. It is estimated that economic losses to the US livestock industry as a result of tall fescue contamination are approximately $1 billion annually (Strickland et al., 2011). Disconcertingly, it is currently unknown how much productivity or performance losses occur to the beef cattle operations due to subclinical C. purpurea ergot toxicosis. Furthermore, concentration thresholds for these changes have not been identified in cattle or other livestock species. Globally, ergot regulations for livestock feed tend to vary. In the European Union, <100 µg/kg (i.e., <0.1% ergot sclerotia by weight) is considered the limit in cereals for livestock consumption. This value is an order of magnitude less in the United Kingdom, i.e., <1 µg/kg (Coufal-Majewski et al., 2016). In the United States, grain is considered contaminated when sclerotia comprise 0.05 to 0.3% of grain on a weight basis, depending on the substrate (Wegulo and Carlson 2011; Miedaner and Geiger 2015). This corresponds approximately to a total ergot alkaloid concentration of 300 µg/kg (Coufal-Majewski et al., 2016). The current Canadian recommended tolerance level for ergot consumption in cattle is 2000 to 3000 µg/kg of feed (CFIA 2017), which is considerably higher than other countries with ergot regulations for livestock feed. The purpose of this study was to investigate changes in arterial response in cows associated with increasing ergot alkaloid exposure near the current Canadian tolerance concentration. We hypothesized that low-concentrations of ergot alkaloids will alter the hemodynamic endpoints of caudal, median sacral and internal iliac arteries in a dose-dependent manner.

2.2 MATERIALS AND METHODS 2.2.1 Statement of animal ethics. This study was carried out in accordance with the recommendations of the University of Saskatchewan University Committee on Animal Care and

Supply and Animal Research Ethics Board. The protocol (animal use protocol #20140044) was approved by the University of Saskatchewan Animal Care Committee prior to commencing any animal work. Animals were monitored throughout the study for health and wellbeing. 2.2.2 Ergot alkaloid extraction and quantification procedure with Liquid Chromatography Mass Spectrometry (LC-MS). Quantification of ergot alkaloids in feed with LC-MS was performed as described by Grusie et al. 2017. Briefly, all calibrations and analyses were conducted with a high-performance liquid chromatography (HPLC) system (Agilent 1100) fitted with an Agilent Zorbax Eclipse XDB-C18 narrow bore (2.1x150 mm; 5 µm) HPLC column that was used in tandem with a mass spectrometer (Micromass Quattro Ultima). The 85/15 (% v/v) extraction solution was prepared using pre-filtered acetonitrile (HPLC grade; EMD Millipore) and 10 mM ammonium acetate (771 mg ammonium acetate in 1L of fresh barnstead water with a resistance of 16.8 MΩ or higher). Ammonium acetate and acetonitrile solutions were also used as the ‘Mobile Phase A’ and ‘Mobile Phase B’ solutions, respectively. A six-point calibration (i.e., standard) curve was generated by serial dilution of each of the working alkaloid solutions of 1 µg/mL (Romer Labs Inc., Newark, DE, USA) in extraction solution to obtain 12.5, 7.5, 2.5, 1.25, and 0.75 ng/mL. The alkaloids evaluated were ergotamine, ergometrine, ergocornine, ergocristine, ergocryptine, and ergosine. Standards were accepted if the standard deviation (i.e., area under the chromatograph curve) did not exceed 15%. Ground grain and pellet samples (5 g) were diluted in 25 mL of the 85/15 extraction solution and mixed on a magnetic stir plate for 10 minutes. Supernatants were filtered through a Whatman 41 filter paper into clean glass beakers. In individual glass test tubes, 50 mg of Bondesil-PSA bulk sorbent (40 µm particle size; Agilent Technologies, Santa Clara, CA, USA) was weighed and 400 µL of supernatant was added. Samples were agitated for 45 seconds and allowed to settle. Supernatant (100 µL) was collected without disturbing the Bondesil-PSA, placed in Agilent auto-sampler vials, a sample volume of 20 µL was injected, and alkaloid detection was performed for 21-minute run-times. Individual and total alkaloid concentrations were reported in units of micrograms per kilogram (µg/kg) for each sample. 2.2.3 Feed formulation and treatment groups. Diets were formulated by diluting highly ergotized pellets with ergot-free pellets, silage, and chopped barley. The ergot-concentrated pellets contained 46520 µg total ergot alkaloids per kg (Ergocristine: 18229, Ergotamine:12000, -Ergocryptine: 6965, Ergocornine: 3394, Ergometrine: 3231, and Ergosine: 2700 µg/kg). The

concentrations of ergot in the experimental diet were selected based on the current Canadian guideline of 2000 to 3000 µg/kg total ration. Four treatments were employed in this study: negative control, low ergot concentration, medium ergot concentration, and high ergot concentration. Of the 4 kg of pellets fed daily, animals in the control, low, medium, and high treatment groups received 0 kg, 0.025 kg, 0.1 kg, and 0.4 kg of the ergot-concentrated pellets, respectively. Concentration and dose data are provided in Table 2.1. The weights of pellets included in the ration corresponded to 0, 70, 280, and 1121 µg total ergot alkaloids per kg ration as fed. On a dry matter basis, these concentrations were 0, 132, 529, and 2115 µg/kg of feed. Animals were fed at 1.5% of their body weight during the pre-treatment, treatment and post-treatment periods (see section 2.2.4 for details), corresponding to 16.6 kg of feed (as fed; 4 kg pellets, 11 kg barley silage, 1.6 kg chopped barley per head) per day. This was approximately 8.8 kg of dry matter. The animals were fed three times daily. The ergotized portion of the ration was fed once daily (during the treatment period of 7 days) at approximately 0800h; all cows were fed equivalent amounts of ergot-free pellets at this time during the pre- (4 days) and post- treatment (3 days) periods. The cows were fed a mixture of ergot-free pellets, barley, and silage for the remaining two feedings, at approximately 1000h and 1400h. Silage used in the total mixed ration contained <15 µg total ergot alkaloids/kg on an as fed basis and <42 µg total ergot alkaloids/kg dry matter. Barley used in the total mixed ration contained <7.0 µg total ergot alkaloids/kg on an as fed basis and <7.0 µg total ergot alkaloids/kg dry matter. 2.2.4 Animal husbandry and experimental design. Lactating Hereford cross beef cows (n=16) and their calves were maintained at the University of Saskatchewan Goodale Research Farm. Cows were 48 ± 3 days (mean ± SEM) post-partum and were 522 ± 22 kg of body weight at the start of the experiment. The experiment was conducted in the months of June and July. Ambient temperature ranged from 1 to 25.4ºC in June and 6 to 28.4ºC in July (Saskatoon data from the Environment Canada monthly climate summary). The study was conducted in two replicates (n=8 cows per replicate) 15 days apart to mitigate space and time constraints. Individual cow-calf pair were housed in pens inside the barn (straw bedding, open air access) starting four days prior to the feeding of the experimental diet to allow cows to become

TABLE 2.1. Treatment ration composition and corresponding total ergot alkaloid concentrations of diets fed to lactating Hereford cows. Cows were fed the total mixed ration daily for one week during the treatment period. Body weight dosage was calculated for each ergot treatment group using the average weight during the treatment period (see Table 2.2). Ergot Amount of ergot Amount of total Feed dose Body weight dose Treatment concentrated pellets (kg) ergot alkaloids (µg) (µg/kg dry (µg consumed /kg matter)‡ BW) Control 0 0 0 0 Low 0.025 1163 132 0.12 Medium 0.1 4653 529 0.58 High 0.4 18610 2115 2.43 ‡Cows were fed the total mixed ration daily for 1 week during the treatment period. Body weight dosage was calculated for each ergot treatment group using the average weight during the treatment period (see Table 2.2).

69 accustomed to the pelleted ration and the surroundings. Indoor barn temperature and outside ambient temperatures were recorded at the time of ultrasound examination. Animals had ad libitum access to water and were monitored daily for general health and for signs of lameness. Rectal temperature was recorded daily between 0900 and 1100h. This study included three experimental periods: pre-treatment, treatment, and post-treatment. The pre-treatment period took place for four consecutive days (i.e., days -4, -3, -2, and -1). The treatment period occurred for seven days (i.e., first day of ergot feeding = day 0) immediately following the pre-treatment period. The post-treatment period incorporated three days immediately following the treatment period (i.e., days 7, 8, and 9) and one day a week later (i.e., day 14).

2.2.5 Plasma samples. Blood was collected via jugular venipuncture daily between 0900 and 1100 hours using heparinized collection tubes. Plasma was separated from whole blood by centrifugation (15 minutes at 10 000 × g). Blood samples were stored at -20ºC until ELISA analysis for prolactin. 2.2.6 B-mode and Doppler Vascular Ultrasonography. Ultrasonography was used to image the caudal, internal iliac, and median sacral arteries on each cow daily. The caudal (coccygeal) artery was selected due to its anatomical location (i.e., the tail) and its examination in similar studies with ergot alkaloids (Aiken et al. 2007; Aiken et al. 2009). The median sacral artery was selected as the internal counterpart of the caudal artery and likely to be affected less by the changes in ambient temperature due to its location. The internal iliac artery was selected because it is a large elastic artery inside the pelvic cavity that can be reliably imaged at the chosen location over time. Caudal artery was imaged using the transcutaneous approach while the median sacral and internal iliac arteries were imaged using the transrectal approach. The MyLab™Five ultrasound system (Esaote S.p.A.) with a 7.5 MHz linear-array transducer for transcutaneous and transrectal use. Cows were restrained in a locking head gate prior to ultrasound examination. The caudal artery was imaged transcutaneously at the fourth coccygeal vertebra. The right internal iliac artery was imaged inside the body cavity cranial to the vaginal artery branchpoint. The median sacral artery was measured internally caudal to the sacral ridge at its highest point. Figure 2.1 depicts the anatomy of the pelvis and measurement locations of the arteries in the present study, as well as ultrasound images captured for hemodynamic variable measurement. The ultrasound transducer was placed on each artery lengthwise to capture a longitudinal section. Videos and images were recorded once a clear image and steady

70 position were achieved. The first set of measurements for each artery was taken in a duplex view of B-mode (brightness mode) and CFM (color flow mode). A ten second video (audio video interleave (avi) format) was recorded of each artery in the longitudinal aspect to image blood flow and maximum arterial diameter. The second set of measurements were taken in spectral Doppler mode. Spectral waveforms of each artery (minimum three consecutive waveforms) were captured as images. For all arteries, B-mode gain and power were set at 64% and 100%, respectively. Gain for CFM was set to 64% for the caudal artery and 64% for the median sacral and internal iliac arteries. For Spectral Doppler, arteries were positioned at a depth of 5cm and Doppler angle, spectral gain, and spectral velocity were set to +75º, 58%, and 95%, respectively and the sample volume gate was set to 1, 4, and 2 for the caudal, internal iliac, and median sacral arteries, respectively. 2.2.7 Hemodynamic variable measurements. Images of Doppler spectra were recorded on each sampling day and saved for later analysis. A minimum of three consecutive waveforms were recorded and analyzed for each time point. The video and image files (Esoate’s proprietary format) were imported into the MyLabDesk software program (Esaote S.p.A.) for analysis. Video recordings of each artery were taken. For analysis, a longitudinal frozen image of the artery was captured at its maximum diameter. The diameter of each artery was measured using the built-in ‘distance’ function of the software by placing calipers at each lumen boundary (perpendicular to the lumen). Three diameter measurements were taken. Measurements of Doppler spectra were completed using the built-in software functions. Manual tracing of each waveform in the software’s built-in ‘vascular FVI mode’ produced a value for peak systolic velocity (PSV; m/s), end diastolic velocity (EDV; m/s), and mean velocity (MnV; m/s), pulsatility index (PI; unitless) and resistivity index (RI; unitless). Pulsatility index is calculated by Systolic Velocity-Diastolic Velocity/Mean Velocity. Resistivity index is calculated by Systolic Velocity-Diastolic Velocity/Systolic Velocity (Timor-Tritsch, Monteagudo 2009). PI and RI are measures of vascular resistance (Jousse-Joulin 2010), with increased values indicating increased resistance to blood flow (Cerdeira and Karumanchi 2011). The pulse rate was measured using the software’s ‘HR’ function by placing measurement calipers at the peak of two consecutive waveforms (units of bpm). Average values from the three consecutive

FIGURE 2.1. Measurement of the hemodynamic endpoints using B-mode and color Doppler ultrasonography of the caudal, median sacral, and internal iliac arteries in cows. A) Diagram indicating branching pattern of major arteries (1 to 7) in the pelvic cavity and arterial measurement locations (green shaded areas). The caudal artery (1) was imaged transcutaneously at the fourth caudal vertebra. The median sacral artery (2) was imaged transrectally caudal to the sacral ridge at the highest point. The internal iliac artery (5) was measured transrectally between the umbilical (4) and vaginal (6) branches. B) Duplex B-mode and Color Flow Doppler mode of the bovine internal iliac artery in longitudinal section was used to measure arterial diameter at 3 locations (+ signs) to obtain a single (average value) for each artery per day. C) Doppler spectrum waveform (bottom part of image) of the bovine caudal artery was recorded by placing the sampling gate (horizontal and oblique green lines in artery lumen) and used to manually trace the waveform (green line along the bottom of the waveform) to obtain hemodynamic parameters including mean arterial velocity (MnV), peak systolic velocity (PSV), end diastolic velocity (EDV), pulsatility index (PI), and resistivity index (RI). Figure A was drawn based on Nickel et al. (1981). Scale bar for Figure B and C = 10mm.

73 waveforms of each endpoint for each day of the experiment were calculated and evaluated statistically. Arterial radius, blood volume per beat, and blood flow per minute were derived using the averages of the above variables in the equations as follows:

Radius (mm) = diameter/2 …………………………………………………………………(1) R pulse duration Blood volume per pulse (mL) = (Mean arterial velocity ∗ 100)(π ( ))( ) …(2) 100 1000 Flow per minute (mL/min) = Blood volume per pulse ∗ Pulse Rate…………………….…(3)

2.2.8 Enzyme-linked immunosorbent assay (ELISA) for bovine prolactin (PRL) (antigen detection). A commercial competitive inhibition ELISA Kit (Cloud-Clone Corp.) for Prolactin (PRL) was purchased from CedarLane (Product number CEA846BO). The assay uses biotin- labelled antibody specific for PRL and the company-reported detection range of the kit is 2.47 to 200 ng/mL (sensitivity <0.98 ng/mL). Standards (0-200 ng/mL) were run on each plate. All samples were assayed in duplicate on the same day as per kit instructions using the provided reagents. Holstein calf serum (previously determined by the laboratory to have high prolactin concentration) was used as reference standards (undiluted and 1:2 dilution). Inter-assay and intra-assay coefficients of variance were 14.1% (n=6) and 5.1% (n=24), respectively. 2.2.9 Statistical analysis – repeated measures analysis of variance. Statistical Analysis Software (SAS) version 9.4 with Enterprise Guide 6.1 (SAS Institute, Cary NC USA) was used for all analyses. The repeated measures Mixed procedure was used to test for the effect of treatment (i.e., four ergot concentrations), experimental period (i.e., pre-treatment, treatment, post-treatment), and interactions (see Appendix A for the complete syntax). Variables analyzed included body weight, rectal temperature, prolactin concentration, and hemodynamic endpoints (both measured and calculated). Day of data collection was included as a repeated variable and animals were nested within treatment groups. Experimental replicate was included in the model as a random factor. The best fit model for the data was selected based on the smallest Akaike information criteria (AICc) value from the nine tested covariance structures (simple, compound symmetry, heterogeneous compound symmetry, Toeplitz, banded Toeplitz, Huynh-Feldt, autoregressive, heterogeneous autoregressive, and ante-dependence). Final analysis of the data (Type 3 Test of Fixed Effects) included least square means for main effects or interaction terms. Statistical significance was =0.05. Multiple comparisons were conducted where applicable using the differences of least square means.

2.3 RESULTS 2.3.1 General. Average ambient temperatures during the pre-treatment, treatment, and post- treatment periods were 17.9 ± 0.3 ºC, 17.2 ± 0.2 ºC, and 19.6 ± 0.5 ºC, respectively. No symptoms of lameness or hyperthermia were observed in any of the animals throughout the duration of the study. 2.3.2 Plasma prolactin concentration, weight, and rectal temperature. The data for plasma prolactin concentration, body weight, and rectal temperature are provided in Table 2.2. There was no Treatment (p=0.554) or Treatment*Experimental Period interaction (p=0.471) for plasma prolactin concentration. Prolactin concentration varied by Experimental Period (p<0.001). Plasma prolactin (averaged across all groups) decreased (p<0.001) from the pre- treatment period (34.7 ± 1.2 ng/mL) to treatment period (31.0 ± 1.0 ng/mL) followed by a further decrease to the post-treatment period (27.3 ± 1.2 ng/mL). No treatment-specific differences in cow body weight were observed during the study period. There was no Treatment*Experimental Period interaction (p=0.668). Weight varied by Experimental Period (p=0.023). Across all treatment groups, cows lost an average of 9.1 kg from the pre-treatment period to the treatment period (p=0.013) and gained an average of 4.5 kg from the treatment period to the post-treatment period (p=0.008). There was no Treatment (p=0.536) or Treatment*Experimental Period interaction for rectal temperature (p=0.984). 2.3.3 Hemodynamic endpoints. Complete data on hemodynamic variables and parameters from ultrasonographic analyses for each of the three arteries are presented in Appendix B (B.1. and B.2.). There was a Treatment*Experimental Period interaction for caudal artery diameter (p=0.009), peak systolic velocity (p=0.024), pulsatility index (p=0.007), blood volume per pulse (p=0.001), and blood flow per minute (p=0.007). End diastolic velocity (p=0.002), resistivity index (p<0.001), and pulse rate (p=0.018) of the caudal artery differed for the Experimental Period only (i.e., no Treatment effect or Interaction). For the median sacral artery diameter, there was a Treatment*Experimental Period interaction (p=0.011). No treatment effects in any other hemodynamic parameters were recorded for the median sacral artery except for the Experimental Period differences in the peak systolic velocity (p=0.027), end diastolic velocity (p=0.023), pulsatility index (p=0.003) and resistivity index (p=0.009). Pulse rate for the internal iliac artery had a Treatment*Experimental Period interaction (p=0.011) and end diastolic

TABLE 2.2. Plasma prolactin concentration, body weight, and rectal temperatures (mean ± SEM) of lactating Hereford cows (n=4 per treatment group) during the pre-treatment (4 days), treatment (7 days), and post-treatment (4 days) experimental periods to increasing concentrations of ergot alkaloids in their feed in Control, Low, Medium and High groups. Plasma for prolactin analysis were collected daily and analyzed by enzyme-linked immunosorbent assay (ELISA). Weights and rectal temperatures were recorded daily. Ergot Treatment

Experimental Period Control Low Medium High (132 µg/kg (529 µg/kg DM) (2115 µg/kg DM) DM)

Prolactin (ng/mL) Pre-treatment 37.8 ± 2.1 35.6 ± 3.3 31.3 ± 2.2 34.3 ± 1.3 Treatment 35.0 ± 2.6 33.7 ± 1.9 25.6 ± 1.5 29.7 ± 1.7 Post-treatment 30.7 ± 3.4 28.8 ± 2.5 24.4 ± 1.5 25.0 ± 1.4

Body weight (kg) Pre-treatment 491 ± 11 567 ± 17 495 ± 10 479 ± 16 Treatment 481 ± 8 565 ± 13 480 ± 7 462 ± 11 Post-treatment 481 ± 12 575 ± 18 480 ± 8 468 ± 17

Rectal temperature (ºC) Pre-treatment 38.9 ± 0.2 38.7 ± 0.1 38.8 ± 0.1 39.0 ± 0.2 Treatment 39.1 ± 0.1 39.0 ± 0.1 38.9 ± 0.1 39.2 ± 0.1 Post-treatment 39.0 ± 0.2 38.8 ± 0.2 38.8 ± 0.2 39.0 ± 0.2

Data analyzed by repeated measures mixed model factorial analysis of variance. Prolactin: Treatment p=0.554; Experimental Period p<0.001; Treatment*Experimental Period interaction p=0.4711. Weight: Treatment p=0.102; Experimental Period p=0.023; Treatment*Experimental Period p=0.668. Rectal temperature: Treatment p=0.536; Experimental period p=0.073; Treatment*Experimental Period p=0.984

velocity varied by Treatment (p=0.024). Other hemodynamic endpoints for the internal iliac artery remained unchanged (i.e., no Treatment effect or Interaction) except for Experimental Period differences in pulsatility index (p=0.02) and resistivity index (p=0.008). 2.3.3.1 Diameter. Changes in diameter for the three arteries during the one-week treatment period are given in Figure 2.2. Each artery was analyzed separately. During the treatment period, caudal artery diameter decreased by 19% (-0.5 mm; p=0.002) in the High ergot treatment compared to the Control group during treatment period (2.9 ± 0.1 mm). Compared to the pre- treatment values, caudal artery diameter during the treatment period decreased by 10% (-0.3 mm; p=0.004) in the Medium ergot group, and by 19% (-0.5 mm; p<0.001) in the High ergot group. No differences (p>0.1) between groups were detected in diameter of the caudal artery during the post-treatment period. Further, the diameter of the caudal artery in the Medium and High groups during the post-treatment period returned to the pre-treatment values (i.e., no difference). During the treatment period, diameter of the median sacral artery was smaller in the Medium ergot group (-0.7 mm, 19%; p=0.006), and in the High ergot group (-0.6 mm, 17%; p=0.017) compared to the Control group. Pre-treatment diameter of the median sacral artery in the Low ergot group was higher (p<0.042) than the Control, Medium and High ergot groups (+0.5 mm, +1.1 mm and +0.7 mm, respectively); therefore, inter-group comparisons for the low group were not performed. Within the Low group, the diameter of the median sacral artery during the treatment decreased compared to the pre-treatment value. For the internal iliac artery diameter, there was no effect of Treatment (p=0.35), Experimental Period (p=0.08), or Treatment*Experimental Period (p=0.92). 2.3.3.2 Peak systolic velocity. During the treatment period, there were no differences between the control group and the Medium (p=0.99), or High (p=0.47) ergot treatment groups in PSV of the caudal artery (Appendix B). Pre-treatment and treatment values did not differ (P>0.22) for PSV value of caudal artery for the Medium and High treatment groups. Caudal artery PSV of the Low ergot group during the treatment and post-treatment period was higher (p=0.015 and p=0.001, respectively) than the pre-treatment value. 2.3.3.3 Blood volume per pulse. Compared to pre-treatment values, blood volume per pulse of the caudal artery (Figure 2.3A) decreased in the Medium (-1.0 mL; p=0.004) and High (-1.1

FIGURE 2.2. Diameter of the a) caudal artery, b) median sacral artery, and c) internal iliac artery of lactating Hereford cows (n=4 per treatment group) before (4 days), during (7 days), and after (4 days) feeding increasing concentrations of ergot alkaloids in Control (<15 µg/kg), Low (132 µg/kg), Medium (529 µg/kg) and High (2115 µg/kg) ergot groups. Each bar represented the mean ± SEM for each experimental period. Repeated measures analysis was used to test for changes in arterial diameter (each artery analyzed individually) for treatment (Tx), experimental period (EP) and their interaction (Tx*EP). Differences among experimental periods within a treatment group (connected bars) are indicated by x and y (p<0.05) and differences among groups during a given treatment period (same colored bars) are indicated by a and b (p<0.05).

79 mL; p=0.004) ergot groups during the treatment period. Blood volume per pulse did not differ in these groups when compared to the Control group during the treatment period (p>0.16).

2.3.3.4 Blood flow. Compared to the Control group during the treatment period, blood flow in the caudal artery was reduced by 29% in the Medium treatment (-43 mL/min; p=0.034), and by 34% in the High treatment (-51 mL/min; p=0.021). Caudal artery treatment period and post- treatment blood flow values for the Medium ergot treatment (Figure 2.3B) were 61% (-69 mL/min; p=0.001) and 63% (-65 mL/min; p=0.007) of the pre-treatment value, respectively. Caudal artery treatment period and post-treatment blood flow values for the High ergot treatment were 59% (-69 mL/min; p=0.006) and 65% (-60 mL/min; p=0.03) of the pre-treatment blood flow. Blood flow was not affected for the median sacral or internal iliac artery (Treatment*Experimental Period p=0.175 and 0.366, respectively). 2.3.3.5 Pulsatility index. Compared to the pre-treatment values, caudal artery PI in the Low ergot treatment group (Figure 2.3C) increased by 23% (p=0.066) and 34% (p<0.001) during the treatment and post-treatment periods, respectively. Post-treatment PI of the caudal artery for ergot treatments was greater than that of the post-treatment PI of the control group. This corresponded to 25% (p=0.002), 18% (p=0.021) and 17% (p=0.033) increases in Low, Medium, and High ergot treatment groups. 2.3.3.6 Pulse rate. In the High ergot treatment, pulse rate measured in the internal iliac artery decreased (p=0.0003) from 62 ± 2 bpm during the pre-treatment to 59 ± 2 bpm during the treatment period (Appendix B), but both periods did not differ from the corresponding periods in control group (p>0.16). Further, internal iliac arterial pulse rate in the control group during the treatment period was higher (p=0.036) than the post-treatment period (62 ± 1 vs. 60 ± 2 bpm). No difference in pulse rate were detectable when measurements from the caudal artery and median sacral artery were compared 2.4 DISCUSSION The present study investigated whether a dose-response relationship existed between total ergot alkaloids in feed and changes in hemodynamic parameters of three arteries in lactating Hereford cows. Ergot alkaloids are known to suppress plasma prolactin concentrations; however, ergot consumption at 132 (low) 529 (medium) and 2115 (high) µg per kg of dry matter intake per day did not affect plasma prolactin, body weight, or rectal temperature during the present study. It is noteworthy that the caudal artery diameter decreased in medium (9.7%) and

p =0.007

/* 81

FIGURE 2.3. Caudal artery hemodynamic parameters of lactating Hereford cows (n=4 per treatment group) before (4 days, white bars), during (7 days; black bars), and after (4 days, gray bars) feeding increasing concentrations of ergot alkaloids in Control (<15µg/kg), Low (132 µg/kg), Medium (529 µg/kg) and High (2115 µg/kg) ergot groups. (A) pulsatility index (unitless) (B) blood volume per pulse (mL), and (C) blood flow (mL/min). Data are presented as the mean ± SEM for each experimental period. Differences among experimental periods within a treatment group (connected bars) are indicated by x and y (p<0.05) and differences among groups during a given treatment period (same colored bars) are indicated by a and b (p<0.05).

82 high (19%) ergot groups during treatment period compared to pre-treatment period. Likewise, median sacral artery diameter during the treatment period decreased in the medium and high ergot groups compared to the control group. In contrast, internal iliac artery diameter was not affected. Further, caudal artery pulsatility index increased during post-treatment period for all groups while the volume per beat during the treatment period was lower for medium and high group than the pre-treatment values. Blood flow in the caudal artery during the treatment and post-treatment periods for the medium and high ergot treatments was reduced to <65% of pre- treatment values. Our results support the notion that peripheral arteries (e.g., the caudal artery) are more sensitive to ergot alkaloids than the more central arteries (e.g., the internal iliac artery). Ergot alkaloid concentrations at 529 and 2115 µg/kg of dry matter intake affected the caudal artery hemodynamics following one week of daily ergot consumption. Specifically, reduced arterial diameter, blood flow, and blood volume per heartbeat were observed. These results are indicative of vasoconstriction and reduced arterial perfusion, both of which are hallmarks of gangrenous ergotism in livestock. Two studies of cattle ingesting endophyte- infected tall fescue detected vasoconstriction in the caudal artery with color Doppler ultrasonography at similar concentrations as in the present study. A decrease in caudal artery cross-sectional area was detectable in beef heifers fed 390 or 790 µg ergovaline per kg dry matter (Aiken et al., 2009) and those that were fed 850 µg ergovaline per kg recorded a 42% reduction (Aiken et al., 2007). The duration of constriction was dose-dependent wherein the 790 µg concentration of ergot resulted in a longer duration of constriction than 390 µg (Aiken et al., 2009). Likewise, goats exposed to fescue ergovaline and ergovalinine (800 µg/kg of dry matter intake) had reduced cross-sectional areas of the carotid and auricular arteries (Aiken and Flythe 2014) and horses fed fescue seed containing 4.93 mg/kg ergovaline had reduced blood flow of the palmar artery (McDowell et al., 2013). These previous studies and the present results suggest that concentrations of ergot alkaloids >390 µg/kg cause vasoconstriction in the caudal artery following short-term exposure in feed in beef cows and heifers. It is worth mentioning that ergocristine (48% of total), ergocryptine (17%) and ergotamine (>11%) are major alkaloids in ergot sclerotia in western Canadian grain (Grusie et al. 2018a) therefore, direct comparison between C. purpurea alkaloids and endophyte alkaloids, and between species should be made with caution due to differing alkaloid profiles and potencies, and species-specific sensitivity to ergot alkaloids.

Our study detected an increased pulsatility index in the low ergot treatment (but not in the medium or high groups) during the treatment period. Further increased PI was recorded during the post-treatment period of all ergot treatments. Our finding indicates an increase in arterial resistance to flow suggesting that some effects of ergot are delayed. Vessel bioassay studies of fescue ergot alkaloids have also noted that there is a persistent effect on the cardiovascular system, despite ergot alkaloid removal (Dyer 1993; Schöning et al. 2001; Klotz et al. 2007, 2008; Pesquiera et al. 2014; Klotz 2015) supporting our notion. This study is the first to report on the effect of ergot alkaloid exposure on bovine median sacral artery. The median sacral artery is the internal counterpart to the caudal artery, and thus served as a test of arterial location for hemodynamic changes. As seen in the caudal artery, constriction was observed (i.e., reduced diameter) in the medium and high ergot treatments. However, reduction in blood flow was not seen in this artery, suggesting that the caudal artery was more responsive to ergot exposure. The difference in response between the two arteries may be related to the influence of ambient temperature, however this remains to be investigated. The internal iliac artery appeared to be the least responsive to ergot treatment. There were no changes in hemodynamic parameters that would indicate vasoconstriction. A potential explanation for these results (compared to the caudal and median sacral arteries) could be related to the relative proportion of smooth muscles in their Tunica Media, size of the vessels, differences in number of bioamine receptors, or variations in receptor sensitivity. Diameter of the internal iliac artery was approximately 2.5 times larger than that of the caudal artery. The internal iliac artery branches off the aorta (Nabors and Linford 2014; Singh 2018), thus receives large volumes of blood for delivering to pelvic organs such as the uterus and vagina. The internal iliac artery is more elastic than more peripheral arteries to compensate for arterial pressure fluctuations during systole and diastole. The median sacral artery is also a branch of the aorta, albeit much smaller than the internal iliac and eventually terminates in the caudal artery supplying the tail. The peripheral location of the caudal artery implies that it has higher smooth muscle content and lower elastic content than the other two arteries (Mescher 2013; Robinson and Robinson 2016). Since ergot alkaloids act on bioamine receptors in vascular smooth muscle (Strickland et al., 1996), it would be reasonable to assume that the effect of the alkaloids would be more prominent in arteries with higher proportion of smooth muscles in their Tunica Media. The observation that the caudal artery is most sensitive to ergot reinforces the well-known

84 feature of ergot as a peripheral vasoconstrictor agent. Peripheral appendages, such as the ear tips, tail tips and hooves are affected by ergotism (Robbins et al., 1986; Botha et al., 2004; Belser-Ehrlich et al., 2013; Dewell and Ensley 2014; Klotz 2015). Reduction in circulating prolactin concentrations in cows exposed to ergot compared to control animals and pre-ergot values were not observed during the present study. Numerous studies indicate that prolactin production is suppressed following ergot alkaloid exposure (Lu et al., 1971; Karg et al., 1972; Schams et al., 1972; Hurley et al., 1980; Browning et al., 1997; Blaney et al., 2000a; Kopinski et al., 2008; Foote et al., 2013). Prolactin secretion is affected by multiple environmental and physical factors, including stress, ambient temperature, photoperiod, mechanical stimulation (i.e., calf suckling, handling), and physiological status (i.e., pregnant, lactating) (Webb and Lamming 1981; Auchtung et al., 2003). The presence of these factors may increase or decrease the basal prolactin concentrations in cattle. It is noteworthy that by design of the experiment, we tested the effect of very low amounts, i.e. subclinical concentrations of ergot consumption. Current Canadian standards permit 2000 to 3000 µg/kg of ergot alkaloids in feed (CFIA 2017). Animals in this study ate between 100 to 2100 µg/kg of dry matter intake for 7 days. It is likely that plasma prolactin was not affected by the amount of ergot fed in this study. The present study suggests that subclinical vasoactive effects of ergot may be a more sensitive bioindicators of exposure as compared to prolactin alterations. It remains to be examined if prolactin suppression may be affected after chronic exposure whereas cardiovascular alterations may be more rapid in nature. The most sensitive endpoint of ergot exposure in cattle may be influenced by both the dose and duration of exposure, not dose alone. It should be noted that no clinical signs of gangrenous ergotism were observed throughout the study. Commonly cited symptoms of gangrenous ergotism include bilateral hindlimb lameness, hoof swelling, and, eventually, loss of the tail switch, ear tips, and, in the most severe cases, hoof loss. The authors did not anticipate adverse clinical effects in the ergot- exposed animals in this study, due to both the moderate summer climate in which this study was conducted and the short duration of the exposure. Had this study been conducted during cold ambient temperatures, however, clinical signs may have developed as cold temperatures exacerbate gangrenous symptoms of ergot alkaloid exposure (Klotz 2015; Craig, Klotz, and Duringer 2015). The current Canadian permissible concentrations of ergot alkaloids in feed do not account for duration of exposure or the season of exposure. Further studies are required to

85 characterize the relationship between ergot alkaloid concentration in feed, ambient temperature, duration of exposure, and development of subclinical and clinical changes in peripheral vasculature. In conclusion, our hypothesis that of increasing ergot concentrations having a dose- related response on arterial hemodynamic parameters was partially supported. Vasoconstriction and reduced perfusion were observed in the caudal artery of beef cows at 529 and 2115 µg/kg dry matter, but not 132 µg/kg dry matter. Increased resistance to flow was observed in all ergot treatments despite removal of ergot from the feed. Constriction was also seen in the median sacral artery at 529 and 2115 µg/kg dry matter, but perfusion was unaffected. The internal iliac artery was largely unaffected by treatment. Plasma prolactin concentrations and rectal temperatures were not affected at these low concentrations of ergot exposure for 7 days. The threshold for vasoconstriction following short term ergot exposure seems to fall between 132 and 529 µg/kg dry matter. Future work should include assessment of arterial responses following long-term ergot alkaloid exposure in feed of cattle. High groups

3. CHAPTER 3 – Arterial responses in periparturient beef cows following a nine-week exposure to ergot (Claviceps purpurea) in feed PREFACE The material described within this chapter was published as a manuscript in Frontiers in Veterinary Science. This chapter has been reformatted from the original published version for inclusion in the thesis. Cowan, V., Grusie, T., McKinnon, J., Blakley, B., Singh, J. (2019). Arterial responses in periparturient beef cows following a 9-week exposure to ergot (Claviceps purpurea) in feed. Front. Vet. Sci. 6 (12 pages). doi: 10.3389/fvets.2019.00262 VC was responsible for conducting the research, data collection and analysis, and manuscript preparation. TG was involved in animal feeding and experimental procedures. JM contributed to ration formulation, nutritional considerations, and feeding considerations. BB was involved in ergot alkaloid concentration selection, experimental design, and consultation. JS was the principle investigator of the grant and contributed toward concept development, hypotheses formulation, experimental design, oversight of the study, statistical analyses and manuscript preparation/revisions. This study was conducted to expand upon the results of Chapter 2, by testing the effect of ergot alkaloids on peripheral vasculature following a long-term exposure and lower ergot alkaloid concentrations near the CFIA guideline. The data from both Chapter 2 and Chapter allowed for interpretation of the interaction between ergot alkaloid dose and duration of exposure.

3.1 ABSTRACT Ergot alkaloids are vasoconstrictors frequently detected in low concentrations in livestock feed. The Canadian Food Inspection Agency permits up to 3000 µg ergot alkaloids per kg cattle feed. The objective of this study was to examine the effects of feeding low concentrations of ergot alkaloids over nine-weeks on vascular dynamics in the caudal and internal iliac arteries of beef cows. A relationship between ergot alkaloid concentration in feed and hemodynamic changes in the caudal and internal iliac arteries was hypothesized. Periparturient beef cows were randomized into four groups and group fed mixed rations containing <15 µg ergot alkaloids per kg of dry matter intake (Control, n=9), 48 µg/kg (Low, n=9), 201 µg/kg (Medium, n=8), 822 µg/kg (High, n=6). Three experimental periods comprised the study: pre-treatment (2 weeks), treatment (9 weeks), and post-treatment (3 weeks). B-mode and Doppler ultrasonography was performed weekly to measure hemodynamic endpoints. Plasma prolactin concentrations and rectal temperatures were measured weekly. Caudal artery diameter decreased (Treatment*Experimental Period i.e. Tx*EP p<0.001) by 14% in the High group during the treatment period. Reductions (Tx*EP p<0.001) in caudal artery blood flow (37%, 29%) and blood volume per pulse (29%, 11%) were recorded during the treatment period in the High and Medium groups. Internal iliac artery diameter and blood flow decreased (Tx*EP p≤0.004) by 13% and 40% during the treatment period in the Medium group. Moderate reductions (Tx*EP p≤0.042; 12-25%) in the mean blood velocity during the treatment and post-treatment periods and decreases (Tx*EP p≤0.01; 12-17%) in the peak systolic velocity of both arteries during the post-treatment period were also detected. Prolactin did not change in any group during the treatment period (p=0.462). Rectal temperatures were within the normal physiological range for beef cows. In conclusion, we documented moderate vasoconstriction in the caudal artery and the internal iliac artery in cows fed 201 to 822 µg ergot alkaloids per kg of dry matter intake for nine-week period near parturition. The pattern of alterations was similar between the caudal and internal iliac arteries. Results of this study suggest that feeding up to 822 µg/kg produce reversible pharmacological changes in beef cow vasculature and warrant reconsideration of current regulations for cattle.

3.2 INTRODUCTION Ergot alkaloid mycotoxins are secondary metabolites produced by the plant fungus Claviceps purpurea that infects cereal crops including rye, wheat, barley, triticale, and oats. Detection of ergot alkaloids in the grain and livestock feed has been on the rise in Western Canada for past 10 years (Menzies and Turkington 2015; Grusie et al. 2018a). Current Canadian Food Inspection Agency standards permit up to 3000 µg of ergot alkaloids per kg of cattle feed (Canadian Feed Inspection Agency 2017). The ergopeptine class of ergot alkaloids are pharmacologically active compounds and are agonists of serotonergic, dopaminergic, and adrenergic receptors (Berde and Stürmer 1978; Müller-Schweinitzer and Weidmann 1978; Dyer 1993; Pertz and Eich 1999). Prolonged consumption of ergot alkaloids has been associated with the development of gangrenous ergotism in livestock (Burfening 1973; Robbins et al. 1986; Shelby 1999; Strickland et al. 2011; Klotz 2015; Craig et al. 2015). Gangrenous ergotism is a manifestation of ergot alkaloid toxicity marked by the progressive loss of circulation to distal limbs and tissues leading to subsequent ischemia and development of dry gangrene (Burfening 1973; Robbins et al. 1986; Shelby 1999; Strickland 2011, Eadie 2003). The principle mechanism of vasoconstriction appears to be through the agonistic activity of ergot alkaloids on smooth muscle serotonin and adrenergic receptors (Dyer 1993; Müller-Schweinitzer et al. 1992; Oliver et al. 1993; Oliver et al. 1998; Villalon et al. 1999; Schöning et al. 2001; Eadie 2003) resulting in decreased blood flow to tissues. Despite this well accepted pathogenesis, there is a lack of research studying the effects of subclinical low concentrations of ergot alkaloids in cattle feed within the permitted tolerance limits on the peripheral vascular system in cattle, particularly under Canadian climatic conditions. In a previous study by our research group, beef cows were fed C. purpurea ergot alkaloids for one week and observed for signs of vasoconstriction in the caudal artery at 529 and 2115 µg total ergot alkaloids (ergocristine, ergocornine, ergocryptine, ergosine, ergotamine, and ergometrine) per kg of dry matter (Cowan et al. 2018). The caudal artery of these animals recovered to pre-treatment status once ergot was removed from the feed. It is not known if prolonged low-concentration long-term exposure to ergot alkaloids will result in cumulative and permanent alterations in vascular flow or increased tolerance at the beginning of lactation. The objective of this study was to examine the effects of feeding ergot alkaloids over a nine-week period on vascular dynamics in the caudal and internal iliac arteries of beef cows during the

89 periparturient and early post-partum period. Based on the results of short-term study, we hypothesized a concentration-response relationship between ergot alkaloid concentration in feed and alterations in hemodynamic parameters in the caudal artery and the internal iliac artery. 3.3 MATERIALS AND METHODS 3.3.1 Statement of animal ethics. This study was carried out in accordance with the recommendations of the University of Saskatchewan University Committee on Animal Care and Supply and Animal Research Ethics Board. The animal use protocol (#20140044) was approved by the University of Saskatchewan Animal Care Committee prior to commencing any animal work. Animals were monitored throughout the study for health and wellbeing using a humane intervention checkpoint. Cows were assessed for food and water intake, appearance of behaviour (including signs of pain and/or distress), and vital, vasoactive, and neurological signs. 3.3.2 Ration formulation and ergot alkaloid quantification in feed. Mixed rations containing pelleted feed (barley, oat hulls, canola, and wheat screenings), barley and hay were formulated to meet the nutritional requirements of the cows using CowBytes computer program (Government of Alberta Agriculture and Rural Development, Canada). Three different concentrations of ergotized pellets were manufactured by the Canadian Feed Research Centre (North Battleford, SK Canada). Six ergot alkaloids (ergocristine, ergocornine, ergocryptine, ergosine, ergotamine, and ergometrine) were solvent extracted from feed samples and analyzed using liquid chromatography mass spectrometry as described previously (Grusie et al. 2017). The lower concentration ergotized pellets (Pellet 1) contained 221 µg total ergot alkaloids per kg of pelleted feed. The intermediate pellets (Pellet 2) contained 731 µg/kg and the higher concentration pellets (Pellet 3) contained 2981 µg/kg. The concentrations of 6 ergot alkaloids in the treatment (ergotized) pellets are given in Table 3.1. Control pellets contained a background of 18 µg total ergot alkaloids per kg of pelleted feed and were purchased from CO-OP® Feeds (Saskatoon, SK Canada). Total concentrations of ergot alkaloids in feed in this study were designed to be within current Canadian guidelines for cattle, corresponding to 2000 to 3000 µg/kg total mixed ration (CFIA 2017). Based on the results of a previous study (Cowan et al. 2018), concentrations were chosen to be conservative of development of vascular alterations as cattle were to be fed for a longer period of time than that study.

TABLE 3.1. Ergot alkaloid concentration (µg/kg; mean ± SEM) and percent of total (in parentheses) in the three pellet formulations. Ergot alkaloids were quantified in pellets using liquid chromatography mass spectrometry (LC-MS; Prairie Diagnostic Services, Saskatoon SK Canada). Ergot Pellets Ergot alkaloid Pellet 1 (%) Pellet 2 (%) Pellet 3 (%) Ergosine 13.2 ± 0.4 (6) 46.8 ± 3.3 (6) 193 ± 6.5 (6) Ergocornine 18.0 ± 2.5 (8) 68.1 ± 4.3 (9) 308 ± 15.2 (10) Ergocristine 123 ± 15.8 (56) 391 ± 26.0 (53) 1550 ± 52.3 (52) Ergocryptine 24.3 ± 4.8 (11) 94.5 ± 5.8 (13) 397 ± 11.4 (13) Ergotamine 41.8 ± 1.4 (19) 130 ± 5.3 (18) 529 ± 16.2 (18) Ergometrine 0.5 ± 0.08 (<1) 1.9 ± 0.14 (<1) 7.9 ± 0.41 (<1) Total ergot alkaloids 221 ± 45 (100) 731 ± 85 (100) 2981 ± 171 (100)

3.3.3 Animal husbandry and experimental design. Periparturient Hereford cows (n=32) were housed in outdoor paddocks with shelter access at the University of Saskatchewan Goodale Research Farm. Calves were housed with the cows in the outdoor pens. Animals had ad libitum access to water, hay, and trace mineral salt blocks (CO-OP® 2:1 beef cattle range mineral block; registration no. 641098; Federated Co-operatives Limited; CO-OP® AgroCentre, Saskatoon, SK Canada) in their pens. Cows and calves were assessed daily for general health and signs of stress. Cows were administered 3 cc of Vétoquinol Vitamin AD-500 (Vitamin A 500 000 I.U./mL, Vitamin D 75 000 I.U./mL, Vitamin E 5 I.U./mL) prior to the study. Cows were randomly assigned to one of the four ergot treatment groups: Control (n=9), Low (n=9), Medium (n=8), or High (n=6) ergot group. Sample size was different for each treatment group due to animals being excluded for various reasons, including loss of pregnancy, late pregnancy (such that it was considered an outlier), death of a cow due to uterine perforation during parturition, and death of a calf during parturition. These factors were unrelated to ergot treatment. Cows were group fed in troughs, with each group being housed in its own paddock. Group feeding was chosen due to facility constraints and practical considerations. Feed was offered daily to animals at a level of 12.7 kg dry matter per day, i.e., approximately 2% of body weight on a dry matter basis. The study consisted of a pre-treatment period (two weeks, i.e., week -2 and -1), a treatment period (nine weeks, i.e, week 0 through 8, where start of ergot feeding = week 0), and a post-treatment period (three weeks, i.e., weeks 9, 10, and 11). Ergot pellets were fed from April 17 to June 19, 2015. Cows in the control, low, medium, and high ergot groups were -9 ± 6, -7 ± 5, 0 ± 5, and 3 ± 9 days post-partum (mean ± standard error), respectively, at the time ergot pellet feeding (where day 0 = parturition). Calving was a potential confounding factor in this experiment and was accounted for in statistical analysis (see section below). In addition, other confounding factors measured include ambient temperature inside and outside of the barn. Ambient temperatures were recorded on sample collection days with digital thermometers. The group fed cows were offered feed to consume a total daily intake of ergot alkaloids for each of the treatment groups based on the total mixed ration (TMR), 0 (control), 48 (low), 201 (medium), and 822 (high) µg/kg (based on dry matter intake). Cows were offered 3.5 kg pellets/head daily. To achieve the desired concentration of ergot alkaloids based on dry matter intake, cows in the low ergot group were offered 2.7 kg of Pellet 1 (221 µg/kg) mixed with 0.8 kg of control pellets (i.e., total of 3.5 kg pellets). The medium ergot group were offered

3.5 kg per head of Pellet 2. The high ergot group was offered 3.5 kg per head of Pellet 3. The control group was fed 3.5 kg per head of the control pellets. The pellets were spread out in concrete outdoor troughs to allow even access to all cows in the treatment group. Cows were also fed 8.5 kg dry chopped hay (grass/alfalfa mix) and 2 kg barley per head per day in their troughs in the afternoon (i.e., after pellet consumption). Mineral mix (1:1 calcium to phosphorus mineral mix, CO-OP®, Saskatoon SK; 70 g per head) was added on top of the pellets daily (at the same time as pellet consumption) in their troughs 3.3.4 Ultrasonography of the caudal and internal iliac arteries. The caudal artery and right branch of the internal iliac artery were evaluated weekly using ultrasonography as described previously (Cowan et al. 2018). The caudal artery was chosen based on its previously documented sensitivity to ergot alkaloids in feed (Aiken et al. 2007, Aiken et al. 2009, Cowan et al. 2018). The internal iliac artery was selected to serve as a negative control based on the results of a previous study in which no hemodynamic changes, aside from altered pulse rate, were observed in the internal iliac following one-week exposure to ergot alkaloids in feed in beef cows (Cowan et al. 2018). Briefly, the caudal artery was imaged at the fourth coccygeal vertebra, while the internal iliac artery was measured between the uterine and vaginal branches. The MyLab™Five ultrasound system (Esaote S.p.A.) with a 7.5 MHz linear-array transducer was used for transcutaneous and transrectal imaging of the caudal and internal iliac arteries, respectively. B-mode (i.e., 2D) ultrasound was used in tandem with color flow Doppler mode to capture longitudinal arterial sections for measuring arterial diameter. Video recordings (10 s) of the artery were saved for later analysis. Color flow Doppler and spectral (power) Doppler mode were used to measure spectral waveforms for each artery. A minimum of three consecutive waveforms were recorded and saved for later analysis. The ultrasonographic endpoints analyzed were arterial diameter (longitudinal section), peak systolic velocity, end diastolic velocity, mean velocity, pulsatility index, resistivity index, and pulse rate. Arterial radius, blood volume per pulse, and blood flow per minute were calculated using equations described previously (Cowan et al. 2018). 3.3.5 Blood collection. Cows were restrained in a locking head gate with halter prior to blood collection. Blood was collected via jugular venipuncture into lithium-heparinized (i.e., green- grey) vacutainer tubes (BD Vacutainer, Becton-Dickinson Canada, Mississauga ON). The side of the neck used for blood collection alternated by week to minimize local trauma. Plasma was

separated from whole blood via centrifugation (> 1500 × g) for 15 minutes at room temperature and stored at -20 ºC until further analysis. 3.3.6 Enzyme-linked immunosorbent assay (ELISA) for bovine plasma prolactin. A commercial competitive inhibition ELISA Kit (Cloud-Clone Corp.) for prolactin (PRL) was purchased from CedarLane (CEA846BO; Burlington ON Canada). The assays were conducted in the Western College of Veterinary Medicine Endocrine Service Lab (Saskatoon SK Canada). The assay uses biotin-labelled antibody specific for bovine PRL and the company-reported detection range of the kit is 2.47 to 200 ng/mL (sensitivity <0.98 ng/mL). Standards (0-200 ng/mL) were run on each plate. All samples were assayed in duplicate on the same day as per kit instructions using the provided reagents. Holstein calf serum (previously determined by the laboratory to have a high prolactin concentration) was used as reference standards (undiluted and 1:2 dilution). Inter-assay and intra-assay coefficients of variance were 26% and 11%, respectively. 3.3.7 Statistical analysis – SAS mixed procedure. Statistical Analysis Software (SAS) version 9.4 with Enterprise Guide 6.1 (SAS Institute, Cary NC USA) was used for all analyses. The repeated measures mixed procedure was used to test for the effect of treatment (i.e., four ergot concentrations), experimental period (i.e., pre-treatment, treatment, post-treatment), and interactions. The data were analyzed as means of each treatment group and of each experimental period. Variables analyzed included rectal temperature, prolactin concentration, and hemodynamic endpoints for the caudal and internal iliac arteries (both measured and calculated). Week of data collection was included as a repeated variable and animals were nested within treatment groups. Random factors included in the model were ambient temperature outside, ambient temperature inside the barn, pasture and month of calving. The best fit model for the data was selected based on the smallest Akaike information criteria (AICc) value from the ten tested covariance structures (simple, compound symmetry, heterogeneous compound symmetry, Toeplitz, banded Toeplitz, Huynh-Feldt, autoregressive, heterogeneous autoregressive, ante- dependence, and unstructured). Final analysis of the data (Type 3 Test of Fixed Effects) included main effects (Tx = treatment, EP = experimental period) or interaction terms (Tx*EP). Statistical significance was considered p<0.05 (=0.05). Multiple comparisons were conducted where applicable using the differences of least square means. The syntax used for the final analysis was as follows:

3.4. RESULTS 3.4.1. General. No signs of ergot toxicity (i.e., lameness, hoof swelling, hoof or tail tip necrosis, hyperthermia) were observed throughout the study. Of the 32 cows in the study, 25 calves were born in April and 7 were born in May. Unilateral lameness was observed in three cows, but each case was determined to be unrelated to ergot by the attending veterinarian and all affected cows were treated with liquamycin (LA-200, ~70 ml subcutaneous injection dependent on body weight; Zoetis Inc., Kalamazoo MI USA). Lameness resolved after treatment. Average outdoor ambient temperature throughout the treatment period was 22 °C (range: 5 to 29 °C). 3.4.2. Hemodynamic endpoints. Complete data on hemodynamic parameters and variables from ultrasonographic analyses of the caudal and internal iliac arteries are presented in Appendix C. Data for arterial diameter, blood flow, and blood volume per pulse for both arteries are displayed in Figure 3.1. Data for blood velocities are displayed in Figure 3.2 and pulse rate, pulsatility index, and resistivity index are presented in Figure 3.3. 3.4.3. Caudal artery hemodynamics 3.4.3.1. Caudal artery diameter. In the High group, caudal artery diameter decreased on average by 0.6 mm during the treatment period (-14%) compared to the pre-treatment period (Tx*EP p<0.001); the diameter returned to pre-treatment value during the post-treatment period (Figure 3.1A). A similar pattern was observed for the Medium group where an increase in caudal artery diameter (+0.3 mm) was recorded during the post-treatment period compared to the treatment period. Caudal artery diameter increased by 0.1 mm in the Low group during the treatment and post-treatment periods compared to the pre-treatment period. 3.4.3.2. Caudal artery blood flow and volume. Blood flow in the caudal artery decreased (Tx*EP p<0.001) during the treatment period by 29% (-112 mL/min) in the Medium and by 37% (-154 mL/min) in the High groups when compared to pre-treatment period values, respectively (Figure 3.1C). Blood flow values during the post-treatment period were intermediate between the pre-treatment and treatment values for the Medium and High groups. Blood flow in the Low group decreased (72 mL/min; -23%) in the post-treatment period from the treatment period. Blood flow was unchanged in the Control group. Compared to the pre- treatment value, blood volume per pulse in the caudal artery also decreased (Tx*EP p<0.001) in the Medium (-0.5 mL; -11%) and High (-1.2 mL; -29%) groups during the treatment period and returned to pre-treatment values by the post-treatment period (Figure 3.1E). In the Low group,

FIGURE 3.1. Diameter (mm), Blood flow (mL/min), and Blood volume per pulse (mL) of the caudal artery (A, D, E) and internal iliac artery (B, D, F) of periparturient Hereford cows (n=32) before (2 weeks), during (8 weeks), and after (3 weeks) feeding increasing concentrations of ergot alkaloids in Control (<15 μg/kg dry matter intake), Low (48 μg/kg), Medium (201 μg/kg), and High (822 μg/kg) ergot groups. Each bar represented the mean ± SEM for each experimental period. Repeated measures analysis was used to test for changes in arterial diameter (each artery analyzed individually) for treatment (Tx), experimental period (EP) and their interaction (Tx*EP). Differences among experimental periods within a treatment group (connected bars) are indicated by f and g for white, x and y for grey, and p and q for black (p<0.05) and differences among groups during a given treatment period (same colored bars) are indicated by a and b (p<0.05).

FIGURE 3.2. Mean velocity (m/s), peak systolic velocity (m/s), and end diastolic velocity (m/s) of the caudal artery (A, D, E) and internal iliac artery (B, D, F) of periparturient Hereford cows (n=32) before (2 weeks), during (8 weeks), and after (3 weeks) feeding increasing concentrations of ergot alkaloids in Control (<15 μg/kg dry matter intake), Low (48 μg/kg), Medium (201 μg/kg), and High (822 μg/kg) ergot groups. Each bar represented the mean ± SEM for each experimental period. All velocities are in units of m/s. Repeated measures analysis was used to test for changes in arterial diameter (each artery analyzed individually) for treatment (Tx), experimental period (EP) and their interaction (Tx*EP). Differences among experimental periods within a treatment group (connected bars) are indicated by f and g for white, x and y for grey, and p and q for black (p<0.05) and differences among groups during a given treatment period (same colored bars) are indicated by a and b (p<0.05).

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FIGURE 3.3. Pulse rate (bpm), pulsatility index, and resistivity index of the caudal artery (A, D, E) and internal iliac artery (B, D, F) of periparturient Hereford cows (n=32) before (2 weeks), during (8 weeks), and after (3 weeks) feeding increasing concentrations of ergot alkaloids in Control (<15 μg/kg dry matter intake), Low (48 μg/kg), Medium (201 μg/kg), and High (822 μg/kg) ergot groups. Each bar represented the mean ± SEM for each experimental period. Repeated measures analysis was used to test for changes in arterial diameter (each artery analyzed individually) for treatment (Tx), experimental period (EP) and their interaction (Tx*EP). Differences among experimental periods within a treatment group (connected bars) are indicated by f and g for white, x and y for grey, and p and q for black (p<0.05) and differences among groups during a given treatment period (same colored bars) are indicated by a and b (p<0.05).

101 blood volume per pulse increased (1.1 mL; +26%) during the treatment period while remaining unchanged in the Control group when compared to the pre-treatment period. 3.4.3.3. Caudal artery blood velocities. Mean velocity was lower in the Medium and High groups during the treatment (-0.13 and -0.06 m/s, respectively) and post-treatment periods (-0.11 and -0.13 m/s) when compared to the pre-treatment values (Tx*EP p=0.027; Figure 2A). Likewise, peak systolic velocity and end diastolic velocities also decreased (Tx*EP p≤0.004 for both endpoints) during the treatment (-0.08 and -0.08 m/s, respectively) and post-treatment (- 0.03 m/s and -0.09 m/s) periods in the Medium group but during the post-treatment period only (-0.08 and -0.07 m/s) in the High group (Figure 3.2C, E). Both peak systolic and end diastolic velocities were lower during the post-treatment in the Low group when compared to the treatment period (-0.07 and -0.05 m/s). In contrast, peak systolic velocity during the post- treatment period was higher (+0.19 m/s) than the pre-treatment value in the Control group. 3.4.3.4. Caudal artery pulse rate, pulsatility index, and resistivity index. Pulse rate of the caudal artery differed by experimental period (p=0.001; 93 ± 2, 77 ± 1, and 68 ± 1 bpm in the pre-treatment, treatment, and post-treatment periods, respectively) and treatment group (p=0.008; 78 ± 1, 74 ± 1, 75 ± 1, and 86 ± 2 bpm in the Control, Low, Medium, and High groups), but no interaction was detected (Figure 3.3A). Average pulse rate of the High group was higher than all other groups. There was decreasing trend in pulse rate for all groups throughout the experiment. Pulsatility index increased progressively over the experimental period (p=0.004; 1.19 ± 0.03, 1.47 ± 0.02, and 1.58 ± 0.04 in the pre-treatment, treatment, and post-treatment periods) but did not differ among groups (Figure 3.3C). Resistivity index in the Medium group increased (Tx*EP=0.05) by 11 and 13% during the treatment and post-treatment periods compared to the pre-treatment period (Figure 3.3E). Similar trends were observed in the other treatments but were not statistically different. 3.4.4. Internal iliac artery hemodynamics 3.4.4.1. Internal iliac artery diameter. Internal iliac artery diameter decreased (Tx*EP p=0.004) by 13% (-1.0 mm) in the Medium group during the treatment period compared to the pre-treatment period (Figure 3.1B). The post-treatment value in the Medium group was intermediate between the pre-treatment and treatment values. Artery diameter remained unchanged in all other groups.

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3.4.4.2. Internal iliac artery blood flow and volume. Blood flow decreased (Tx*EP p=0.001) by 40% in the Medium group from the pre-treatment to treatment period and remained reduced in the post-treatment period (Figure 3.1D). Blood flow decreased by 26% from the treatment to the post-treatment period in the High group. Internal iliac artery blood volume per pulse (Figure 3.1F) decreased over the experimental period (EP p=0.027) and was 28 ± 2, 24 ± 1, and 22 ± 1 mL per pulse during the pre-treatment, treatment, and post-treatment periods (averaged among treatment groups), respectively. 3.4.4.3. Internal iliac artery blood velocities. Compared to the pre-treatment period, mean velocity decreased (Tx*EP p=0.042) in the treatment period for the Control (-0.22 m/s) and Medium (-0.19 m/s) groups (Figure 3.2B). Further, the mean velocity decreased from pre- treatment to post-treatment periods in the Control (-0.32 m/s) and Low (-0.37 m/s) groups. Mean velocity decreased from treatment to post-treatment periods in the Low (-0.16 m/s) and High (- 0.23 m/s) groups. Peak systolic velocity decreased (Tx*EP p=0.01) from the treatment to post- treatment period in the Medium (-0.19 m/s) and High (-0.27 m/s) groups (Figure 3.2D). End diastolic velocity varied by experimental period (p=0.001; 0.40 ± 0.02, 0.29 ± 0.01, and 0.22 ± 0.01 m/s in the pre-treatment, treatment, and post-treatment periods, respectively) and treatment groups (Tx p=0.045; 0.29 ± 0.01, 0.27 ± 0.01, 0.32 ± 0.01, and 0.28 ± 0.02 m/s in the Control, Low, Medium, and High groups, respectively) (Figure 3.2F). 3.4.4.4. Internal iliac artery pulse rate, pulsatility index, and resistivity index. Pulse rate decreased (Tx*EP p=0.045) between the pre-treatment and treatment periods in the Control (-20 bpm), Medium (-21 bpm), and High (-19 bpm) groups (Figure 3.3B). A decrease in the pulse rate was also recorded during the post-treatment (versus the pre-treatment period) for all groups. Internal iliac artery pulsatility index differed between treatment groups (p=0.026; 1.83 ± 0.05, 1.97 ± 0.05, 1.80 ± 0.04, and 1.77 ± 0.05 in the Control, Low, Medium, and High groups, respectively) and increased progressively during the experimental Period (p<0.001; 1.32 ± 0.04, 1.83 ± 0.03, and 2.25 ± 0.05 in the pre-treatment, treatment, and post-treatment periods, respectively) (Figure 3.3D). On average, the Low group pulsatility index value was greater than the Control (+7%) and Medium (+9%) groups. Internal iliac artery resistivity index differed between treatment groups (p=0.029; 0.79 ± 0.01, 0.83 ± 0.01, 0.79 ± 0.01, and 0.81 ± 0.01 in the Control, Low, Medium, and High groups, respectively) and increased progressively during the

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experimental period (p=0.002; pre-treatment, treatment, and post-treatment RI were 0.74 ± 0.01, 0.81 ± 0.01, and 0.84 ± 0.01, respectively) (Figure 3.3F). 3.4.5. Plasma prolactin concentration and rectal temperature. Data for plasma prolactin concentration and rectal temperature are presented in Table 3.2. Prolactin data were previously reported (Grusie et al. 2018b) but was analyzed differently (i.e., by percentage decrease from baseline; weekly values). In the present study, prolactin data was analyzed using absolute values of concentrations per animal averaged over the pre-treatment, treatment period and post- treatment periods. No difference was detected in prolactin values between the treatment groups (p=0.114) or during the experimental periods (p=0.841). These results mirror those of the previously published results (Grusie et al. 2018b). Rectal temperature data were also reported previously (Grusie et al. 2018b) but are presented here based on the average of pre-treatment, treatment and post-treatment periods. Rectal temperature decreased by 0.6 ºC and 0.7ºC from the pre- to post-treatment period in the Control and Low groups, respectively (p<0.001). Post- treatment rectal temperature was also 0.8ºC lower during the treatment period for the Control group. No differences were detected in rectal temperature between experimental periods in either the Medium or High groups. 3.5. DISCUSSION This study evaluated the effects of prolonged low-concentration ergot alkaloid exposure on hemodynamic responses in the caudal and internal iliac arteries of beef cows around the time of parturition. The three concentrations of ergot alkaloids (48, 201, 822 µg total ergot alkaloids per kg of dry matter intake offered) were chosen to be well below the Canadian permissible concentrations of 2000 to 3000 µg/kg of feed (CFIA 2017) and lower limit was decreased compared to our previous work (Cowan et al. 2018) in order to more accurately represent and examine an on-farm feeding scenario. Concentration-dependent hemodynamic changes in the caudal artery indicative of moderate vasoconstriction were observed at the medium (201 µg/kg) and high (822 µg/kg) ergot alkaloid concentrations. Recorded reductions in caudal artery diameter (14% versus none), blood flow (37% vs. 29%) and volume per pulse (29% vs. 11%) were more pronounced in the high than the medium treatment group. In contrast, increased resistivity index was more pronounced in the medium group versus the high group (11% vs. none). Likewise, the diameter of the internal iliac artery and blood flow decreased (10% and 40%) during the treatment period in the medium treatment. The mean blood velocity and peak

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TABLE 3.2. Plasma prolactin concentration (ng/mL; mean ± SEM) and rectal temperature (°C; mean ± SEM) of periparturient Hereford cows (n=32) during the pre-treatment (2 weeks), treatment (9 weeks), and post-treatment (3 weeks) experimental periods to increasing concentrations of total ergot alkaloids in their feed (i.e., Control, Low, Medium and High groups). Plasma for prolactin analysis was collected weekly and analyzed by enzyme-linked immunosorbent assay (ELISA). Rectal temperature was measured weekly by handheld digital thermometer.

Ergot Treatment Control Medium High (<15 µg/kg Low (201 µg/kg (822 µg/kg DM*) (48 µg/kg DM) DM) DM) n 9 9 9 6 Plasma prolactin (ng/mL) P-values: Tx=0.114, EP=0.841, Tx*EP=0.462 Pre-treatment 50.5 ± 3.5 57.2 ± 6.0 65.6 ± 8.2 54.9 ± 5.3 Treatment 45.6 ± 2.4 51.2 ± 2.3 51.6 ± 2.2 42.9 ± 2.2 Post-treatment 33.6 ± 2.2 40.1 ± 3.0 44.4 ± 3.5 38.1 ± 4.7

Rectal temperature (°C) P-values: Tx=0.630, EP<0.001, Tx*EP<0.001 Pre-treatment 39.0 ± 0.1axy 39.2 ± 0.1ax 38.9 ± 0.1y 38.9 ± 0.1xy Treatment 39.2 ± 0.1ax 39.0 ± 0.1ax 38.9 ± 0.1x 38.7 ± 0.1y Post-treatment 38.4 ± 0.1bx 38.5 ± 0.1bx 38.8 ± 0.1y 38.7 ± 0.1xy

* DM = dry matter Superscripts “ab” indicate differences in columns whereas “xy” indicate differences in rows. Values with uncommon alphabets are different at p≤0.05

105 systolic velocity of the caudal and internal arteries decreased between 12 to 25% during the treatment and post-treatment periods. Changes in the internal iliac artery in the medium treatment were comparable to those in the caudal artery. Most hemodynamic endpoints in both arteries recovered to pre-treatment values following removal of ergot from feed. The exceptions to this were the blood flow velocities, internal iliac artery pulse rate, and caudal artery resistivity index. Our results clearly document a relationship between the ergot alkaloid concentration in feed and alterations in hemodynamic parameters in the caudal artery. This was partially observed in the internal iliac artery. Plasma prolactin concentrations did not change, rectal temperatures changes were within normal ranges for cattle, and no clinical symptoms of ergotism were observed during the study period. These findings indicate that hemodynamics changes are sensitive bioindicators of ergot exposure and pharmacological effect. Commonly reported hemodynamic endpoints in various arteries of livestock exposed to ergot alkaloids (originating from cereal grain ergot, tall fescue grass or perennial ryegrass) include reduced arterial diameter (McDowell et al. 2013; Cowan et al. 2018), decreased arterial luminal area (Aiken et al. 2007, 2009, 2011, 2016; McDowell et al. 2013; Aiken and Flythe 2014; Klotz et al. 2016; Poole et al. 2018), decreased blood flow rate (Aiken et al. 2007, 2009, 2016; Cowan et al. 2018), and decreased blood flow volume (Aiken et al. 2007, 2009; McDowell et al. 2013; Cowan et al. 2018). Heart rate (i.e., pulse rate) has been reported to decrease reflexively in livestock exposed to ergot alkaloids to maintain blood pressure during vasoconstriction (Oliver 1997; McLeay et al. 2002; Aiken et al. 2007; Strickland 2011) with subsequent recovery within approximately one week of initial exposure. In the present study, internal iliac artery pulse rate decreased over the course of the experiment. Blood flow was reduced in the medium and high ergot treated groups, with decreased diameter being recorded in the medium group, suggesting that pulse rate changes may have been compensating for vasoconstriction. As expected, changes in the pulse rate were similar between the internal iliac and caudal arteries. The caudal artery is clinically significant to ergot poisoning as tail tip sloughing is a frequently observed symptom of gangrenous ergotism in cattle (Burfening 1973; Robbins et al. 1986; Shelby 1999; Strickland 2011; Craig et al. 2015). Similar vasoconstrictive alterations to those reported in the present study were also observed in the caudal artery of cows following one-week exposure to 529 and 2115 µg ergot alkaloids/kg of dry matter intake (Cowan et al.

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2018). The highest concentration of ergot in feed in that study was ~2.5x higher than the highest concentration in the present study, however the degree of change in hemodynamic parameters are comparable between studies. Caudal artery diameter, blood flow, and blood volume were decreased during the one-week treatment period at 2115 µg/kg and an intermediate response was observed in the 529 µg/kg treatment. Considering together the results of the present and previous study, there is observable peripheral arterial response between 200 to 800 µg ergot alkaloids per kg of dry matter following exposure of cows to ergot for one week to nine weeks followed by recovery and compensation of most of the hemodynamic parameters depending on the concentration of ergot alkaloids in the feed. In contrast to the changes in arterial diameter, blood flow, and blood volume per pulse reported herein, other studies have not found consistent effects of treatment on blood flow velocities and indices of resistance to flow (Aiken et al. 2007, 2009; McDowell et al. 2013). The present study observed a general decrease of caudal artery blood flow velocities in the post- treatment period, most predominantly in the animals fed 500 to 800 µg ergot alkaloids per kg of dry matter. Blood velocities changes were less consistent in the internal iliac artery. Decreased velocities of flow during the treatment period were not anticipated, as velocities are expected to increase in response to decreased arterial diameter and vasoconstriction (Skinner 1978; Stoner et al. 2004). However, decreased flow velocity following removal of ergot from the feed could indicate compensatory mechanisms to maintain blood pressure and flow. Conversely, decreased blood flow coupled with increased resistivity index (an indicator of vasoconstriction and increase vascular impedance to blood flow (Maulik 1993; Pontremoli et al. 1999) observed during treatment and post-treatment period at 500 µg/kg ergot alkaloid concentration could indicate delayed effects of ergot. It is noteworthy that there were no concentration-dependent treatment effects on pulsatility index to accompany the changes in peak systolic velocity, similar to the effects observed in cows that consumed toxic tall fescue grass containing related ergot alkaloids (reported by Aiken et al. (2007)) during the treatment period. The progressive increase in pulsatility index in all groups including control animals over the course of experiment with highest values in post-treatment period was interpreted to have originated due to unrelated metabolic changes (e.g. postpartum length) or variations in environment factors. In contrast to effects during short-term (one-week) ergot exposure in cows (Cowan et al. 2018), decreased arterial diameter and blood flow in the internal iliac artery were observed

107 around 500 µg/kg ergot alkaloid concentration in the present study. As the internal iliac artery supplies blood to the organs of the pelvis, reduced blood flow to the uterus and placenta could negatively affect ovarian function (Matsui and Miyomoto 2009) and fetal development (Reynolds et al. 2006). A fescue ergot alkaloid study from Poole et al. 2018 found decreased uterine and ovarian artery luminal areas on days 10 and 17 of the estrous cycle in treatment heifers fed 500 µg ergovaline and ergotamine per kg feed for 63 days. However, ovarian function, as indicated by antral follicle counts, corpus luteum area, and progesterone concentration were unaffected by the fescue ergot alkaloid treatment in that study. It is interesting to note that that postpartum ovarian function was also not affected by ergot treatment in our group of animals (reported earlier by Grusie et al. 2018b) and cows maintained normal pregnancy (i.e., no pregnancy loss during first two months of gestation during observation period). Overall, it appears that caudal artery is more responsive to ergot alkaloids than the internal iliac artery, as a greater number of hemodynamic endpoints were altered in the caudal artery. We speculate that this may be related to anatomic location and histological structure of the arteries (i.e., elastic versus smooth muscle content, internal versus subcutaneous location) and/or the differences in abundance of serotonin and adrenergic receptors (Horn et al. 1982; Nakane and Chiba 1986; Martin 1994; Masu et al. 2008). It is unclear why an effect of ergot was present in the medium treatment versus the high treatment group in this study. We suspect this is related to inter-animal variability within the different treatment groups. This observation needs to be confirmed. In vitro studies of bovine arteries have demonstrated that arterial contractility is present despite removal of ergot alkaloids from the treatment medium (Dyer 1993; Klotz et al. 2007; Pesquiera et al. 2014). This raises concerns regarding the withdrawal times of ergot alkaloids in tissues and persistent pharmacological effects. In the present study, indicators of vasoconstriction in the caudal and internal iliac arteries of cows recovered to pre-treatment values following removal of ergot from the feed, thereby indicating no apparent persistent effect on hemodynamic parameters. There was no effect of ergot treatment on circulating prolactin concentration in the present study. Studies of ergot alkaloid mediated fescue toxicosis in cattle commonly cite depressed prolactin concentration in the blood as a bioindicator (Hurley et al. 1980; Lipham et al. 1989; Aldrich et al. 1993; Paterson et al. 1995; Aiken et al. 1998; Strickland et al. 2011;

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Klotz 2015), due to the dopaminergic activity of ergot alkaloids (Berde and Stürmer 1978; Sibley and Creese 1983; Pertz and Eich 1999). This has been observed in other studies of fescue toxicosis, in which ergot alkaloids, principally ergovaline, are implicated in symptoms similar to C. purpurea ergotism. Our results, in addition to those of previous studies from our laboratory (Grusie et al. 2018b; Cowan et al. 2018), do not demonstrate that prolactin response is measurable when beef cattle are fed ergot alkaloids in the range 50 to 800 µg per kg of dry matter. Prolactin may be a more relevant and sensitive bioindicator in pigs, horses, and potentially in dairy cattle but is not a useful diagnostic indicator for subclinical ergot exposure in beef cows. The vasoconstrictive activity of ergot alkaloids is known to increase susceptibility of livestock to heat or cold stress (Aldrich et al. 1993; Strickland et al. 2011). In the present study, despite arterial constriction, rectal temperatures in ergot-exposed cattle were within normal physiological ranges (Robertshaw 2004; Fielder 2019). The moderate climatic conditions of this study in addition to restricting cattle to pens during treatment period (i.e., no grazing and pasture activity) made the development of hyperthermic ergotism unlikely. In conclusion, hemodynamic alterations suggestive of vasoconstriction was demonstrated in the caudal artery and partially in the internal iliac artery of periparturient cows exposed to up to 822 µg ergot alkaloids per kg of dry matter intake for a period of nine weeks. These included reduced diameter, blood flow in the caudal and internal iliac arteries, changes which are consistent with an altered hemodynamic state associated with ergot exposure. Prolactin concentration and rectal temperature are not useful indicators of subclinical ergot exposure in beef cows. The results of this study suggest that the current regulatory permissible concentrations of ergot in cattle feed may be re-examined to account for observed subclinical hemodynamic changes.

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4. CHAPTER 4 – Development and partial validation of a method to detect and quantify ergopeptine alkaloids in bovine plasma with Liquid Chromatography Tandem Mass Spectrometry PREFACE This research was conducted to generate pharmacokinetic information on ergot alkaloids in cattle following exposure in feed (i.e., the most toxicologically relevant route of exposure). In turn, a sensitive method to detect ergot alkaloids in bovine blood samples was developed and partially validated to accompany the pharmacokinetic studies. However, ergot alkaloids were not detected in experimental samples by the method developed. This lack of detection is likely due to the reported low oral bioavailability of ergot alkaloids. The data described in this chapter may be published later. The proposed citation for this chapter is as follows: Cowan, V., Michel, D., Blakley, B., Alcorn, J., and Singh, J. (2020). Development and partial validation of a method to detect and quantify ergopeptine alkaloids in bovine plasma with Liquid Chromatography Tandem Mass Spectrometry. VC was responsible for conducting the pharmacokinetics studies, sample preparation and extraction for analysis with LC-MS, analysis, and chapter preparation. DM instructed laboratory and analytical procedures, provided technical and analytical supports, and contributed to chapter preparation. BB was involved in ergot alkaloid concentration selection, experimental design, and consultation. JA contributed to concept development and pharmacokinetic study design (starting in 2017), including the theoretical background on pharmacokinetic principles, provided laboratory space and technical support for sample extraction and analysis, and chapter preparation and revisions. JS was the principle investigator of the grant and contributed toward chapter preparation/revisions.

In order to understand the duration of vascular effects observed in Chapters 2 and 3, generation of pharmacokinetic information (such as half-life of elimination) was necessary. In turn, a sensitive analytical method was required for detecting ergot alkaloids in bovine plasma.

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4.1. ABSTRACT Ergopeptine alkaloids are common adulterating substances in livestock feed and may lead to poisoning of exposed animals. The objective of this work was to develop and partially validate a method to quantify ergopeptine alkaloids (ergocornine, ergocryptine, ergosine, and ergocristine) in bovine plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) to enable generation of pharmacokinetic information from feeding trials in cows. Bovine plasma samples (190 µL) were spiked with ergocristine, ergocornine, ergocryptine, and ergosine and extracted by protein precipitation with acetonitrile. Extracts were analyzed by LC-MS/MS. Method development and partial validation included linearity, determination of limit of detection and limit of quantitation, accuracy, precision, and autosampler stability. Chromatographic separation was achieved by gradient elution with a mobile phase consisting of acetonitrile and ammonium acetate on a ZORBAX C18 column (2.1150 mm, 5 m). LC-MS/MS detection was performed on an Applied Biosystems SCIEX 4000 hybrid triple quadrupole linear ion trap mass spectrometer instrument with positive electrospray ionization operated in multiple reaction monitoring mode with a 21-minute run time. Ergocornine, ergocryptine, and ergosine were linear (R2≥0.9990) between 0.98 and 125 ng/mL. Ergocristine was linear between 1.95 and 250 ng/mL (R2=0.9992). Inter-day precision for the lower limit of quantification (LLOQ), low quality control (LQC), medium quality control (MQC), and high quality control (HQC) was ≤8.3% for each alkaloid. Inter-day accuracy for the LLOQ, LQC, MQC, and HQC ranged from 92.4-103% for each alkaloid. The QCs for each alkaloid met passing criteria on days 1 and 2, but sample failure was observed on day 3 for ergocristine (LLOQ, LQC) and ergocryptine (LLOQ). Intraday precision for each QC on day 1, 2, and 3 was ≤8.8%, ≤9.3%, and ≤9.2%, respectively. Intraday accuracy each QC on day 1, 2, and 3 ranged from 92.8-108%, 93.3-111%, and 89.9-105%, respectively. Analytes were stable under autosampler conditions. Two replicates of pharmacokinetic studies were conducted in beef cows following a one-time high concentration exposure to ergot alkaloids in pelleted feed (27000-29000 µg/kg feed; 250 µg/kg body weight). Analysis of experimental samples from both studies did not reveal any chromatographic peaks for any of the ergot alkaloids, leading to the conclusion of low oral bioavailability and lack of analytical sensitivity.

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4.2 INTRODUCTION Ergot alkaloids are toxic secondary metabolites produced by the pathogenic plant fungus Claviceps purpurea. These mycotoxins are contained within the characteristic sclerotium of the fungus and are harvested alongside grains and cereals, leading to contamination of cereal and grass-based feeds. As a result, ergot alkaloids are commonly detected in and quantified in livestock feed. The C. purpurea fungus produces four types of ergot alkaloids, including simple lysergic acid derivatives (e.g., ergometrine), clavine alkaloids (e.g., agroclavine), lactam alkaloids (e.g., ergocristam), and ergopeptine alkaloids (e.g., ergotamine) (Flieger, Wurst and Shelby 1997; Komorova and Tolkachev 2001; Krska and Crews 2008; EFSA 2012; Arroyo- Manzanares et al. 2017). The ergopeptine alkaloids are generally accepted as the most toxicologically important of the ergot alkaloids. Historically, ergopeptine alkaloids have been associated with poisoning in humans but remain an important modern veterinary problem due to increased mold contamination in livestock feeds and difficulties in mycotoxin mitigation. The most toxicologically relevant ergopeptine alkaloids are ergotamine, ergocornine, ergocryptine, ergocristine, and ergosine (EFSA 2012). Symptoms of ergot poisoning, i.e., ergotism, in livestock varies from feed refusal, hypoprolactinemia, poor offspring performance, impaired thermoregulation, peripheral ischemia and gangrene, and, most rarely, neurologic signs (Robbins et al. 1986; Shelby 1999; Klotz 2015). Despite the availability of some data regarding the intravenous pharmacokinetic behaviour of ergot alkaloids in livestock, the oral pharmacokinetics are largely unknown. It is generally accepted that the ergopeptine alkaloids have a low oral bioavailability based on studies of ergotamine (Ala-Hurula et al. 1979a,b; Ekbom, Paalzow, Waldenlind 1981; Waldenlind et al. 1982; Ibraheem, Paalzow, Tfelt-Hansen 1983); however, these compounds are highly potent and possess high vasoconstrictive activity through binding to peripheral serotoninergic and adrenergic receptors (Berde and Stürmer 1978; Pertz and Eich 1999). In addition, the ergopeptine alkaloids appear to have a moderate elimination half-life of minutes to hours (Aellig and Nüesch 1977; Orton and Richardson 1982; Ibraheem, Paalzow, Tfelt-Hansen 1982; Perrin 1985; Sanders et al. 1986; Jaussaud et al. 1998; Durix et al. 1999; Bony et al. 2001) that may contribute to the progressive development of symptoms in humans and livestock. These factors contribute to a concentration-effect relationship where low or non-detectable concentrations of

112 the ergopeptine alkaloids are associated with pharmacologic effect (Tfelt-Hansen, Eickhoff, Olesen 1980; Perrin 1985; Tfelt-Hansen 1988; Bigal and Tepper 2003). Ergopeptine alkaloids have been detected in food- and feedstuffs using numerous technologies. This topic has been extensively reviewed by Scott (2007), Krska and Crews (2008), Crews (2015), and Arroyo-Mantanzares et al. (2017). Modern, sensitive methods of detection for ergot alkaloids in food and feed include liquid chromatography mass spectrometry (LC-MS), (ultra) high performance liquid chromatography mass spectrometry ((U)HPLC- MS/MS), and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although ergot alkaloids and their various derivatives have been quantified in plasma from livestock subjects (Moubarak et al. 1996; Jaussaud et al. 1998; Durix et al. 1999, Bony et al. 2001), quantification has generally following intravenous administrations in animals. Quantification of common ergopeptine alkaloids in plasma of livestock, including ergocristine, ergocornine, ergocryptine, and ergosine, has not been achieved. Sensitive and accurate methods to detect the ergopeptine alkaloids in livestock plasma following oral exposure are unavailable at present due to analytical difficulties and kinetic challenges. The objective of this work was to develop a method to detect and quantif y ergot alkaloids in bovine plasma for subsequent use in pharmacokinetics studies. The rationale was that, with highly sensitive technologies available such as LC-MS/MS, detection of common ergopeptine alkaloids in cow plasma following exposure to these compounds in feed may be possible. The overarching goal of this work was to generate basic pharmacokinetic information on ergopeptine alkaloids, as such information is not widely available in the scientific literature. Applications of this information could include diagnostic implications (i.e., detection of ergot alkaloids in aborted fetuses or assessing livestock that consumed suspect feed), therapeutic implications (i.e., testing for unexplained feed withdrawal in livestock), food safety (i.e., withdrawal times of ergot alkaloids from food animal tissues). Lastly, information on the correlation between the pharmacology and toxicology of ergot alkaloids with feed concentrations is not well-established in livestock. 4.3. MATERIALS AND METHODS 4.3.1. Chemicals and reagents. Analytical grade acetonitrile and water were used for this study (Fisher Chemical, USA). Mobile phase A (i.e., aqueous) was comprised of 10 mM ammonium acetate in Millipore water (0.22 µm Millipak® 40, Milli-Q Q-POD® Advantage

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A10). Mobile phase B (i.e., organic) was HPLC grade acetonitrile (Optima® LC-MS A955-4, Fisher Chemical, USA). Ergot alkaloid standards were diluted in a solution of 85:15 (%v/v) acetonitrile:water with ammonium acetate. This 85:15 solution was also used to prepare the blank. The 85:15 (%v/v) solution is employed by Prairie Diagnostic Services to extract ergot alkaloids from cereals and grasses and was demonstrated by Krska et al. (2008) as a suitable solvent for mycotoxin extraction. 4.3.2. Ergot alkaloid standards. Ergot alkaloid standards were provided by Prairie Diagnostic Services (Saskatoon SK Canada). Ergocornine (Art. No. 002064), ergocristine (Art. No. 002065), and ergocryptine (Art. No. 002066) were purchased and imported from Romer Labs (Romer Labs Division Holding GmBH, Austria). Ergosine (CAS No. 561-94-4) was purchased from LGC Standards (LGC Limited, USA). The ergot alkaloids standards were each diluted to from 100 µg/mL (0.5 mg) to 20 µg/mL in 85:15 (%v/v) acetonitrile:water with ammonium acetate solution. The alkaloid stock solutions were stored at -80 °C when not in use and warmed to room temperature (approx. 22 °C) on the day of use. The ergot alkaloids were pooled in a master standard stock solution that was used to make the calibration curve. The ratio at which the alkaloids were added changed throughout the method development procedure but was either 1:1:1:1 (%v/v) (earlier work) or 1:1:1:2 (with ergocristine being the more concentrated alkaloid; later work). The stock concentrations of the pooled alkaloids varied from 2500 to 5000 ng/mL pre-extraction concentration. The pre-extraction calibration curve was prepared by parallel dilution using the 85:15 extraction solution as the diluent. Details for preparation of the calibration curve are given in Appendix E. 4.3.3. Spiked plasma preparation. Bovine plasma was thawed at room temperature on the same day of analysis. Plasma was centrifuged for 5 minutes at 3000 rpm before use with Beckman Coulter Allegra 25R Centrifuge to pellet any debris or fibrin in the sample. Spiked plasma samples were prepared for extraction by adding 10 µL of standard or quality control (QC) to 190 µL of plasma. A blank was prepared by adding 10 µL of 85:15 extraction solution to 190 µL of plasma. For experimental samples, a volume of 200 µL was extracted. Cow plasma samples from pharmacokinetics studies were stored at -80 °C prior to extraction. Experimental design and procedures for the two pharmacokinetic studies are described below. Samples were mixed by vortex prior to protein precipitation procedure.

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4.3.4. Quality control sample preparation. Quality control (QC) stock solutions were prepared from the 5000/2500 ng/mL pooled alkaloid mix. Instructions for QC preparation are described in Appendix V. The concentration of the low, medium, and high QCs (LQC, MQC, and HQC, respectively) were selected such that they were not calibration points on the standard curve but would test the quantifiable range of the curve. The QCs were prepared in replicates of six. Accuracy and precision of QCs was assessed by taking the average %accuracy of the analytical run for each QC and the coefficient of variation of the calculated concentration. The equations for accuracy and precision are as follows: 푡푎푟푔푒푡푒푑 푐표푛푐푒푛푡푟푎푡푖표푛 퐴푐푐푢푟푎푐푦: % 푎푐푐푢푟푎푐푦 = ∗ 100 ……………………………………(4) 푐푎푙푐푢푙푎푡푒푑 푐표푛푐푒푛푡푟푎푡푖표푛 (푎푣푒푟푎푔푒 푐표푛푐푒푛푡푟푎푡푖표푛) 푃푟푒푐푖푠푖표푛: % 퐶푉 = ∗ 100……………………………………………(5) (푆푇퐷퐸푉 표푓 푐표푛푐푒푛푡푟푎푡푖표푛)

The concentrations of the QCs used in the method development procedure varied. The high quality control (HQC) sample was chosen to be 4000/2000 ng/mL (pre-extraction concentration) to satisfy the requirement of 75% of the calibration curve range. The medium quality control (MQC) sample is defined as midpoint of the calibration range and was chosen to be 2400/1200 ng/mL (pre-extraction concentration). The low quality control was defined as up to 3x the LLOQ and was set to 100/50 ng/mL (pre-extraction concentration). The lowest limit of quantitation (LLOQ) is the lowest concentration of the calibration curve having a minimum response of 5x signal-to-noise ratio (S:N) of the baseline. Each QC was prepared in replicates of six. 4.3.5. Sample extraction by protein precipitation. Protein precipitation extraction procedures were carried out in 1.5 mL microcentrifuge tubes. Samples (including calibrants, QCs, and experimental samples) were precipitated by adding three volumes of cold acetonitrile (i.e., 600 µL) per sample. The rationale for protein precipitation was to liberate any ergot alkaloids bound to plasma proteins that could affect alkaloid recovery and to remove potential interfering endogenous compounds. Precipitated samples were mixed by vortex for 30 seconds. Precipitation debris were pelleted by centrifugation for 10 minutes at 14 000 rpm (4°C; VWR Micro 18R centrifuge). Supernatant (approx. 500 L) was added to amber autosampler vials (2 mL; Agilent Technologies part no. 5182-0716) for subsequent LC-MS/MS quantification. 4.3.6. Instrument and liquid chromatography-mass spectrometry (LC-MS) conditions. Note: Prior to March 29th, 2018, LC-MS analysis was performed using an Agilent 1200 series High Performance Liquid Chromatography (HPLC) system (Model # 1022643 P; Mississauga,

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ON, Canada) interfaced to an Applied Biosystems SCIEX 4000 hybrid triple quadrupole linear ion trap mass spectrometer (Concord, ON, Canada) equipped with a TurboionsprayTM interface. A new HPLC was installed on March 29th, 2018. This was an Agilent 1260 Infinity II HPLC (Model # G7112B) with a 1260 vial sampler. The MS was the same as before. All analytical work was carried out using the College of Pharmacy and Nutrition Core Mass Spectrometry Laboratory. The system was fitted with an Agilent ZORBAX Eclipse ZDB- C18 Narrow-Bore column (2.1x150 mm, 5 µm; P.N. 993700-902, S.N. USNM006257, L.N. B13203) that was gratefully provided by Prairie Diagnostic Services. Sample was injected using the HPLC autoinjector (volume=10 µL) at 4°C. A gradient of mobile phases A and B (95% A, 5% B) was used to deliver sample to the column at a flow rate of 0.300 mL/minute. The gradient sequence was as follows: 5% B at 0 minutes, 17% B at one minute, 46% B at 2 minutes, 54% B at 8 minutes, 8% B at 13 minutes, 5% B at both 16 and 20 minutes. Multiple reaction monitoring (MRM) was accomplished by electrospray ionization in positive mode. The monitored precursor ion to product ion transitions for each of the four alkaloids are given below in Table 4.1. Source temperature was set to 700 °C. Ion spray voltage was set to 5500 V. Curtain gas was set to 50. Nebulizer gas and heater gas were set at 55 and 60 PSI, respectively. Collision gas was set to 8. Exit potential for all transitions was 10. Dwell time for all transitions was 150 ms. Nitrogen gas was used as the gas for all cases. Interface heater was on. Declustering potential was 106, 131, 121, and 111 V for ergocornine, ergocristine, ergocryptine, and ergosine, respectively. Collision energy was 37, 39, 37, and 47 volts for the Q1 to Q3 transitions of ergocornine, ergocristine, ergocryptine, and ergosine, respectively. Collision exit potential was 16, 18, 18, and 14 V for the Q1to Q3 transitions of ergocornine, ergocristine, ergocryptine, and ergosine, respectively. Chromatographic separation for each analyte was achieved with 21-minute run times. The blank was run before and after the calibration curve. The software used for quantification was Applied Biosystems/MDS SCIEX Analyst version 1.6 (prior to 2019) and version 1.7 (2019). Calibration curves for each alkaloid were constructed by plotting the concentration (ng/mL) against analyte intensity (counts per second). A weighted 1/x linear regression of the data was performed to generate the equation of the line and the associated coefficient of determination (R2). Accuracy was automatically calculated by the software for samples designated as standards or quality controls. Calculated concentration of samples generated by the software was used to assess sample precision (by %CV, as indicated

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TABLE 4.1. Q1 to Q3 Transitions of ergot alkaloids analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) by Multiple Reaction Monitoring (MRM). Ergot alkaloid analysis was conducted with an Agilent 1200 series High Performance Liquid Chromatography (HPLC) system interfaced to an Applied Biosystems SCIEX 4000 hybrid triple quadrupole linear ion trap mass spectrometer equipped with a TurboionsprayTM interface. Quantifier Qualifier Ergot Alkaloid Q1 mass (Da) or Q3 mass (Da) or Q3 mass (Da) or Product Precursor Ion [M+H+] Product Ion (m/z) Ion (m/z) (m/z) Ergocornine 562.475 268.300 208.000 Ergocristine 610.450 268.200 208.100 Ergocryptine 576.508 268.200 208.200 Ergosine 548.479 223.100 208.100

117 above in equation (5)). 4.3.7. Acceptance criteria for calibration curve and quality control samples. The acceptance criteria for calibration curves and QCs was based on FDA Bioanalytical Method Validation guide for Industry (U.S. Food and Drug Administration 2018). Calibration curves were considered to have good linearity when R≥0.98 (i.e., R2≥0.96) with a minimum of 6 non- zero standards. Calculated concentrations for individual standards had to be within ±15% of the nominal concentration (i.e., 85-115% accuracy). Pass/fail criteria for QCs are listed in Table 4.2. The LLOQ, i.e., the lowest standard on the calibration curve, was allowed 20% variation from the nominal concentration but could not fail on the calibration curve during validation. Quality control samples were analyzed in replicates of six with no more than one replicate having > ±15% accuracy. More than one replicate not passing the accuracy criteria (>15%) was considered a failure for the entire QC and required repeating the validation day. Precision of the quality control samples was assessed with %CV, which was required to be ≤15% for low, medium, and high QCs and ≤20% for the LLOQ. During the validation, intraday and inter-day accuracy and precision were assessed by quantification of each QC on each of the three validation days. Therefore, there were three separation calculations for each of the three validation days for both accuracy and precision. For inter-day accuracy and precision, all values that met acceptance criteria for individual days were included in this overall calculation. This is described in greater detail in section 4.3.9.6. Linearity and range of standard curves was tested for each of the four alkaloids. Chromatograms were checked for signal-to-noise ratio, where peaks having S:N>3 were considered detectable and represented the approximate limit of detection (LOD). Chromatogram peaks with S:N between 5-10 were considered quantifiable and were considered the LLOQ. Only samples with S:N>5 (i.e., LLOQ or greater) were included in linear regression analysis for calibration curves. 4.3.8. Practical applications – oral pharmacokinetics studies in beef cows 4.3.8.1. Animal ethics statement. The following studies and procedures used therein were reviewed and approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use (Animal Use Protocol #20160068). All researchers received formal animal care and use training before working with the animals.

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TABLE 4.2. Definition of quality control samples and associated passing criteria for method validation. Quality control Definition Pass/Fail Criteria LLOQ S:N>5 >20% nominal concentration Low Up to 3× LLOQ >15% nominal concentration Medium Mid-range >15% nominal concentration High 75% of range >15% nominal concentration Abbreviation: LLOQ, lowest limit of quantitation

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4.3.8.2. Animal husbandry. Cows and their calves were housed individually in indoor pens in a barn at the Goodale Research Farm of the University of Saskatchewan Livestock and Forage Center of Excellence. The pens contained straw bedding (that was cleaned regularly), open air and sunlight access, and ad libitum access to water. In replicate 1 (summer 2016), the study was comprised of Simmental (n=2; non-lactating/no calf) and Hereford cows (n=8, lactating). In replicate 2 (summer 2017), the sample population included Hereford cows (n=11; lactating). Ergot-free pellets were offered to the cows in the individual pens to introduce them to the treatment medium to be used in the study. 4.3.8.3. Ergotized pellet preparation and ergot alkaloid quantification with liquid chromatography mass spectrometry (LC-MS). The ergotized pellets used for these experiments were manufactured by the Canadian Feed Research Centre (North Battleford, Saskatchewan). Control (i.e., ergot-free) pellets were formulated to meet the nutrition requirements of the cattle, including 80% barley and 15% oat hulls. The total concentration of ergot alkaloids in the ingredients used in the pellets were as follows: barley 22 µg/kg; canola 41.4 µg/kg; and oat hulls 86.1 µg/kg. Highly contaminated wheat screenings (previously obtained from near Weyburn, Saskatchewan) were used to spike treatment pellets with ergot alkaloids. The screenings comprised 5% of the final pellets. The rest of the pellets were made with 80% barley and 10% oat hulls. The total ergot alkaloid concentration of the pellets (ergotamine, ergometrine, ergosine, ergocornine, ergocryptine, and ergocristine) was determined following solvent extraction and quantification with Liquid Chromatography Mass Spectrometry. This analysis was conducted in Prairie Diagnostic Services (Saskatoon, Saskatchewan) as described by Grusie et al. (2017). The ergot alkaloid concentration in the treatment pellets is given in Table 4.3. 4.3.8.4. Jugular catheterization 4.3.8.4.1. Replicate 1. Jugular catheters were placed on morning of the experiment. Each cow was restrained in a locking head gate with pen and haltered prior to catheter placement. The skin was scrubbed with 70% isopropyl alcohol prior to catheter placement. Catheters were made and placed according to laboratory protocol. Catheters were made using polyethylene tubing (PE-160 0.045” internal diameter × 0.062” outer diameter; SAI Infusion Technologies, Lake Villa, IL USA) that was placed in isopropyl alcohol to sterilize minimum of 24 hours

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TABLE 4.3. Concentration of ergot alkaloids (µg/kg) in pelleted rations fed to cattle for two oral pharmacokinetics studies. Ergot alkaloids were quantified in feed following solvent extraction by liquid chromatography mass spectrometry (LC-MS) in Prairie Diagnostic Services (Saskatoon SK Canada). Cows (replicate 1 n=9, replicate 2 n=11) were given feed corresponding to 250 µg/kg body weight dose. Alkaloid concentration (µg/kg; % of total) Ergot alkaloid Replicate 1* Replicate 2** Ergocornine 4065 (13.9) 1259 ± 60 (4.6) Ergocristine 12437 (42.5) 18983 ± 917 (69.6) Ergocryptine 4230 (14.5) 3115 ± 115 (11.4) Ergometrine 2113 (7.2) 76 ± 16 (0.3) Ergosine 1513 (5.2) 990 ± 37 (3.6) Ergotamine 4852 (16.6) 2860 ± 92 (10.5) TOTAL 29210 (99.9) 27283 ± 947 (100.0) * Samples analyzed at dilution of 1/100 and 1/1000; ergocristine was analyzed at 1/1000 as per Prairie Diagnostic Services protocol for highly contaminated samples ** Two samples analyzed – concentration is mean ± standard deviation

121 prior to use. The researcher occluded the jugular vein manually and punctured it upon filling with a 12-gauge stainless steel needle. Once placement was correct, the tubing was pushed through the needle and blood flow through the tubing was checked. Once placed, bandage material (Elastoplast Beiersdorf; Hamburg DE EU) with Kamar adhesive (Kamar Inc. H.B. Fuller Co.; Vadnais Heights MN USA) secured the catheter to the placement site. Patency was maintained with installation of heparinized saline solution. Vetrap self adhesive dressing wrap (3M Animal Care Products; St. Paul MN USA) was used to keep the catheter in place by wrapping around the neck of the cow. 4.3.8.4.2. Replicate 2. Jugular catheters were placed on the day before the experiment. Each cow was restrained in a locking head gate with pen and haltered prior to catheter placement. The surgical area was clipped and washed with betadine solution and 70% isopropanol. The researcher placing the catheters practiced clean glove technique. Lidocaine (5 cc, neat) was injected subcutaneously over the jugular vein in two blebs (2.5 cc per bleb; ordered from the Western College of Veterinary Medicine pharmacy). Following vein palpation, the technician performed a stab incision in the skin (in one of the lidocaine blebs) overlying the jugular vein. The catheter was placed and secured with suture material. Vetrap self adhesive dressing wrap was used to further secure the catheter and extension tubing in place. The catheter was flushed with normal saline. Acid citrate dextrose (ACD) solution B (G-Biosciences; St. Louis MO USA) was instilled into the catheter (void volume = 2.5 mL from first port). Patency of the catheter was checked in the evening of the placement day by flushing with saline and re-instilling with ACD solution B. 4.3.8.4.3. Dose regimen and experimental protocol. 4.3.8.4.3.1. Replicate 1. The body weight of the cow recorded the day before the experiment was used to calculate the amount of ergotized feed required to achieve a one-time dose of 250 µg ergot alkaloids per kg body weight. Cow body weight and dose information is presented in Appendix F. Feed was withheld overnight. Calves and cows were separated prior to ergotized feed being offered (i.e., only the cow had access to ergotized feed). The time at which the cows started eating the pellets was recorded. If the cow consumed the entirety of the pellets offered, the time at which she finished them was considered time zero. If the cow did not consume all the pellets, but naturally stopped eating at a certain point, this point of time was considered time zero for that cow. The average consumption time for the cows was 14 ± 2 minutes. Whole blood

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(30 mL, when possible) was collected at 0, 15, 30, 45 minutes, 1, 2, 4, 6, 8, 12, 16, 20, 24, 32, 40, 48, and 56 hours after dosing. Catheters were filled with heparinized saline after each blood collection and initial 5mL of fluid was discarded at each collection. If the catheters were not working, blood was collected using 18-gauge vacutainer needles and lithium heparinized vacutainers with PST inserts (BD Vacutainer, Becton Dickinson Canada; Mississauga ON CAN). Plasma was separated from whole blood using centrifugation (1000 × g for 15 minutes at room temperature). Plasma was stored on-site at -20°C and was later transferred to -80°C at the Western College of Veterinary Medicine. During the most intense collection period, cows were given measured quantities of alfalfa hay and pellets in the individual pens (starting approximately 8 hours post-ergot). During longer intervals between collections (i.e., 8 hours), cows and calves were taken to an outdoor pen with ad libitum water, hay, and shade access. The cows were walked to the barn for the collections during this time period. 4.3.8.4.3.2. Replicate 2. A different type of anticoagulant was chosen for this replicate – ACD solution B versus heparin – to mitigate any potential kinetic differences due to ergot alkaloid interaction with heparin. We also found that the method of catheterization used in the first replicate worked poorly for our study and we elected to use real catheters. The time course of samples was also changed to ensure that we well characterized the distribution/elimination phase of the alkaloids from the system. We also found that our technique of washing the catheters with heparinized saline was incorrect so we ensured that we only instilled the void volume of the catheter needed, and replaced volume with neat (non-heparinized) saline in the cows. The body weight of the cow recorded the day before the experiment was used to calculate the amount of ergotized feed required to administer a one-time dose of 250 µg/kg BW. Cow body weight and dose information is presented in Appendix F. Feed was withheld overnight. Calves were separated from the cows prior to dosing (i.e., only the cow had access to ergotized feed). The time at which the cows started eating the pellets was recorded. If the cow consumed all her pellets, the time at which she finished them was recorded as time zero. If the cow did not consume all the pellets, but naturally stopped eating at a certain point, this point of time was considered time zero for that cow. Any remaining pellets were weighed back when possible to determine the dose she received. Whole blood (30 mL, where possible) was collected at 0, 10, 20, 30, 45 minutes, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48, and 72 hours after dosing. Following sample collection, when there was enough time between collections (i.e., after 45 minutes post-

123 dose), the catheters were flushed with normal saline (approximately 30 mL). The ACD solution B was instilled in catheters (1X void volume to 2X void volume) only when clotting became an issue. Plasma was collected from whole blood using heparinized vacutainers with PST inserts (BD Vacutainer, Becton Dickinson Canada; Mississauga ON CAN) using centrifugation (10 000 × g for 15 minutes at room temperature). Plasma was stored on-site at -20 °C and was later transferred to -80 °C. Catheters were removed by 12 hours post-dosing if still intact. Cow-calf pairs were taken back to their home pen with ad libitum hay and water access at this time. Collections at 24, 28, and 72 hours were done via jugular venepuncture using 18 G vacutainer needles. If the catheters failed before 12 hours, jugular venepuncture was used to collect samples. The calves were re-introduced to the cows at 4 hours post-dosing to allow for suckling as these cows were lactating. At 8 and 10 hours post-ergot, measured quantities of hay, oats, and ergot-free pellets were given to the animals. At 12 hours post-ergot, cows and calves were moved to an outdoor pen with ad libitum water, shelter, and hay access. Cows were walked to the barn for collections following this point. 4.3.9. Method development procedures 4.3.9.1. Detection limit test of ergot alkaloids in spiked plasma based on Prairie Diagnostic Services method for ergot alkaloids in feedstuffs. The purpose of this experiment was to determine the detection limit for ergot alkaloids spiked in plasma based on the method used by Prairie Diagnostic Services for ergot alkaloids solvent extracted from feed samples (described by Grusie et al. 2017). Ergot alkaloid standards were added to a master solution at 1:1:1:1 to achieve a final pooled concentration of 2000 ng/mL, i.e., 500 ng/mL per alkaloid pre- extraction. This corresponded to post-extraction concentrations of 100 ng/mL, i.e., 25 ng/mL per alkaloid post-extraction. The standard curve was prepared by serially diluting samples two-fold from 12.5 ng/mL to 0.049 ng/mL. A blank was run on the LC-MS prior to loading the samples. The calibrants were analyzed on the machine in order from least to most concentrated. 4.3.9.2. Linearity test based on detection limits using six calibration points. In order to meet the definition of a standard curve (as per FDA guidelines), a minimum of six calibrants was required. Results from section 4.3.9.1 informed us that our limit of detection was near 0.80 ng/mL for the analytes. Thus, a six-point calibration curve was prepared for each analyte based on the results of the detection limit test. The ratio of ergot alkaloids in the master stock remained

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the same as in section 4.3.9.1. The post-extraction calibration curve prepared to be from 25 ng/mL to 0.78 ng/mL for each alkaloid with two-fold dilutions. 4.3.9.3. Extended calibration curve to expand upper quantitation range. This test involved extending the standard curve further for linearity over a greater range from 250 ng/mL to 1.95 ng/mL. Results from section 4.3.9.2 indicated that there were sensitivity issues at the 0.78 ng/mL concentration for the analytes (i.e., the required six points to meet the criteria for a calibration curve were not achieved), therefore it was decided to test more concentrated calibrants at the low range of the curve. The pooled stock solution (1:1:1:1) of ergot alkaloids used was 20 000 ng/mL (20 µg/mL), corresponding to a post-extraction concentration of 1000 ng/mL pooled, i.e., 250 ng/mL per alkaloid. The calibration curve was prepared by two-fold parallel dilution from 250 ng/mL to 1.95 ng/mL. As 1.95 ng/mL was the lowest concentration of the calibrants, this was chosen was the preliminary LLOQ to test. For analysis following quantification, a weighted 1/x linear regression was used (as the calibration curve had a 100-fold change in concentration over its range). 4.3.9.4. Additional calibration curve test and preliminary low, medium, and high quality control sample analysis. Based on the previous results indicating that ergocristine was less reliably quantified below 1.95 ng/mL (versus the other three alkaloids), the concentration of ergocristine was increased 2-fold in the stock solution. This corresponded to a ratio ergocristine, ergocornine, ergocryptine, and ergosine of 2:1:1:1. Ergocristine standards were prepared by parallel dilution from 500 ng/mL to 3.91 ng/mL (post-extraction concentration). As in previous analyses, the calibration curve of ergocornine, ergocryptine, and ergosine was between 250 ng/mL to 1.95 ng/mL (post-extraction concentration). Linear regression for the calibration curve was weighted using 1/x because the range of the curve was approximately 100-fold. The first attempt to establish quality controls samples was made for this analysis to assess accuracy and precision of quantitation. The LQC, MQC, and HQC for ergocornine, ergocryptine, and ergosine were chosen to be 3.5, 112, and 186 ng/mL. The LQC, MQC, and HQC for ergocristine were chosen to be 7, 224, and 372 ng/mL, respectively. The QCs were prepared from the 500 ng/mL (ergocristine) and 250 ng/mL (other three EAs) stock solution. The order of analysis (and loading into the autosampler) was as follows: calibration curve (from lowest to highest concentration), blank, QCs (from lowest to highest concentration).

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4.3.9.5. Full QC test including first LLOQ set and analysis of experimental cow samples (replicate 1). Based on promising results that the LLOQ was below 1.95 but above 0.78 ng/mL for the alkaloids (and below 3.91 but above 1.56 for ergocristine), an additional lower concentration (0.98 or 1.95 ng/mL) was analyzed to further characterize the lower range of the calibration curve. This included running this lowest concentration in (n=6) as a dedicated LLOQ. The loss of sensitivity observed in the previous run was attributed to high machine use by other users. To offset this, the LC-MS/MS and column were primed thoroughly prior to use. The calibration curve for ergocristine ranged from 250 to 1.95 ng/mL while for the other three alkaloids ranged from 125 to 0.98 ng/mL. Quality control samples were included in this test for accuracy and precision; however, the concentrations were lowered from the previous run to accommodate for the altered range. The LQC, MQC, and HQC for ergocornine, ergocryptine, and ergosine were chosen to be 1.75, 66, and 93 ng/mL. The LQC, MQC, and HQC for ergocristine were chosen to be 3.5, 112, and 186 ng/mL, respectively. The QCs were prepared from the 250 ng/mL (ergocristine) and 125 ng/mL (other three EAs) stock solution. As the method had linearity and reproducibility, cow samples from the first replicate of the pharmacokinetics study were analyzed. Samples from two cows (pharmacokinetics study replicate 1) were used to assess if ergot alkaloids could be detected and/or quantified following a one-time high-dose oral ergot alkaloid exposure in feed. The following timepoints were analyzed: 0 (one cow), 0.25, 0.75, 1, 2 (one cow), 4, 6, 8, 12, 16 (one cow) 20, 24 (one cow) hours post-ergot. These time points were chosen based on the Cmax and tmax data available for ergot alkaloids (Tables 1.5 and 1.6). The run order for this analysis was as follows: blank, LLOQ (n=6), calibration curve (from lowest to highest concentration), blank, QCs (n=6 each, from lowest to highest concentration), blank, cow samples (samples from one cow first, from earliest to latest time points, followed by second cow). 4.3.9.6. Partial method validation with refined QCs. Note: A new HPLC instrument was installed in the Mass Spectrometry laboratory in March 2018. As ergot alkaloids were not detected in plasma samples from ergot exposed cows of the 2016 study, the potential to investigate in vitro pharmacokinetics studies became an avenue of interest. In addition, a more refined pharmacokinetics study was conducted in 2017 and these samples were not yet analyzed. Continued use of this method for either in vitro or in vivo applications required method validation. The partial method validation was conducted in accordance with the FDA

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Bioanalytical Method Validation guide for Industry (U.S Food and Drug Administration 2018). This included linearity, accuracy, precision, and autosampler stability. Plasma used for the three-day partial validation was collected from adult Angus bulls via jugular venepuncture using lithium heparin Vacutainer® tubes with PST inserts (BD Vacutainer, Becton Dickinson Canada; Mississauga ON CAN). Plasma was separated from whole blood via centrifugation and was pooled. Plasma aliquots were stored frozen (-20 °C) until use. Plasma for calibrant and QC preparation was the same as described above (section 4.3.3 to 4.3.5). Linear regression of the calibration curve was weighted using 1/x. Curve performance was assessed daily for three days for each analyte by assessing deviation of the standard concentration from the nominal concentration in addition to examination of the slope, y- intercept, and coefficient of determination (R2). Precision and accuracy of the calibration curve were evaluated using four quality control samples in replicates of six. The concentration of the QCs for ergosine, ergocornine, and ergocryptine following protein precipitation were as follows: LLOQ: 0.977 ng/mL; LQC: 2.5 ng/mL; MQC: 60 ng/mL; and HQC: 100 ng/mL. For ergocristine, these were LLOQ: 1.95 ng/mL; LQC: 5 ng/mL; MQC: 120 ng/mL; and HQC: 200 ng/mL. Accuracy and precision were calculated on each day (i.e., intraday accuracy and precision) and collectively over the three-day period (i.e., inter-day accuracy and precision). Coefficients of variation (%) were also calculated each QCs’ precision data. The acceptance criteria for the QCs was the same as described earlier (Table 4.1). 4.3.9.7. Autosampler stability. Autosampler stability was assessed by reinjecting the QCs from day 1 that were left overnight at 4 °C in autosampler conditions. This corresponded to re-injection and analysis of QCs approximately 42 hrs after first injection. 4.3.9.8. Statistical analysis – linear regression for comparison of slopes and Y- intercepts from three-day partial validation. Calibration curves were compared statistically using linear regression (GraphPad Prism Version 7; GraphPad Software, San Diego CA USA). The built-in linear regression analysis compared if slopes were different and if Y-intercepts were different (p<0.05) for each iteration (i.e., Day 1 versus Day 2, Day 1 versus Day 3, Day 2 versus Day 3). 4.3.9.9. Experimental cow samples from 2017 pharmacokinetics study in cows. The rationale for testing samples from this 2017 replicate was that the study was better designed and refined than the 2016 study, therefore minimizing potential erroneous results due to technical

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errors that may have occurred. The time points (post-ergot exposure) analyzed were as follows: 30 minutes (3/4 cows), 1, 1.5, 2, 4, 12, 24, and 72 hours post-exposure. The calibration curve was the same as section 4.3.9.6. The LQC, MQC, and HQC were run in replicates of four, in which half were ran before the cow samples and half were run afterwards (following a blank). The LLOQ was run in replicates of two (not including the lowest concentration calibrant on the curve). The QC concentrations were the same as section 4.3.9.6. 4.4. RESULTS 4.4.1. Detection limit test of ergot alkaloids in spiked plasma based on Prairie Diagnostic Services method for ergot alkaloids in feedstuffs. The retention times for ergocornine, ergocristine, ergocryptine, and ergosine were 10.0, 11.5, 11.0, and 8.5 minutes, respectively. Linear regression analysis for each analyte is given in Table 4.4. No chromatographic peaks were detected for any analytes below 0.40 ng/mL. Ergocornine had S:N >3 starting at 0.8 ng/mL (S:N=3.6). The S:N for 1.6 ng/mL was 7.9 and represented a potential LLOQ for ergocornine. Linear regression for the four points between 1.6 ng/mL and 12.5 ng/mL demonstrated good linearity for ergocornine (R2=0.9984). Accuracies ranged from 95.7 to 110%. Ergocristine had S:N=3.9 at 1.6 ng/mL and S:N=5.9 at 3.13 ng/mL. A three-point line was fitted between 3.13 ng/mL and 12.5 ng/mL, with accuracies ranging from 82.3 to 124%. Linearity was acceptable for these points (R2=0.96). Ergocryptine had S:N=3.2 at 0.8 ng/mL and S:N=5 at 1.6 ng/mL, thus a line was fitted for points between 1.6 ng/mL and 12.5 ng/mL. Accuracies for these samples ranged from 95.1 to 108%. Ergocryptine concentration displayed good linearity (R2=0.9982) across this range of concentrations. Ergosine had S:N >3 starting at 1.6 ng/mL (S:N=7) and represented a possible LLOQ. Linear regression analysis of the points between 3.13 ng/mL and 12.5 ng/mL produced linearity (R2=0.999). Calculated concentrations for each of the points of the standard curve had accuracies between 96.1 and 107%. Chromatograms for each alkaloid displaying the LOD and LLOQ are presented in Figures 4.1 and 4.2, respectively. Based on the concentrations analyzed and the respective signal-to-noise ratios, the limits of detection for ergocornine and ergocryptine were 0.8 ng/mL. These alkaloids were quantifiable at ≥1.6 ng/mL. The limit of detection for ergosine was greater than 0.8 ng/mL but less than 1.6

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TABLE 4.4. Analysis information and calculated concentrations of ergopeptine alkaloids spiked in bovine plasma following a detection limit test. Spiked plasma samples were extracted by protein precipitation with acetonitrile and were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The horizontal line in each ergot alkaloid column indicates the samples excluded (above) and included (below) in the calibration curve for that alkaloid. Ergot alkaloid Ergocornine Ergocristine Ergocryptine Ergosine Targeted Calculated concentration (ng/mL); concentration (ng/mL) (%Accuracy)* 0.049 0.343 (N/A) 0.628 (N/A) 0.451 (N/A) < 0 (N/A) 0.098 0.343 (N/A) 0.517 (N/A) 0.396 (N/A) < 0 (N/A) 0.195 0.318 (N/A) 0.648 (N/A) 0.551 (N/A) 0.064 (N/A) 0.39 0.61 (N/A) 0.821 (N/A) 0.664 (N/A) 0.294 (N/A) 0.78 1.02 (N/A) 1.14 (N/A) 1.14 (N/A) 0.872 (N/A) 1.56 1.73 (111) 1.86 (N/A) 1.70 (109) 1.67 (107) 3.13 3.12 (99.5) 3.87 (124) 3.17 (101) 3.17 (101) 6.25 5.97 (95.5) 5.15 (82.3) 5.93 (94.9) 6.0 (96.1) 12.5 12.6 (101) 12.9 (103) 12.6 (101) 12.6 (101)

n 4 3 4 4 Slope 2940 1190 2630 2660 Intercept -101 -368 -437 524 R 0.9991 0.9798 0.9990 0.9994 R2 0.9982 0.9600 0.9980 0.9988 * Accuracy = (calculated concentration/targeted concentration)  100 Abbreviations: N/A, not applicable; R, correlation coefficient; R2, coefficient of determination

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FIGURE 4.1. Representative chromatograms for four ergopeptine alkaloids spiked in bovine plasma having signal-to-noise ratios (S:N) at or near the limit of detection (LOD) as quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The S:N for ergocornine (0.78 ng/mL), ergocristine (1.56 ng/mL), and ergocryptine (0.78 ng/mL) were 3.6, 3.9, and 3.2, respectively; these all represented the LOD for that alkaloid. The S:N for ergosine at 0.78 ng/mL was 2.2, therefore below detection.

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FIGURE 4.2. Representative chromatograms for four ergot alkaloids spiked in bovine plasma having signal-to-noise ratios (S:N) at the lowest limit of quantitation (LLOQ) as quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The S:N for ergocornine (1.56 ng/mL), ergocristine (3.13 ng/mL), and ergocryptine (1.56 ng/mL), and ergosine (1.56 ng/mL) were 7.9, 6.8, 6.5, and 7, respectively.

131 ng/mL, as 1.6 ng/mL met LLOQ criteria based on its S:N. Ergocristine met detection criteria at 1.6 ng/mL and was quantifiable at 3.13 ng/mL. Ergocornine, ergocristine, and ergosine demonstrated good linearity between 1.6 and 12.5 ng/mL as all R2 were ≥0.998. Linear regression analysis of the three standards for ergocristine demonstrated linearity but more calibrants were required to meet calibration criteria (i.e., a minimum of six calibrants). 4.4.2. Linearity test based on detection limits using six calibration points. Retention times for ergocornine, ergocristine, ergocryptine, and ergosine were 10.0, 11.5, 11.0, and 8.50 minutes, respectively. The 0.78 ng/mL S:N for ergocornine, ergocristine, ergocryptine, and ergosine was 3.8, <3, 3.7, and 3.8, respectively. These concentrations were limits of detection for each analyte in this analysis. The 1.56 ng/mL S:N for ergocornine, ergocristine, ergocryptine, and ergosine was 9.9, 4.2, 5.8, and 5.4, respectively, indicating the LLOQ was 1.56 ng/mL for three of the four alkaloids for this analysis. Linear regression analysis of the points between and including 1.56 ng/mL to 25 ng/mL for ergocornine, ergocryptine, and ergosine displayed good linearity (R2≥0.996; Table 4.5); however, these regression curves only had five calibrants and did not meet the definition of calibration curve. For ergocristine, the curve was fitted between 3.13 ng/mL and 25 ng/mL and had good linearity (R2=0.9980), however, the regression curve only contained four calibrants. Representative chromatograms for each alkaloid at its limit of detection for this test are given in Figure 4.3. Ergocornine, ergocryptine, and ergosine were detectable at 0.78 ng/mL and quantifiable at 1.56 ng/mL. The LOD for ergocristine was between 0.78 ng/mL and less than 1.56 ng/mL. Ergocristine was nearly quantifiable at 1.56 ng/mL. The results of this analysis for ergocornine and ergocryptine agreed with the first analysis (4.4.1), which indicated that the LOD was 0.78 ng/mL. The LOD for ergosine varied between the first test (4.4.1) and the current section. In contrast with the first test (4.4.1), ergosine was detectable at 0.78 ng/mL in this run whereas it was below detection criteria in the first run. This indicated that the method was not robust at the lower range of the calibration curve. Ergocristine was less ionizable than the other three alkaloids. 4.4.3. Extended standard curve to extend upper range of quantitation. Retention times for ergocornine, ergocristine, ergocryptine, and ergosine were 10.0, 11.5, 11.0, and 8.5 minutes, respectively. The 1.95 ng/mL S:N for ergocornine, ergocristine, ergocryptine, and ergosine were 8.0, 3.1, 8.9, and 9.0, respectively. The LLOQ for ergocristine was 1.56 ng/mL whereas the LLOQs for ergocornine, ergocryptine, and ergosine were 1.95 ng/mL. Ergocristine was

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TABLE 4.5. Linearity information for ergopeptine alkaloids spiked in bovine plasma following a test of six calibration points. Spiked plasma samples were extracted by protein precipitation and quantified by liquid chromatography tandem mass spectrometry (LC- MS/MS). The horizontal line in each ergot alkaloid column indicates the samples excluded (above) and included (below) in linear regression analysis of the data for that alkaloid (as these were below the limit of detection, i.e., LOD). Nominal Ergot alkaloid concentration Ergocornine Ergocristine Ergocryptine Ergosine (ng/mL) Calculated concentration (ng/mL); Accuracy (%)* 0.78 0.818 (N/A) 1.30 (N/A) 0.822 (N/A) 0.717 (N/A) 1.56 1.79 (115) 1.74 (N/A) 1.91 (122) 1.70 (109) 3.13 3.31 (106) 3.45 (110) 2.96 (94.7) 3.21 (103) 6.25 5.46 (87.3) 5.67 (90.7) 5.43 (86.8) 5.56 (88.9) 12.5 12.9 (103) 12.8 (103) 13.4 (107) 13.1 (105) 25 24.9 (99.8) 24.9 (99.8) 24.8 (99.1) 24.8 (99.4)

n 5 4 5 5 Slope 3470 1380 2940 2730 Intercept 961 -15.1 420 799 R 0.9988 0.9990 0.9978 0.9987 R2 0.9976 0.9980 0.9956 0.9974 * Accuracy = (calculated concentration/targeted concentration)  100 Abbreviations: N/A, not applicable; R, correlation coefficient; R2, coefficient of determination

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FIGURE 4.3. Representative chromatograms of four ergot alkaloids spiked in bovine plasma with signal-to-noise (S:N) ratios >3 for intensity (counts per second, cps) versus run time (minutes) as quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Ergocornine, ergocryptine, and ergosine had S:N>3 at 0.78 ng/mL (post-extraction). Ergocristine had S:N>3 at 1.56 ng/mL (post-extraction). Chromatographic separation was achieved with 21- minute run times.

134 detectable at 1.95 ng/mL and quantifiable at 3.9 ng/mL (S:N=8.5). The 250 ng/mL concentration was accurately detected and quantified for each alkaloid (Figure 4.4). All calculated accuracies (except for the lowest calibrant for ergocristine) met acceptance criteria of being within 15% of the nominal concentration. As a minimum of six calibrants were used, the method met the definition of a calibration curve. Calibration curve quantification accuracy and linearity are given in Table 4.6. All calibrations curves contained a minimum of seven points with good linearity (R2≥0.999). 4.4.4. Accuracy and precision for preliminary low, medium, and high quality control (QC) sample analysis. Retention times were 10.0, 11.5, 11.0, and 8.5 minutes for ergocornine, ergocristine, ergocryptine, and ergosine, respectively. The 1.95 ng/mL S:Ns for ergocornine, ergocristine, ergocryptine, and ergosine were 4.6, 3.6, 3.3, and 2.7, respectively. This concentration was included in the calibration curve for ergocornine (as it nearly met criteria for LLOQ), but not the other three alkaloids as they were considered limits of detection for this analysis. The 3.91 ng/mL S:N for ergocornine, ergocristine, ergocryptine, and ergosine were 8.7, 5.7, 7.6, and 6, respectively, and were all considered LLOQ for this analysis. There was a clear loss of sensitivity for all ergot alkaloids in this analysis. Calibration curve information is given in Table 4.7. Linearity was less robust in this analysis compared to section 4.4.3 as R2 ranged from 0.988 to 0.992. Quality control information for each alkaloid is given in Table 4.8. For ergocornine, each of the LQC, MQC, and HQC passed, with only one sample failure occurring in the HQC replicates (accuracy 84.1%). Overall, the QCs for ergocornine were accurate (99-105%) and precise (%CV≤9.4). For the other three alkaloids, however, all the LQCs failed. This is due to the removal of the 1.96 ng/mL or 3.91 ng/mL standard from the calibration curve for these alkaloids, thereby placing the LQC outside of the calibration range. It should be noted, however, that the S:N for the LQCs of these alkaloids were all above 5 and indicated possible LLOQs. The MQC and HQCs passed for ergocristine, ergocryptine, and ergosine, with one sample failure occurring in the HQC for ergocristine (accuracy 81.4%) and ergocryptine (accuracy 82.2%). This was the same sample that failed for ergocornine, thereby indicating systematic error (e.g., pipetting) in this sample. The MQC and HQC for ergocristine, ergocryptine, and ergosine were accurate (101 to 108%) and precise (%CVs 2.6 to 6.2). The QCs chosen, aside f rom the LQCs (the outcome which was unforeseen at the time of concentration selection) were appropriate.

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FIGURE 4.4. Representative liquid chromatography tandem mass spectrometry (LC-MS/MS) chromatogram of four ergopeptine alkaloids spiked in bovine plasma each at post-extraction concentrations of 250 ng/mL. Retention times for each alkaloid were as follows: 8.5 minutes for ergosine, 10 minutes for ergocornine, 11 minutes for ergocryptine, and 11.5 minutes for ergocristine.

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TABLE 4.6. Calibration curve information of ergopeptine alkaloids spiked in bovine plasma over an extended range of concentrations (1.95 to 250 ng/mL). Spiked plasma samples were extracted by protein precipitation and quantified with liquid chromatography tandem mass spectrometry (LC-MS/MS). The horizontal line in the ergocristine column indicates the sample(s) excluded (above) and included (below) in linear regression analysis of the data. Nominal Ergot alkaloid concentration Ergocornine Ergocristine Ergocryptine Ergosine (ng/mL) Calculated concentration (ng/mL); (%Accuracy) 1.95 1.71 (87.7) 1.63 (N/A)* 1.92 (98.3) 1.82 (93.5) 3.91 3.76 (96.1) 3.55 (90.8) 3.50 (89.4) 3.55 (90.8) 7.81 8.0 (102) 7.93 (102) 8.09 (104) 8.34 (107) 15.6 17.4 (112) 17.1 (110) 16.9 (108) 16.9 (108) 31.3 33.5 (107) 31.8 (102) 32.4 (104) 32.4 (104) 62.5 61.3 (98.1) 61.7 (98.8) 62.6 (100) 62.4 (99.9) 125 120 (96.1) 119 (95.5) 119 (95) 120 (96.1) 250 252 (101) 255 (102) 254 (102) 252 (101)

n 8 7 8 8 Slope 4610 1970 3940 3310 Intercept 2550 709 1240 1580 R 0.9993 0.9993 0.9993 0.9995 R2 0.9986 0.9986 0.9986 0.9990 Abbreviations: N/A, not applicable; R, correlation coefficient, R2, coefficient of determination

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TABLE 4.7. Calibration curve information for ergot alkaloids spiked in bovine plasma. Spiked plasma samples were extracted by protein precipitation and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The horizontal line in each ergot alkaloid column indicates the samples excluded (above) and included (below) in the calibration curve for that alkaloid. Nominal Ergot alkaloid concentration Ergocornine Ergocristine Ergocryptine Ergosine (ng/mL)* Concentration (ng/mL; %accuracy) 1.95 / 3.91 2.06 (106) 4.7 (N/A) 2.46 (N/A) 2.2 (N/A) 3.91 / 7.81 3.83 (97.9) 7.58 (97) 3.82 (97.7) 4.06 (104) 7.81 / 15.6 7.72 (98.8) 16.1 (103) 7.84 (100) 7.7 (98.6) 15.6 / 31.3 16.5 (106) 33.5 (103) 17.5 (112) 16.4 (105) 31.3 / 62.5 29.3 (93.7) 58.8 (94.1) 29.3 (93.7) 30 (95.7) 62.5 / 125 66.6 (107) 134 (107) 66.1 (106) 65.8 (105) 125 / 250 108 (86.3) 214 (85.6) 105 (84.2) 107 (85.5) 250 / 500 264 (106) 528 (106) 266 (107) 265 (106)

n 8 7 7 7 Slope 2080 960 1980 1650 Intercept 589 -112 -588 -178 R 0.9959 0.9951 0.9941 0.9952 R2 0.9918 0.9902 0.9882 0.9904 * the targeted concentration for ergocristine was higher than the other three alkaloids Abbreviations: N/A, not applicable; R, correlation coefficient; R2, coefficient of determination

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TABLE 4.8. Preliminary quality control precision and accuracy information for ergopeptine alkaloids spiked in bovine plasma. Spiked plasma samples were extracted by protein precipitation and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Calculated Quality control (nominal Precision Ergot alkaloid n* concentration (mean Accuracy (%) concentration; ng/mL) (%CV) ± SD; ng/mL)

Ergocornine LQC (3.5) 6 3.61 ± 0.34 9.4 103 MQC (112) 6 118 ± 3.4 2.9 105 HQC (186) 5 184 ± 6.11 3.3 99.0

Ergocristine LQC (7) - - - - MQC (224) 6 242 ± 7.3 3.0 108 HQC (372) 5 380 ± 9.2 2.4 102

Ergocryptine LQC (3.5) - - - - MQC (112) 6 120 ± 5.2 4.4 107 HQC (186) 5 188 ± 8.8 4.7 101

Ergosine LQC (3.5) - - - - MQC (112) 6 121 ± 3.1 2.6 108 HQC (186) 6 187.2 ± 11.7 6.2 101

* quality control samples must have minimum 5/6 passing replicates (i.e., accuracies within ±15%) in order to pass overall. Quality control samples with more than one failure do not pass Abbreviations: CV, coefficient of variation; HQC, high quality control; LQC, low quality control; MQC, medium quality control; SD, standard deviation

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4.4.5. Full QC test including first LLOQ set and analysis of experimental cow samples (replicate 1). Calibration curve information and quality control information for this analysis is given in Table 4.9 and Table 4.10, respectively. Linearity was achieved between 1.95 and 125 ng/mL (ergocornine, ergocryptine, and ergosine) and between 3.91 and 250 ng/mL (ergocristine). The S:N for the chosen LLOQ concentrations for each alkaloid varied from being below detection to being above detection. The majority of samples, however, were below the required S:N>5. Ergosine at 0.98 ng/mL had S:N for the six replicates that ranged from 2.9 to 4.7. Ergocornine at 0.98 ng/mL S:N ranged from 3.8 to 5.6. Ergocryptine at 0.98 ng/mL S:N ranged from 2.6 to 3.4. Lastly, ergocristine at 1.95 ng/mL S:N ranged from 2.6 to 4.6. Although these samples did not have sufficient signal to meet LLOQ criteria, it is interesting to note that ergocristine and ergocryptine met criteria for accuracy and precision. For ergocornine, 5/6 samples failed low (i.e., <15%). For ergosine, 4/6 samples failed low compared to the targeted concentration of 0.977 ng/mL. For ergocristine, 1/6 samples failed high with an accuracy of 122% (sample concentration was 2.37 ng/mL). Similarly, 1/6 ergocryptine LLOQ samples failed high (143% accuracy) while the passing samples were accurate and precise. All LQC and MQC samples failed low for ergocornine. These samples were precise (%CV for LQC and MQC were 13 and 2.1%, respectively) but not accurate (%accuracy 74% and 84% for LQC and MQC, respectively). The LQC (4/6 samples) failed low for ergosine (83% accuracy). As with ergocornine, these samples were precise (12% CV). In contrast, the LQC samples (4/6) failed high for ergocryptine (average accuracy of 116%). As with ergocornine and ergosine, the LQC samples were precise (11% CV). The HQC samples were precisely and accurately quantified for all four alkaloids. For experimental samples, no detectable peaks were present for any of the ergot alkaloids in any of the timepoints analyzed. A representative chromatogram for each alkaloid is given in Figure 4.5. 4.4.6. Partial method validation 4.4.6.1. General. Chromatographic peaks for each ergot alkaloid were observed on all three validation days. A representative chromatogram of the four alkaloids on each validation day are presented in Figure 4.6. There is a clear loss of sensitivity on day three of the partial validation. In comparison to the other two days, a much lower intensity was detected and

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TABLE 4.9. Calibration curve information for ergopeptine alkaloids spiked in bovine plasma. Spiked plasma samples were extracted by protein precipitation and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Ergocristine was twice as concentrated as the other alkaloids. The horizontal line in each ergot alkaloid column indicates the samples excluded (above) and included (below) in the calibration curve for that alkaloid. Nominal Ergot alkaloid concentration Ergocornine Ergocristine Ergocryptine Ergosine (ng/mL)* Calculated concentration (ng/mL; %accuracy) 0.975 / 1.95 0.561 (N/A) 2.0 (N/A) 1.07 (N/A) 0.522 (N/A) 1.95 / 3.91 1.90 (97.2) 4.0 (102) 2.06 (106) 2.17 (112) 3.91 / 7.81 3.92 (100) 7.93 (102) 3.78 (96.6) 3.70 (94.6) 7.81 / 15.6 8.37 (107) 16.6 (106) 8.12 (104) 8.15 (104) 15.6 / 31.3 15.1 (96.6) 28.9 (92.2) 14.8 (95.2) 14.5 (92.9) 31.3 / 62.5 30.1 (96.3) 60.6 (96.9) 31.4 (100) 29.9 (95.6) 62.5 / 125 64.5 (103) 123 (98.4) 59.7 (95.6) 61.6 (98.5) 125 / 250 124 (99.3) 255 (102) 128 (102) 128 (102)

n 7 7 7 7 Slope 4290 1660 3510 3110 Intercept 2480 248 183 1550 R 0.9996 0.9994 0.9994 0.9993 R2 0.9992 0.9988 0.9988 0.9986 Abbreviations: N/A, not applicable; R, correlation coefficient; R2, coefficient of determination

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TABLE 4.10. Preliminary quality control precision and accuracy for ergot alkaloids spiked in bovine plasma. Spiked plasma samples were extracted by protein precipitation and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Dashes (-) indicate sample failure. Quality control Calculated (nominal Precision Accuracy Ergot alkaloid n* concentration (mean concentration; (%CV) (%) ± SD; ng/mL) ng/mL)

Ergocornine LLOQ (0.977) - - - - LQC (1.75) - - - - MQC (66) - - - - HQC (93) 6 92.4 ± 5.10 5.5 99.4

Ergocristine LLOQ (1.95) 5 2.10 ± 0.12 5.5 108 LQC (3.5) 5 3.70 ± 0.41 11.0 106 MQC (112) 6 116 ± 2.0 1.7 104 HQC (186) 6 194 ± 6.1 3.1 104

Ergocryptine LLOQ (0.977) 5 1.05 ± 0.09 8.9 108 LQC (1.75) - - - - MQC (66) 6 60.1 ± 0.7 1.2 91.1 HQC (93) 5 97.1 ± 3.1 3.2 104

Ergosine LLOQ (0.977) - - - - LQC (1.75) - - - - MQC (66) 5 57.3 ± 0.9 1.6 86.9 HQC (93) 6 96.5 ± 1.1 1.1 103

* quality control samples must have minimum 5/6 passing replicates (i.e., accuracies within ±15%) in order to pass overall. Quality control samples with more than one failure do not pass. Abbreviations: HQC, high quality control; LLOQ, lowest limit of quantitation

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143

144 background noise was apparent. The maximum intensity for days 1 and 2 is approximately 6000 and 6200 cps, respectively, while it is 1250 cps for day 3. Retention times for ergocornine, ergocristine, ergocryptine, and ergosine were approximately 7.8, 9.1, 8.7, and 6.3 minutes, respectively, for all three days of the partial validation. 4.4.6.2. Linearity. Calibration curve information for each alkaloid on each day of the validation is given in Table 4.11. All standards for all alkaloids passed on days 1 and 2 of the validation. On day 3, however, some calibrants failed (i.e., accuracies >15% of nominal concentration) for ergocornine, ergocristine, and ergocryptine. None of the calibrants failed for ergosine. The R2 values throughout the partial validation for ergocornine, ergocristine, ergocryptine, and ergosine were ≥0.997, 0.990, 0.995, and 0.998, respectively. 4.4.6.3. Slope. Slopes for calibration curves for each alkaloid were compared using the linear regression function in GraphPad Prism. The slopes of the linear regression lines from day 1 and day 2 were not different for ergocornine and ergocristine (p≥0.716). In contrast, the day 1 slope for ergocryptine was greater than day 2 (p=0.029) but was lower for ergosine than day 2 (p=0.0005). Slopes of the regression lines for day 1 and day 3 were not different for ergosine (p=0.41), but differed between the days for ergocornine, ergocristine, and ergocryptine (p≤0.0002). Day 2 and day 3 slope were different for all four alkaloids (p≤0.0005).

4.4.6.4. Y-intercepts. For ergocornine, the Y-intercepts of the day 1 and day 2 calibration curves were not statistically different from one another (p=0.25). As the slopes of days 1 and 2 differed from that of day 3, the y-intercepts of these linear regression lines could not be compared. The y-intercepts for ergocristine on days 1 and 2 were not different (p=0.875). For ergocryptine, Intercepts could not be compared all the slopes were different from one another. For ergosine, the y-intercepts of day 1 and 3 did not differ (p≥0.999). 4.4.6.5. Precision and accuracy. Intraday accuracy precision data for each alkaloid on each day of the partial validation is listed in Table 4.12. Passing criteria was met on each of the three days for ergocornine and ergosine for each QC. Passing criteria for all QCs was met for ergocristine and ergocryptine on days 1 and 2 of the validation, however sample failure was observed on day 3. Specifically, the LLOQ and LQC failed for ergocristine on day 3 whereas the LLOQ failed for ergocryptine. On day 1 of the validation, precision and accuracy for each ergot alkaloid was ≤8.8% and ≤±8%, respectively. On day 2, precision and accuracy for all analytes were ≤9.3% and ≤±11%, respectively. On day 3 (except for the QC failures), precision

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TABLE 4.11. Calibration curve information for ergot alkaloids on each of a partial three-day validation. Ergopeptine alkaloids calibration curves were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) over three consecutive days. Ergot alkaloid Concentration (ng/mL) Day 1 Day 2 Day 3 Ergocornine 0.977 0.847 (86.8) 0.887 (90.9) 0.936 (95.8) 1.95 1.83 (93.9) 1.96 (100) 1.65 (84.7)* 3.91 4.03 (103) 4.02 (103) 3.73 (95.4) 7.81 8.41 (108) 8.21 (105) 8.82 (113) 15.6 17.2 (110) 16.4 (105) 16.7 (107) 31.3 32.4 (104) 30.4 (97.3) 34.1 (109) 62.5 59.1 (94.6) 60.8 (97.3) 60.6 (96.9) 125 125 (100) 126 (101) 122 (98)

Slope 2700 2650 2360 Intercept 335 -84 540 R 0.9989 0.9996 0.9986 R2 0.9978 0.9992 0.9972

Ergocristine 1.95 1.77 (90.7) 2.06 (106) 1.44 (74)* 3.91 3.65 (93.5) 3.62 (92.5) 3.3 (84.4)* 7.81 7.91 (101) 7.56 (96.7) 7.66 (98.1) 15.6 17 (109) 16.8 (108) 19.2 (123)* 31.3 34.1 (109) 32.3 (103) 36.3 (116)* 62.5 63.5 (102) 59.8 (95.6) 72 (115) 125 117 (93.7) 120 (95.9) 115 (91.7) 250 253 (101) 256 (102) 243 (97.4)

Slope 1070 1050 921 Intercept 176 12.8 854 R 0.9989 0.9993 0.9950 R2 0.9978 0.9986 0.9900

Ergocryptine 0.977 0.973 (99.6) 1.01 (103) 0.709 (72.5)* 1.95 1.71 (87.9) 1.76 (90.1) 1.92 (98.3) 3.91 4 (102) 3.93 (101) 3.8 (97.1) 7.81 8.57 (110) 8.00 (102) 9.07 (116)*

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15.6 16.3 (105) 16.7 (107) 17.6 (113) 31.3 31.2 (99.8) 30.6 (97.7) 34.8 (111) 62.5 58.8 (94.1) 62.1 (99.4) 59 (94.4) 125 127 (102) 125 (100) 122 (97.7)

Slope 2140 2070 1870 Intercept 82.3 -162 615 R 0.9990 0.9997 0.9973 R2 0.9980 0.9994 0.9946

Ergosine 0.977 0.953 (97.6) 0.985 (101) 1.02 (104) 1.95 1.83 (93.7) 1.90 (97.3) 1.77 (90.7) 3.91 3.75 (96) 3.89 (99.4) 3.67 (93.8) 7.81 8.39 (107) 8.19 (105) 8.38 (107) 15.6 17 (109) 15.9 (102) 15.9 (102) 31.3 31.4 (100) 30.4 (97.3) 31.9 (102) 62.5 59.3 (94.8) 60.6 (96.9) 63.8 (102) 125 126 (101) 127 (102) 123 (98.1)

Slope 3590 3720 3590 Intercept 407 -444 449 R 0.9991 0.9997 0.9996 R2 0.9982 0.9994 0.9992 * Indicates sample failure (accuracy >±15% for standards 7 through 1; >±20% for standard 8) Abbreviations: R, correlation coefficient; R2, coefficient of determination

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TABLE 4.12. Intraday accuracy and precision of four ergopeptine alkaloids spiked in bovine plasma by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Samples were quantified daily over three days consecutively. Dashes (-) indicate QC failure. Day 1 Day 2 Day 3 ------Ergot Quality Observed n Precision Accuracy Observed n Precision Accuracy Observed n Precision Accuracy Alkaloid Control concentration (%CV) (%) concentration (%CV) (%) concentration (%CV) (%) (mean±SD; (mean±SD; (mean±SD; ng/mL) ng/mL) ng/mL) Ergocornine LLOQ 0.91 ± 0.03 5 2.8 92.8 0.92 ± 0.07 6 8.2 94.2 0.88 ± 0.08 5 9.2 89.9 LQC 2.40 ± 0.14 5 5.8 95.9 2.43 ± 0.23 6 9.3 97.3 2.32 ± 0.14 6 6.2 92.8 MQC 61.8 ± 3.3 5 5.3 103 56.2 ± 1.0 5 1.8 93.6 61.5 ± 1.0 6 1.7 102 HQC 95.4 ± 1.7 6 1.8 95.4 90.7 ± 0.9 6 0.9 90.7 101 ± 1.7 6 1.7 101

Ergocristine LLOQ 1.83 ± 0.14 6 7.4 94.0 1.82 ± 0.15 6 8.0 93.3 - - - -

1 LQC 4.80 ± 0.24 6 5.1 95.8 4.67 ± 0.21 6 4.6 93.5 - - - - 4

8 MQC 122 ± 5.63 5 4.6 101 114 ± 2.2 5 1.9 95.3 126 ± 2 6 1.6 105 HQC 188 ± 3.21 6 1.7 93.9 185 ± 2.5 6 1.3 92.4 210 ± 2.9 6 1.4 105

Ergocryptine LLOQ 1.02 ± 0.09 6 8.8 105 0.99 ± 0.08 6 8.4 102 - - - - LQC 2.44 ± 0.15 6 5.9 97.9 2.48 ± 0.14 6 5.6 99.1 2.39 ± 0.14 6 5.9 95.6 MQC 63.4 ± 4.0 6 6.3 106 56.8 ± 1.1 5 1.9 94.7 61.9 ± 0.9 6 1.4 103 HQC 95.9 ± 2.2 6 2.2 95.9 93.0 ± 1.4 6 1.5 93.0 103.5 ± 1.5 6 1.5 104

Ergosine LLOQ 0.95 ± 0.08 5 8.1 97.5 1.08 ± 0.06 5 5.6 111 0.96 ± 0.02 6 2.4 99.3 LQC 2.55 ± 0.14 6 5.5 104 2.55 ± 0.12 6 4.8 102 2.46 ± 0.06 6 2.5 98.5 MQC 64.6 ± 2.9 5 4.4 108 56.8 ± 0.64 5 1.1 94.7 59.8 ± 1.3 6 2.1 99.7 HQC 102 ± 1.9 6 1.8 102 92.4 ± 0.8 6 0.9 92.4 98.5 ± 1.7 6 1.7 98.5

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and accuracy for each analyte was ≤9.2% and ≤10%, respectively. Data for inter-day accuracy and precision of each alkaloid is listed in Table 4.13. Overall, inter-day precision of the QCs that could be analyzed for each alkaloid was acceptable due to coefficients of variation less than 9%. All inter-day accuracies of the QCs were less than ±9%. 4.4.6.6. Autosampler stability. Concentration data at 0 hr and 42 hr injections are listed in Table 4.14. The coefficients of variation for each average for each QC were all ≤7.4%, indicating that autosampler conditions did not affect quantitation. 4.4.7. Experimental samples (replicate 2 - 2017 pharmacokinetics study). Calibration curve information is given in Table 4.15. There was good linearity for all alkaloids between 0.977 and 125 ng/mL (ergocornine, ergocryptine, and ergosine) and between 1.95 and 250 ng/mL (ergocristine). The LLOQs met S:N and accuracy acceptance criteria for all the alkaloids, however there was some variability in the other QCs. For ergocornine, the LQCs ran before the experimental samples passed whereas the ones ran after the experimental samples failed high (accuracies of 132, 123%). Three quarters of the MQCs failed high (accuracies between 121 138%). All the HQCs failed high (accuracies between 118 and 138%). For ergocryptine and ergosine, three quarters of the LQCs and MQCs failed high (accuracies between 117 and 164%). All the HQCs failed high (accuracies between 116 and 144%). For ergocristine, three quarters of the LQCs and MQCs failed high (accuracies between 116 and 129%). Both HQCs ran before the cow samples passed, whereas the two HQCs ran after the cow samples failed high (accuracies of 133%). For experimental samples (replicate 2), no detectable peaks were observed for any of the timepoints analyzed for all four cows. A representative chromatogram for each alkaloid is given in Figure 4.7. 4.5. DISCUSSION The work described in this paper culminated in a partially validated method to detect four ergopeptine alkaloids spiked in bovine plasma. Discrete peaks for each ergot alkaloid were consistently produced in calibration curves and quality control samples with this method, however chromatographic peaks were not observed in any samples from oral pharmacokinetics studies. This method lacked robustness and sensitivity at the low range of the calibration curve. The limits of detection for ergocornine, ergocryptine, and ergosine were approximately 0.98 ng/mL before installation of the new HPLC. The LOD for ergocristine was 1.95 ng/mL. Analytical capacity improved after installation of the new HPLC as retention times were

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TABLE 4.13. Inter-day precision and accuracy following a three-day partial validation to quantify ergot alkaloids in bovine plasma. Plasma samples with ergopeptine alkaloids were quantified consecutively over a three-day period with liquid chromatography tandem mass spectrometry (LC-MS/MS). Quality Observed Precision Accuracy Ergot alkaloid n control concentration (ng/mL) (%CV) (%) Ergocornine LLOQ 0.90 ± 0.06 16 7.1 92.4 LQC 2.38 ± 0.17 17 7.2 95.3 MQC 59.9 ± 3.2 16 5.4 100 HQC 95.7 ± 4.6 18 4.8 95.7

Ergocristine LLOQ 1.83 ± 0.1 12 7.3 93.7 LQC 4.76 ± 0.2 15 4.6 95.1 MQC 121 ± 6.0 16 4.9 101 HQC 194 ± 12.0 18 6.2 97.1

Ergocryptine LLOQ 1.01 ± 0.08 12 8.3 103 LQC 2.44 ± 0.14 18 5.7 97.5 MQC 60.9 ± 3.7 17 6.0 102 HQC 97.5 ± 4.8 18 5.0 97.5

Ergosine LLOQ 1.00 ± 0.08 16 7.8 102 LQC 2.52 ± 0.11 18 4.5 101 MQC 60.3 ± 3.6 16 6.0 101 HQC 97.6 ± 4.3 18 4.4 97.6 Abbreviations: CV, coefficient of variation; HQC, high quality control; LLOQ, lowest limit of quantitation; LQC, low quality control; MQC, medium quality control

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TABLE 4.14. Autosampler stability of ergot alkaloid quality control samples at zero and 42-hour injections. Following the zero-hour injection, samples were left overnight at 4 °C under autosampler conditions and reinjected at 42 hours. Ergot Alkaloid Quality Control Observed Concentration (ng/mL) %CV mean ± SD (n) 0 hr injection 42 hr injection* Ergocornine LLOQ 0.84 ± 0.04 (5) 0.92 ± 0.08 (6) 6.4 LQC 2.35 ± 0.04 (5) 2.37 ± 0.17 (5) 0.6 MQC 61.8 ± 3.3 (5) 58.5 ± 4.3 (6) 3.9 HQC 95.5 ± 1.7 (6) 90.9 ± 2.6 (6) 3.5

Ergocristine LLOQ 1.73 ± 0.09 (5) 1.88 ± 0.13 (6) 5.9 LQC 4.70 ± 0.24 (6) 4.71 ± 0.29 (6) 0.2 MQC 121.8 ± 5.6 (5) 118.8 ± 10.0 (6) 1.8 HQC 187.8 ± 2.9 (6) 188.7 ± 4.8 (6) 0.3

Ergocryptine LLOQ 0.99 ± 0.11 (6) 1.07 ± 0.04 (5) 5.5 LQC 2.42 ± 0.14 (6) 2.57 ± 0.10 (6) 4.3 MQC 63.4 ± 4.0 (6) 60.4 ± 5.1 (6) 3.4 HQC 95.9 ± 2.2 (6) 94.5 ± 3.0 (6) 1.0

Ergosine LLOQ 0.95 ± 0.1 (6) 1.03 ± 0.03 (6) 5.7 LQC 2.6 ± 0.1 (6) 2.44 ± 0.6 (6) 4.5 MQC 64.5 ± 2.9 (5) 59.0 ± 3.4 (6) 6.3 HQC 101.8 ± 1.9 (6) 91.7 ± 2.8 (6) 7.4 *analyzed with different calibration curve Abbreviations: CV, coefficient of variation; HQC, high quality control; LLOQ, lowest limit of quantitation; LQC, low quality control; MQC, medium quality control; SD, standard deviation

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TABLE 4.15. Calibration curve information for ergot alkaloids spiked in bovine plasma. Samples were extracted by protein precipitation and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Nominal Ergot alkaloid concentration Ergocornine Ergocristine Ergocryptine Ergosine (ng/mL)* Concentration (ng/mL; %accuracy) 0.977 / 1.95 1.02 (104) 1.92 (98.3) 0.894 (91.5) 1.15 (118) 1.95 / 3.91 1.88 (96.4) 3.51 (89.6) 2.11 (108) 1.76 (90.5) 3.91 / 7.81 3.78 (96.8) 7.99 (102) 4.00 (102) 3.96 (101) 7.81 / 15.6 8.16 (104) 16.7 (107) 8.12 (104) 7.48 (95.8) 15.6 / 31.3 15.1 (97) 31.7 (101) 15.0 (96.2) 15.4 (99.0) 31.3 / 62.5 31.8 (102) 65.4 (105) 31.0 (99.2) 29.4 (93.9) 62.5 / 125 61.9 (99.1) 122 (97.4) 60.8 (97.2) 62.5 (100) 125 / 250 125 (100) 249 (99.7) 127 (102) 127 (102)

n 8 8 8 8 Slope 2050 739 1470 2270 Intercept 528 229 70.7 -122 R 0.9999 0.9996 0.9997 0.9995 R2 0.9998 0.9992 0.9994 0.9990 Abbreviations: R, correlation coefficient; R2, coefficient of determination

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Intensity (cps)Intensity

Time (min) FIGURE 4.7. Representative chromatograms for experimental cow plasma samples analyzed for ergot alkaloids by liquid chromatography tandem mass spectrometry (LC-MS/MS) after a one- time high concentration exposure in feed. No peaks were observed for any of the cow samples analyzed.

153 shortened and former LODs became LLOQs for each of the alkaloids. Linearity was achieved for ergocornine, ergocryptine, and ergosine between 0.98 and 125 ng/mL. Ergocristine, however, was apparently less well ionized than the other compounds. This was addressed by increasing the concentration of ergocristine two-fold in the working solutions. As a result, ergocristine was linear and accurately quantified between 1.95 and 250 ng/mL. Ergocristine has slightly different moieties attached to its ergoline ring than the other three ergopeptines under study (see Komarova and Tolkachev 2001, Arroyo-Manzanares et al. 2017). Structural differences and different stereochemistry may partially describe its behaviour in this method. Epimerization of the ergot alkaloids to their -inine epimers may have played a role as well. Interconversion between the R (-ine) to S (-inine) forms is rapid and can occur during storage (Buchta and Cvak 1999; Komarova and Tolkachev 2001). Hafner et al. (2008) described a greater tendency of ergopeptine alkaloids to epimerize in acetonitrile and acetonitrile/buffer solutions, however the behaviour of ergocristine was like the other ergopeptine alkaloids. In contrast, Merkel et al. (2012) indicated that ergotoxine type ergopeptines, i.e., ergocornine, ergocryptine, and ergocristine, tend to shift towards the S-form after digestion. To the authors’ knowledge, the present study is the first report to describe differences in detection abilities between ergocristine and other ergot alkaloids. It is interesting to note that, in Western Canada, ergocristine the most abundant alkaloid detected in feed samples (Grusie et al. 2018a). Therefore, ergocristine would be expected to be at the highest concentration in plasma compared to the other ergot alkaloids. These observations and the degree of epimerization during sample preparation warrant further investigation. In terms of practical applications tested, this method was unable to detect ergot alkaloids in plasma of exposed cattle. This observation confirms the low oral bioavailability of ergopeptine alkaloids cattle. Studies of oral pharmacokinetics of ergot alkaloids in livestock are minimal at the present time. Results from the present study indicate that feeding a one-time concentration of 27 000 to 29 000 µg total ergot alkaloids per kg feed to cows did not result in plasma concentrations greater than 0.98 ng/mL. It is important to note that this concentration fed to the cattle in this present study represents a very high degree of ergot alkaloid contamination and is unlikely to be encountered commonly in a farm scenario. As such, it is an important practical conclusion that ergot alkaloids will not be detected in plasma if blood is collected from exposed or purportedly exposed animals. Although depressed plasma prolactin is a widely

154 accepted biomarker for ergot alkaloid exposure (Klotz 2015), feeding trial results in cows from our laboratory do not support this notion (Cowan et al. 2018; Grusie et al. 2018b; Cowan et al. 2019). Therefore, analytical detection of ergot alkaloids in feed remains the most reliable method for diagnosing ergot exposure in cattle. Low oral bioavailability in cattle is consistent with pharmacokinetics studies in humans (Eckert et al. 1978; Ala-Hurula et al. 1979a,b; Perrin 1985; Ekbom, Paalzow, and Waldenlind 1981; Ibraheem, Paalzow, and Tfelt-Hansen 1983; Sanders et al. 1986; de Groot et al. 1994). These studies estimate that oral bioavailability of ergotamine is < 2%. Pharmacokinetic information on ergotamine is widely available due to its long history of use as an anti-migraine treatment (Tfelt-Hansen et al. 2000). Low oral bioavailability of the ergot alkaloids could be related to high first pass metabolism or poor gastrointestinal absorption (Ibraheem, Paalzow, Tfelt-Hansen 1983; EFSA 2012). A study found that plasma clearance of ergotamine administered intravenously to human migraine patients was similar to hepatic blood flow, thereby concluding that low bioavailability of ergotamine was related to high first pass metabolism (Ibraheem, Paalzow, and Tfelt-Hansen 1982). First pass metabolism is reportedly up to 90% for ergotamine (Silberstein and McCrory 2003). Additionally, intestinal absorption of ergot alkaloids is variable in humans, with some studies reporting low intestinal absorption (Franz, Vonderscher and Voges 1980) and others reporting high (60 to 70%) oral absorption (Tfelt-Hansen et al. 2000). Certainly, the absorption characteristics between monogastrics and ruminants is expected to be different. Gastrointestinal absorption in ruminants is reported to be high (Piper and Moubarak 1992; Westendorf et al. 1993; Stuedemann et al. 1998; Hill et al. 2001). One study postulated that, if a cow were fed 5.9 mg ergotamine per day (i.e., feed contaminated with 473 µg ergotamine/kg), the concentration in blood would be no higher than 0.215 g/mL (215 ng/mL) if bioavailability was 100% (Craig, Klotz, and Duringer 2015). Based on a maximum 2% oral bioavailability, the highest concentration of ergotamine in blood that could be expected would be 2.2 ng/mL. This theoretical concentration is at the lowest range of the current calibration range described in the present study. With this information in mind, and due to continued sensitivity issues experienced during these development procedures, more sensitive instrumentation and techniques that increase response (i.e., larger sample volume, improved sample clean up procedures) should be investigated.

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Other method development studies with subsequent generation of pharmacokinetic data have been conducted with plasma from ergot alkaloid exposed livestock. These studies administered ergot alkaloids intravenously. One study developed an HPLC method to detect ergotamine, ergosine, and ergine in bovine plasma following intravenous administration to Holstein steers (Moubarak et al. 1996). Ergotamine, ergosine, and ergine (1.8 to 14 µg/kg BW) were detected at 0.5 to 1.0 ng/mL concentrations. Another study quantified ergovaline in ovine plasma with HPLC and fluorometric detection following intravenous administration of 17 µg/kg BW (Jaussaud et al. 1998). The LOQ and LOD for this method were 3.5 and 15 ng/mL, respectively. Pharmacokinetic parameters generated from these data were clearance and elimination half-life and were 1.2 L/hr-kg and 23.6 minutes, respectively. Another study developed an HPLC method to detect ergovaline in plasma and milk following intravenous administration (32 µg/kg BW) to lactating goats. (Durix et al. 1999). In milk, the authors determined the LOD and LLOQ to be 0.2 ng/mL and 0.7 ng/mL, respectively, with mean recovery of ergovaline being 99.8%. For plasma, the authors found the LOQ to be 3.5 ng/mL. Pharmacokinetic modelling of the data demonstrated that ergovaline followed a two- compartment model. The clearance and elimination half-life for the goats in this study were 2.0 L/hr-kg and 32.41 minutes, respectively. Overall, the results from these intravenous ergot alkaloid pharmacokinetics studies in livestock indicate that ergot alkaloids follow a biphasic or triphasic pattern of elimination and have a relatively short half-life. In addition, ergot alkaloids administered intravenously to livestock can be detected at ng/mL concentrations in plasma with HPLC. The work described above appeared to have better analytical sensitivity than our method, as indicated by lower limits of detection and quantitation. In our tests with HPLC and fluorometric detection using spiked concentrations in plasma (250 to 1.95 ng/mL), we did not observe any peaks for any of the concentrations (data not shown). More recently, Rudolph et al. (2018) used UHPLC-MS to detect ergotamine, ergovaline, ergocristine, and ergocryptine in horse serum. This method employed solid phase extraction for mycotoxin extraction from spiked serum samples (1.5 mL). This method achieved limits of detection and limits of quantification from 0.05 to 0.5 ng/mL and 0.1 to 1.0 ng/mL, respectively. Although intravenous administration is ideal for pharmacokinetics studies and would be expected to result in quantifiable plasma concentrations, oral mycotoxin administration is the only relevant exposure route for livestock.

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Detection of ergopeptine alkaloids in plasma of livestock continues to be a challenge for researchers, as analytical sensitivity to overcome low oral bioavailability and high first pass metabolism is not yet available. As such, ergot alkaloids will not be detected in plasma of poisoned or exposed animals. Linearity, accuracy, and precision were achieved in spiked plasma samples; however, quantitation at the low range of the curve – concentrations most relevant to livestock exposures – could be unreliable. Further work is required to fully refine and validate the method described herein. Possible applications for this method could include in vitro pharmacokinetic studies (e.g., plasma binding and metabolic stability assays, Caco-2 permeation assays). Instrumentation with analytical sensitivity at the low ng/mL (parts per billion) level is necessary for pharmacokinetic studies with ergot alkaloids. 4.6. FUTURE DIRECTIONS Due to the analytical difficulties faced with in vivo ergot alkaloid pharmacokinetic studies, in vitro avenues to generate pharmacokinetic information using the method described herein remain an active area of interest. Plasma protein binding studies (i.e., ultrafiltration) would enable determination of the extent of plasma protein binding of the ergopeptine alkaloids. Data for the bound and unbound fractions in plasma of ergot alkaloids is currently not available in the literature. In addition, metabolic stability assays (e.g., the substrate depletion approach) using bovine hepatic microsomes would provide information not currently available in the literature. These assays would enable estimation of the intrinsic clearance of the ergot alkaloids. The biotransformation of the ergot alkaloids appears to be principally by hepatic metabolism followed by biliary excretion (Eckert et al. 1978; Tfelt-Hansen and Johnson 1993; Perrin 1985); however, ergot alkaloids have been detected in both the urine and feces of exposed livestock (Stuedemann et al. 1998). Biotransformation reactions appear to include cytochrome P450 enzyme superfamily (Ball et al. 1992; Peyronneau et al. 1994; Moubarak and Rosenkrans 2000; Settivari et al. 2006; Settivari et al. 2008). Other directions that are planned for further refinement of this method include determination of alkaloid recovery, assessment of matrix effects, and effects of repeated freeze/thaw cycles on quantification (similar to the work of Rudolph et al. 2018). The study of matrix effects was partially conducted in tandem with the partial validation. The QCs were prepared in both plasma and UHPLC grade water. The QCs prepared in water represented the pre-extraction spiking of analytes in water analysis, whereas the QCs prepared in plasma were

157 pre-extraction spiked samples for the matrix. Equations given in Appendix G indicate the other factors that we need in order to calculate ergot alkaloid extraction efficiency, extraction recovery, and matrix factor. The LC-MS output for pre-extraction spiked plasma and water QCs is given in Appendix G. Some evidence suggests that the metabolites of parent ergopeptine alkaloid compounds are bioactive as well, which may explain the lack of correlation between plasma concentrations of the parent compounds and pharmacologic effect (Perrin 1985). Epimers of ergopeptine alkaloids have not been widely studied. Ergopeptine alkaloids have a double bond at C9 and C10 that allows these compounds to undergo epimerization into -inine compounds (i.e., ergotamine converts to ergotaminine). Epimerization occurs in nature, during prolonged sample storage, and during sample extraction (Krska et al. 2008; Arroyo-Manzanares et al, 2017). This conversion occurs especially in aqueous acidic or alkaline conditions (Komarova and Tolkachev 2001) and is also favoured by exposure to direct light and solvents with extreme pH (Arroyo-

Manzanares et al. 2017). The physicochemical properties (i.e., pKa) of the -inine epimers vary slightly from their -ine counterparts (Maulding and Zoglio 1970), which would be expected to influence their pharmacokinetic behaviour. The main -inine epimers of ergotamine, ergosine, ergocristine, ergocornine, and ergocryptine have been detected in feedstuffs by LC-MS/MS (Krska et al. 2008; Kokkonen and Jestoi 2010; Malysheva et al. 2013). These compounds have longer retention times than the parent ergopeptine alkaloids. The generally accepted notion is that the ergopeptine forms are more biologically active than the -inine epimers (Berde and Stürmer 1978; Pierri et al. 1982). Some studies suggest minimal biological activity (Merkel et al. 2012). Currently, the -inine epimers are considered analytically important by the European Food Safety Authority due to interconversion (EFSA 2012). It is unclear whether the -inine epimers contribute to toxicity in livestock, thus warrants further investigation. Clearly, there are numerous avenues that can be explored to generate valuable pharmacokinetic information on ergot alkaloids for livestock exposures. The addition of any pharmacokinetic information, whether it be for single ergot alkaloids, mixtures, or exploration of bioactive metabolites, would be important for advancing the understanding of ergot alkaloid disposition in cattle.

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5. CHAPTER 5 – Plasma prolactin, scrotal circumference, and semen quality following long-term exposure to ergot alkaloids (Claviceps purpurea) in adult Angus bulls PREFACE The material described within this chapter was submitted for publication in Toxins. This chapter has been reformatted from the original published version for inclusion in the thesis. Cowan, V., Chohan, M., Blakley, B., McKinnon, J., Anzar, M., Singh, J. Plasma prolactin, scrotal circumference, and semen quality following long-term exposure to ergot alkaloids (Claviceps purpurea) in adult Angus bulls. Submitted June 11, 2020. VC was responsible for conducting the research, data collection and analysis, and manuscript preparation. MC was involved in animal feeding and experimental procedures. BB was involved in ergot alkaloid concentration selection, experimental design, and consultation. JM contributed to ration formulation, nutritional considerations, and feeding considerations. MA contributed to experimental design and provided the means for all semen assays. JS was the principle investigator of the grant and contributed toward concept development, hypotheses formulation, experimental design, oversight of the study, statistical analyses and manuscript preparation/revisions.

As the other studies described in this dissertation were conducted in cows, it was logical to then investigate the effects of ergot alkaloids on bulls. In particular, the effects on bull reproduction and breeding potential were of interest, as reproductive effects of ergot alkaloids in females are much more widely studied than in males.

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5.1. ABSTRACT Canadian standards allow ≤3000 µg ergot alkaloids/kg cattle feed. Moderate to high concentrations of ergot alkaloids in feed have been associated with negative fertility outcomes in bulls, however this area of study is much less understood compared to reproductive effects of ergot alkaloids on cows. A concentration-response relationship was hypothesized between ergot in feed and reductions in plasma prolactin, sperm motility, sperm function, and increase in sperm abnormalities. The study consisted of pre-treatment (12 weeks), treatment (9 weeks), and post- treatment periods (10 weeks). Adult bulls were fed 1113 (n=8; low-) or 2227 (n=6; high ergot group) µg/kg of dry matter intake. Endpoints were measured every two weeks. Ejaculates were analyzed for sperm concentration, total and progressive motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and sperm abnormalities. Data were analyzed by repeated-measures MIXED PROC. Plasma prolactin decreased markedly during treatment (-52.4%; Experimental period p<0.01). Rectal temperature was higher during the treatment and post-treatment periods (EP p<0.01) but was within normal physiological range. Bull weight increased during the study (EP p<0.01). Scrotal circumference in low ergot group increased during treatment (+0.8 cm; Tx*EP p=0.05). Progressive motility in high ergot group decreased during treatment (-7%; Tx*EP p=0.05), however, no other semen endpoints were affected (p≥0.11). Live sperm with high and medium MMP decreased during treatment (-1.4 and -3.7%; EP p<0.01). Results suggest that feeding ≤2227 µg ergot alkaloids/kg has only minor effects on adult bull semen quality.

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5.2. INTRODUCTION Ergot alkaloids are pharmacologically active secondary metabolites produced by the plant fungus Claviceps purpurea. The fungus infects numerous cereal grains and grasses across Canada, including rye, wheat, barley, and triticale. As these grains are widely used feed ingredients for livestock, domestic animals may become poisoned if their feeds are contaminated with ergot alkaloids. In the United States, ergot alkaloids are more commonly encountered in endophyte- contaminated tall fescue, in which the symbiotic relationship between the fungal endophyte (Epichloë coenophiala) and tall fescue grass (Lolium arundinaceum) produces ergopeptine alkaloids. The ergot alkaloids produced by E. coenophiala and C. purpurea are similar, however the principle toxic alkaloid in endophyte-infected tall fescue is ergovaline, which is not produced by C. purpurea. Toxicity in domestic livestock due to consumption of ergot alkaloids is common. Ergopeptine alkaloids act as agonists and partial agonists of serotonin, dopamine, and norepinephrine receptors in multiple tissues (Berde and Stürmer 1978; Pertz and Eich 1999). The principle tissues affected appear to be vascular smooth muscle, as ergot alkaloids are well known vasoconstrictive agents. In C. purpurea ergotism, severe intoxication results in persistent vasoconstriction that progresses to ischemic gangrene of tail tips, ear tips, and hooves. Animals in the early stages of gangrenous ergotism tend to be lame on the hindlimbs (Robbins, Porter, and Bacon 1986). Less severe symptoms of poisoning include decreased feed intake, reduced weight gain, heat intolerance in high ambient temperatures, decreased prolactin secretion, and reduced reproductive efficiency (Robbins, Porter, and Bacon 1986). Poisoning from endophyte- infected tall fescue may also result in gangrenous lesions (“fescue foot”), but also includes hyperthermia during the summer, decreased feed intake, and reduced weight gain, and decreased prolactin secretion (Robbins, Porter, and Bacon 1986; Schmidt and Osborn 1993; Strickland, Oliver, and Cross 1993). Ergot alkaloids have been associated with reduced fertility in female animals. Although the two classical forms of ergot poisoning are the convulsive and gangrenous forms, reproductive ergotism, specifically for female reproduction, has more recently been included (Porter and Thompson 1992; Strickland et al. 2009; Klotz 2015; Poole and Poole 2019). The reported effects of ergot alkaloid exposure from tall fescue toxicosis on cow reproduction are numerous. These include delayed onset of puberty (Washburn, Green, and Johnson 1989; Washburn and Green 1992), decreased prolactin secretion (Hurley et al. 1980; Walner et al. 1983; Mizinga et al. 1990;

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Browning et al. 1998), decreased progesterone secretion and altered ovarian function (Bolt and Bond 1989; Burke et al. 2001; Burke and Rorie 2002; Jones et al. 2003; Seals et al. 2005), reduced milk production and agalactia via prolactin suppression (Hemken et al. 1979; Schmidt et al. 1986; Strahan et al. 1987; Blaney et al. 2000b; Craig, Klotz, and Duringer 2015), reduced birth weights (Beers and Piper 1987; Watson et al. 2004), reduced calving rate (Boling 1985; Schmidt et al. 1986; Gay et al. 1988; Brown et al. 1992), and abortion (Mantle and Gunner 1965; Appleyard 1986; Craig, Klotz, and Duringer 2015). In studies of cows fed known concentrations of C. purpurea ergot alkaloids, hemodynamic changes in the caudal artery have been reported (Cowan et al. 2018, 2019). It is commonly advised that pregnant and lactating cattle do not consume ergot alkaloid contaminated feed. Decreased reproductive efficiency in cattle consuming endophyte-infected tall fescue reportedly causes billions of dollars of economic losses to producers annually in the United States (Kallenbach 2015; Poole and Poole 2019). In bulls, however, the effects of prolonged ergot alkaloid exposure on reproduction are less well defined. Ergot alkaloids are vasoconstrictive in the testicular artery of bulls (Aiken et al. 2015), which could impair spermatogenesis through altered thermoregulation and/or tissue hypoxia. Impaired testicular thermoregulation is a known deleterious factor to spermatogenesis (Kastelic et al. 1996; Kastelic, Cook, and Coulter 1997). Some in vitro studies have demonstrated deleterious effects of ergot alkaloids on sperm viability and motility (Wang et al. 2009; Page et al. 2018), indicating that ergot alkaloids may act directly on sperm through altered signaling pathways. Other negative impacts of tall fescue ergot alkaloids on bull fertility have also been reported (Jones et al. 2004; Schuenemann et al. 2005a,b; Looper et al. 2009; Stowe et al. 2013; Pratt et al. 2015; Burnett, Bridges, and Pratt 2017; Burnett et al. 2018). However, there is no consistently reported adverse effect of ergot alkaloids on sperm function or morphological parameters. Further, there are no studies presently available that assess the effect of C. purpurea ergot alkaloid consumption on bull reproduction. The effects of ergot alkaloids from C. purpurea contaminated feed on bull reproductive capacity remain to be investigated. The objective of this study was to examine the effects of feeding two permissible concentrations of ergot alkaloids on the scrotal circumference, semen characteristics, body temperature and plasma prolactin concentrations in adult beef bulls. We hypothesized a concentration-response relationship between ergot in feed and reductions in

162 plasma prolactin concentrations, sperm motility, sperm function, and increase in sperm abnormalities. 5.3. MATERIALS & METHODS 5.3.1. Animals. 5.3.1.1. Animal Ethics Statement. The following studies and procedures used therein were reviewed and approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use (Animal Use Protocol# 20170032). All researchers involved in the study received University- approved animal care and use training before working with the animals. 5.3.1.2. Animal Husbandry. Adult Angus bulls (n=14) were maintained at the Goodale Cattle Research Farm of the University of Saskatchewan Livestock and Forage Centre of Excellence under uniform housing and management conditions. Two studies were conducted – a short-term pilot feeding study to assess ergot alkaloid intake and a long-term ergot alkaloid feeding study to test our hypotheses regarding the effect of ergot alkaloids on bull breeding potential. The sample population for the pilot study included 3 Red Angus bulls and 11 Black Angus bulls. The sample population of the long-term study included 1 Red Angus bull and 13 Black Angus bulls. Some bulls from the pilot study were replaced due to aggressive temperament; the two studies shared 11 bulls. The age (mean ± standard deviation) of bulls at the time of the two studies was 3±1 year (range: 2 to 5 years). The ergot exposure in the long- term feeding study was started after 236 days of the end of the exposure in the pilot study. The body weight for bulls in the pilot study was 857 ± 117 kg (range: 735 to 1049 kg) as of the first day of the study. The body weight for the bulls in the long-term study was 864 ± 111 kg (range: 717 to 1085 kg). Bulls were housed in outdoor group paddocks with ad libitum access to water, shelter, and total mineral salt blocks (CO-OP® Saskatoon, SK). Prior to the study, bulls were subjected to the standardized breeding soundness evaluation (BSE) at the Large Animal Clinical Sciences Department in the Western College of Veterinary Medicine. All bulls included in this study passed breeding soundness evaluations as stipulated by the Western Canadian Association of Bovine Practitioners (Barth 2013). Bulls were observed daily for any changes in health and fitness. For the pilot and long- term studies, bulls were group housed in two pens (6 and 8 in each pen). Bulls acclimated to their surroundings prior to the study to minimize stress due to fighting. Bulls were given an

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intramuscular injection of Vétoquinol Vitamin AD-500 in January (Vitamin A 500 000 I.U./mL, vitamin D 75 000 I.U./mL, vitamin E 5 I.U./mL; approx. 7-8 cc per bull as per manufacturer’s instructions). A vitamin and mineral pre-mix (CO-OP® 2:1 beef cattle range mineral; 100 g/hd/day) was offered to bulls in their feed starting mid-point of the long-term study. 5.3.2. Experiment 1: Pilot ergot alkaloid feeding study. 5.3.2.1. Experimental design. The purpose of the pilot study was to assess intake of ergotized pellets in adult bulls after individualized feeding based on the body weight. Bulls were randomly allocated to three treatment groups for this pilot study based on the summed ranks of scrotal circumference and body weight. The intended protocol of this study was to feed ergot alkaloid concentrations of 40 µg/kg body weight (n=4) and 80 µg/kg body weight (n=5), with the remaining bulls (n=5) consuming control pellets (<2.4 µg/kg). Control (i.e., ergot-free) and ergotized pellets were manufactured at the Canadian Feed Research Centre (North Battleford, Saskatchewan, Canada). Control pellets were comprised of 79% barley, 15% oat hulls, and 6% canola meal. Ergotized pellets were comprised of 74% barley, 15% oat hulls, 6% canola meal, and 5% ergotized wheat screenings (contained 27276 µg ergot alkaloids/kg of pellets). Feed samples were analyzed for ergot alkaloid and mycotoxin concentration prior to feeding using liquid chromatography mass spectrometry in Prairie Diagnostic Services (Saskatoon, SK) as described (Grusie et al. 2017). Concentrations of six main ergot alkaloids in the ergotized pellets are summarized in Table 5.1. The ration that the bulls were offered during the pilot study is described in Table 5.2. The pilot feeding study was conducted in July 2017. The pre-treatment period of control (i.e., non-ergotized pellet) feeding was from days -7 through -1 (i.e., 7 days). The treatment period of ergotized pellet feeding was from days 0 through 9 (i.e., pellets containing ergot alkaloids were offered for 10 days). The post-treatment period of control (i.e., non-ergotized pellets) feeding was from days 10 through 13 (i.e., 4 days). Bulls were separated into individual outdoor pens prior to feeding. Bulls were given 1 hour to consume the feed in absence of the researchers to minimize stress. Bulls in the 80 µg/kg treatment group were individually offered between 2.1 to 3.1 kg of ergotized pellets per day whereas bulls in the 40 µg/kg ergot treatment were individually offered between 1 to 1.4 kg of ergotized pellets per day, dependent on the body weight of each bull. After the one-hour feeding period, any remaining feed was weighed and recorded. Percent pellet consumption was calculated based on

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TABLE 5.1. Ergot alkaloid concentration (µg/kg) of control and treatment pellets used in Experiment 1 (pilot ergot alkaloid feeding study) in adult Angus bulls as determined by liquid chromatography mass spectrometry (LC-MS) analysis (Prairie Diagnostic Services, Saskatoon SK Canada). Ergot alkaloid Concentration (µg/kg) % Composition Concentration (µg/kg) % Composition Ergocornine <1.25 - 1260 5 Ergocristine <1.25 - 18980 70 Ergocryptine <1.25 - 3110 11 Ergometrine <1.25 - 76 0 Ergosine <1.25 - 990 4 Ergotamine <1.25 - 2860 10 TOTAL <7.5 N/A 27276 100 N/A = not applicable <1.25 µg/kg = limit of detection

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TABLE 5.2. Total mixed ration of adult Angus bulls used in Experiment 1 (pilot ergot alkaloid feeding study). Ergotized pellets were offered to individual bulls daily for the ten-day treatment period. Bulls were fed the same amount of control pellets (i.e., 2.5 kg/head) during the pre- (seven days) and post-treatment (four days) periods. Control Low dose High dose (40 µg/kg BW) (80 µg/kg BW) n=14 5 4 5 Ingredients Control pellets 2.5 kg 1.1-1.4 kg N/A Ergotized pellets* N/A 1.1-1.4 kg 2.1-3.1 kg Silage 0.4 kg 0.4 kg 0.4 kg Oats** 0.4 kg 0.4 kg 0.4 kg Grass hay Free choice in pen Free choice in pen Free choice in pen * Ergotized pellets were fed on a body weight basis, therefore a range of pellets is indicated. ** Oats were offered starting day 3 of the experiment to enhance palatability Abbreviations: BW, body weight; N/A, not applicable

166 the feed (pellets, oats, and silage) offered and the amount of unconsumed feed. Pellet consumption (including the silage and oats, as these were offered at the same time as the pellets and mixed with the pellets) was calculated by measuring the amount of pellet mixture before and after the bulls were given an hour to eat. Free choice hay was offered ad libitum to the bulls in their home pens, thus the amount of hay consumed was not measured. Consequently, hay consumption was not included in the calculation of consumption. Results of this feeding trial informed the exposure regimen for the long-term study. 5.3.3. Experiment 2: Long term ergot alkaloid feeding study 5.3.3.1. Experimental design. In the long-term feeding study, a repeated measures design was chosen to test two concentrations of ergot alkaloids in feed by utilizing bulls as their own controls. Instead of three treatment groups with smaller sample sizes (as in the pilot feeding study), two treatment groups with more animals per group were chosen. This enabled an assessment of concentration-response between ergot alkaloid concentration and the various breeding potential endpoints chosen. The pre-treatment period served as the control period for the animals. Bulls were randomly allocated into two treatment groups: low ergot (1113 g ergot alkaloids /kg of dry matter intake; n=8) and high ergot (2227 g/kg DMI; n=6). The two ergot alkaloid treatments were chosen for this study to mimic moderate and high concentration ergot exposure in feed near the current Canadian permissible concentrations of 2000 to 3000 g/kg (CFIA 2017). The experiment consisted of a pre-treatment (12 weeks), treatment (9 weeks), and a post-treatment (10 weeks) period. This study was conducted from December 19th, 2017 through July 19th, 2018. The pre-treatment period took place from December 19th, 2017 to March 12th, 2018 (i.e., 84 days or 12 weeks). The ergot-contaminated feed was fed for 61 days from March 13th, 2018 through May 12th, 2018 (i.e., 9 weeks). The post-treatment period was from May 13th, 2018 to July 19th, 2018 (i.e., 68 days or 10 weeks). The nine-week exposure period was chosen based on the length of one spermatogenic cycle in bull i.e., 61 days (Amann 1962a,b), to track potential changes in sperm function and morphology due to ergot feeding. Note: the pre-treatment period for flow cytometry endpoints was six weeks. The targeted feeding for bulls was 1.5-2% body weight (based on 1000 kg average body weight). Barley pellets, alfalfa silage, and grass hay were offered at 5, 6, and 6 kg per head per day, respectively. Results of the pilot feeding study indicated that bulls were averse to individual feeding. Due to animal behavior, limited facilities, and concerns around researchers’

167 safety, individual feeding was not feasible. Therefore, group feeding was chosen for this long- term trial. Ergotized pellets were manufactured using heavily ergot-contaminated concentrated wheat screenings at the Canadian Feed Research Centre (North Battleford, Saskatchewan, Canada). The control pellets were comprised of barley (74%), oat hulls (15%), canola meal (6%), and molasses (5%). The treatment pellets were comprised of barley (72%), oat hulls (14%), canola meal (6%), molasses (5%), and ergotized wheat screenings (4%). Control pellets were determined to have <391 μg total ergot alkaloids/kg of pellet weight. The concentration and composition of ergot alkaloids in the treatment pellets is given in Table 5.3. The total mixed ration and associated ergot alkaloid concentrations offered to bulls is given in Table 5.4. Bulls were pen-fed the treatment pellets daily in the morning. To give each bull an equal opportunity to consume the pellets, the pellets were spread out in a long trough. In addition, the bulls were limited to one hour to consume the pellets on sampling days. Total daily dry matter intake was approximately 16.5 kg/head (dry matter content of silage ~35%). The ‘low’ ergot treatment group was group fed in one pen (n=8) whereas the ‘high’ treatment group was fed in a separate pen (n=6). Each bull in the low ergot treatment were offered 1.1 kg of ergotized pellets and 3.9 kg of control pellets (i.e., 8.8 kg ergot pellets + 31.2 kg control pellets for group feeding 8 bulls). Bulls in the high treatment were offered 2.2 kg ergot pellets and 2.8 kg control pellets (i.e., 13.2 kg ergot pellets + 16.8 kg control pellets for group feeding 6 bulls). The remainder of the ration (i.e., barley silage and hay) was fed in the afternoon by feed truck (see Table 5.4). Farm barley was analyzed for ergot alkaloids concentration and contained <70 μg/kg. 5.3.3.2. Sample collection and animal handling. Blood and semen samples were collected at two-week intervals from all bulls. Bulls from each pen were sampled on each collection day (i.e., either Tuesdays or Thursdays) to provide blocking criteria for subsequent statistical analysis. Eight bulls were sampled on Tuesdays (i.e., five from low ergot group, three from high ergot group) whereas six bulls were sampled from on Thursdays (i.e., three from low ergot group, three from high ergot group). 5.3.3.3. Rectal temperature, body weight, and scrotal circumference. Rectal temperature, body weight, and scrotal circumference were recorded at 2-week intervals prior to semen collections. Each endpoint was measured starting an hour post-feeding and took place before semen collection. Rectal temperature was measured using a handheld digital

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TABLE 5.3. Concentration (mean ± standard deviation; µg/kg) of six ergot alkaloids in control and treatment pellets as determined by liquid chromatography mass spectrometry (LC-MS) following solvent extraction (Prairie Diagnostic Services, Saskatoon SK Canada). Ergotized pellets were included in the total mixed ration of bulls in Experiment 2 (long term ergot alkaloid feeding study). Control pellet Treatment pellet Ergot alkaloid Concentration (μg/kg*) % Composition Concentration (μg/kg*) % Composition Ergocornine 22 ± 16 8 1571 ± 278 9 Ergocristine 73 ± 51 25 8465 ± 2203 51 Ergocryptine 33 ± 25 11 3089 ± 373 18 Ergometrine 128 ± 30 44 431 ± 28 3 Ergosine 10 ± 2 3 917 ± 105 5 Ergotamine 24 ± 16 8 2285 ± 377 14 TOTAL 291 ± 140 100 16757 ± 3364 100 *µg/kg=parts per billion (ppb)

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TABLE 5.4. Total mixed ration offered to adult Angus bulls (n=14) in Experiment 2 (long term ergot alkaloid feeding study). Ergotized pellets and the rest of the ration were offered daily for the 9-week treatment period (i.e., 61 days). Bulls were fed the same weight of control pellets during the pre- (12 weeks; 84 days) and post-treatment (10 weeks; 68 days) periods. Feed ingredients below are given per head per day. Ingredient Amount (per head per day) Grass hay 6 kg (~6 kg DM) Barley silage* 18 kg (~6 kg DM) Trace mineral salt block Ad lib. (approx. 2 oz) Pellets (control or ergotized) 5 kg (~4.5 kg DM) Total approximate dry matter intake 16.5 kg

Ergot concentration (μg/kg, DM)** Low treatment (n=8) 1113 High treatment (n=6) 2227 * Analysis determined silage contained 35% dry matter ** Bulls in the low treatment offered 1.1 kg ergot pellets + 3.9 kg control per bull Bulls in high treatment offered 2.2 kg ergot pellets + 2.8 kg control per bull Abbreviations: DM, dry matter

170 thermometer. Bull weights were measured with the weighing scale built-in the chute system at the farm. Weights for heavy bulls (i.e., over 1000 kg) could not be recorded between February 15th through March 15th, 2018 until the scale was recalibrated. Scrotal circumference was measured by one researcher (VC) throughout the study period using a Reliabull scrotal circumference tape measure. 5.3.3.4. Semen collection. Semen was collected once every two weeks from each bull when possible. Semen was collected after measurement of rectal temperature, body weight, scrotal circumference, and after blood collection. Certain bulls were noncooperative during restraint and electroejaculation processes, leading the researchers to avoid collection for personal and animal safety. In those cases, semen was collected on an alternate day if possible. Bulls were restrained in a locking head gate and chute prior to semen collection. Semen was collected via electroejaculation using a commercial equipment with manual electrical impulse control (Pulsator IV model; Lane Manufacturing Inc., Denver CO USA). Feces were removed manually from rectum and transrectal massage was given to minimize the electrical stimulation. An electric probe was inserted in the rectum and electric current was given. Following penile erection and protrusion, the penis was diverted towards collection tube for semen collection. Seminal plasma was not collected as to not dilute the semen sample and distort subsequent concentration analysis and production calculation. Collection began when opaque semen was observed and stopped when the bull did not produce further ejaculate. Samples were transferred into 15 mL conical tubes and transported to the laboratory in an insulated container (37°C) within a maximum of 2 hr of collection. Semen samples were transferred directly to a 37°C water bath upon arrival in the laboratory. Transport from the farm to the laboratory was approximately 20-30 minutes. 5.3.3.5. Blood collection and plasma separation. Blood was collected at two-week intervals from each bull. Blood collection took place after rectal temperature, body weight, and scrotal circumference measurement, but before semen collection. This ensured that blood collection occurred within the same frame of time for the bulls every two weeks to minimize prolactin variation due to time of day. Blood was collected from the caudal vein using 18-gauge Vacutainer® needles (1.5”) and lithium heparin BD Vacutainer® tubes with PSTTM inserts (Becton Dickinson Canada, Mississauga, ON). Approximately 16 mL whole blood was collected (2 × 8 mL Vacutainer® tubes). Plasma was separated from whole blood by

171 centrifugation (3000 rpm, 10 minutes) at room temperature on-farm and stored at -20°C until prolactin analysis. 5.3.3.6. Semen assays. All semen assays were carried out in the WestGen Research Suite at the Western College of Veterinary Medicine (Saskatoon, SK). Before semen collection, tris- citric-acid buffer (TCA; .03% Tris base, 1.74% citric acid monohydrate, 1.2% fructose, Milli-Q distilled H2O, adjusted to pH 7.1), eosin-nigrosin morphology stain (purchased from the Western College of Veterinary Medicine Pharmacy), and CASA slides (Leja® 20 μm depth slides; Leja Products B.V., Nieuw-Vennep, The Netherlands; or 20 μm depth MicroTool chamber slides; Cytonix, Beltsville, MD USA) were warmed using a water bath (TCA buffer, stain) or a slide warmer (slides). The TCA buffer was used as a semen diluent for subsequent assays. Buffer was added slowly to semen samples to prevent dilution shock to sperm. 5.3.3.6.1. Computer assisted sperm analyzer (CASA). Fresh semen was diluted 1:20 in TCA buffer. If samples were highly concentrated or very dilute, samples were prepared at 1:40 or 1:10 dilution ratio respectively. Samples (2.5 µL) were loaded in a chamber of the CASA slide and analyzed under 20× power on a Zeiss Axioskope 40 microscope using SpermVision software (Version 3.0; Minitube Canada, Ingersoll ON). At least 200 sperm from seven fields were randomly selected for analysis. Sperm concentration (billion/mL), motility (%), and progressive motility (i.e., the percentage of sperm moving in a straight line, >10 µm radius at >4.5µm/s) were recorded. Sperm number per ejaculate (x109) was calculated by multiplying ejaculate volume (mL) and concentration (billion/mL). Note: A different CASA system (Hamilton Thorne IVOS II) was used on June 19, 2018. The parameters for progressive motility were different than the normal SpermVision system, therefore progressive motility data for this day were excluded. 5.3.3.6.2. Flow cytometer analysis (PI, FITC-PNA, MT Deep Red). A simultaneous assessment of fresh bull sperm plasma membrane, mitochondrial membrane potential, and acrosomes was conducted using triple stains as described (Anzar, Kroetsch, and Boswall 2011) with slight modifications. Samples were prepared in duplicate per bull. The following fluorescent probes were used: propidium iodide (PI; Invitrogen, stock 2.4 mM in water), Mitotracker Deep Red (MtDR; Invitrogen, stock 2 µM in dimethylsulfoxide), and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; stock 1 mg/mL in 1X phosphate buffered saline). Aliquots of each dye were thawed at room temperature in the dark. Semen

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samples were diluted to a final sperm concentration 1x106 cells/mL in TCA buffer (i.e., 1 mL of TCA). PI (1 μL), MtDR (2 μL), and FITC-PNA (3 μL) were added to 500 µl of diluted semen sample. Each sperm-dye mixture was incubated with the dyes for 20 minutes at room temperature. Sperm were fixed using 10 μL of 10% formalin per sample. The remaining 500 μL of TCA buffer was added in each sample before analysis. Sperm were identified and gated (R1) using forward and side light scatter (SSC versus FSC) as previously described (Anzar, Kroetsch, and Boswall 2011) with a Partec Cyflow Space flow cytometer (Partec GmBH, Munster, Germany) equipped with a 400 mW argon laser and a red diode laser. Briefly, the peaks of distribution of fluorescence dictated where to place each gate. For PI and FITC-PNA, this was approximately 10. For MtDR, gates were approximately set to 10 and 100 to delineate low and medium and medium and high mitochondrial membrane potential, respectively. There was some bull-to-bull variation in gating, however, gating was fixed for each bull. Data were acquired and analyzed using the Partec FloMax software (version 2.4). Samples were ran at a flow-rate of 1 μL/s. Run times were approximately 1 minute per sample. At least 9900 cells were analyzed per run. The FITC-PNA and PI probes were excited with a 488 nm blue laser. The emission spectra from these dyes were detected with photo-multiplier detectors at FL-1 (527 BP filter) and FL-3 (680 LP filter), respectively. The MtDR probe was excited with a 635 nm red diode laser and its emission spectra were detected with a photo-multiplier detector at FL-6 (670 BP filter). A representative set of histograms and the various cell populations detected is presented in Figure 5.1. Simultaneous fluorescence for all probes was recorded on logarithmic scales. In histograms with PI and FITC-PNA staining, four cell populations were separated: intact plasma membrane and intact acrosome (IPM/IACR (QB3)), intact plasma membrane and compromised acrosome (IPM/CACR (QB4)), compromised plasma membrane and intact acrosome (CPM/IACR (QB1)), and compromised plasma membrane and compromised acrosome (CPM/CACR (QB2)). Detection of PI and MtDR revealed six populations: intact plasma membrane and low mitochondrial membrane potential (IPM/LMMP), intact plasma membrane and medium mitochondrial membrane potential (IPM/MMMP), intact plasma membrane and high mitochondrial membrane potential (IPM/HMMP), compromised plasma membrane and low mitochondrial membrane potential (CPM/LMMP), compromised plasma membrane and medium mitochondrial membrane potential (CPM/MMMP), and compromised plasma membrane and

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FIGURE 5.1. Representative histograms of triple-stain flow cytometric analysis of fresh adult bull sperm exposed to ergot alkaloids. (A) Sperm specific events were identified based on forward light scatter versus side light scatter characteristics and gated as R1. (B) One-dimension histogram of propidium iodide (PI) fluorescence intensity (x-axis) and number of sperm specific events (counts; y-axis). The left peak represents sperm with intact plasma membranes (IPM). The right peak represents sperm with compromised plasma membranes (CPM). (C) Two- dimensional dot plot of FITC-Peanut Agglutinin intensity versus PI intensity. Each quadrant is as follows: QB1 – sperm with CPM and intact acrosomes (IACR); QB2 – sperm with CPM and compromised acrosomes (RACR); QB3 – sperm with IPM and IACR; QB4 – sperm with IPM and CACR. (D) One-dimensional histogram of MitoTracker® deep red (MtDR) fluorescence intensity (x-axis) versus sperm specific events (counts; y-axis). Sperm with fluorescence intensity <10 were considered to have low mitochondrial membrane potential (LMMP). The central peak represents sperm with medium MMP (MMMP). The right peak represents sperm with high MMP (HMMP). (E) Two-dimensional dot plot of MtDR fluorescence intensity versus

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PI fluorescence intensity to distinguish sperm with intact or compromised plasma membrane and LMMP, MMMP, and HMMP. Each quadrant is as follows: QA1 – sperm with CPM and LMMP; QA2 – sperm with CPM and both MMMP and HMMP; QA3 – sperm with IPM and LMMP; QA4 – sperm with IPM and both MMMP and HMMP (F) Two-dimensional dot plot of MtDR fluorescence intensity versus PI fluorescence intensity. Each quadrant is as follows: Q1 – sperm with CPM and both LMMP and MMMP; Q2 – sperm with CPM and HMMP; Q3 – sperm with IPM and both LMMP and MMMP; Q4 – sperm with IPM and HMMP. Sperm with MMMP were calculated by subtracting QA4-QA3 (IPM) and QA2-QA1 (CPM). Quadrant data from C, E, and F were extracted from FlowMax software as “Percentage [of cells] Gated [in R1]”.

175 high mitochondrial membrane potential (CPM/HMMP). Data extracted were “Percentage (of cells) Gated”. 5.3.3.6.3. Sperm morphology (eosin-nigrosin vital staining). A drop of stain was placed on a warm slide (approx. 5 mm in diameter). A drop of semen (approx. 2-5 mm in diameter, depending on concentration of sample) was placed on the slide next to the stain droplet using a plastic cryopreservation straw. The stain and sample were lightly mixed on the slide with the straw. A smear was then made to have areas of high and low sperm concentrations by alternating pressure held against the slide with the straw as the semen/dye mixture was moved down the slide. Sperm were evaluated using phase contrast microscope at 1000x magnification with oil immersion (Zeiss Immersol with Zeiss Axioskop 40 Microscope). Minimum of 100 sperm were counted per slide. In smears with apparently elevated morphological defects, at least 300 cells were counted. The percentages of live versus dead sperm and normal versus abnormal sperm were determined. Live sperm were those unstained by eosin-nigrosin. Sperm were examined for the following morphological defects: head defects, midpiece defects, principle piece defects, proximal droplets, acrosome defects (i.e., knobbed acrosomes, including flattened and indented forms), and detached heads (both normal and abnormal). Specific head defects included pyriform and tapered heads, nuclear vacuoles, micro- and macrocephalic sperm. Midpiece defects included distal midpiece reflexes and mitochondrial sheath defects as indicated (Barth 2013). Spermiogram evaluation was conducted as per the Western Canadian Association of Bovine Practitioners Bull Breeding Soundness Manual (Barth 2013). Briefly, ejaculates containing at least 70% morphologically normal sperm with <20% nuclear abnormalities or proximal droplets and <25% acrosomal or tail abnormalities were considered satisfactory quality. One researcher (VC) carried out all differential sperm counts and morphology classifications. 5.3.3.7. Plasma prolactin quantification by radioimmunoassay. Plasma prolactin concentration was determined by double antibody radioimmunoassay. Assays were carried out in the Endocrine Research Laboratory (Western College of Veterinary Medicine, Saskatoon SK). Reagents were obtained from the Dr A.F. Parlow, National Hormone and Pituitary Program (NHPP; Harbor-UCLA Medical Center, Torrance CA USA). Concentrations are

176 expressed in terms of bovine Prolactin (AFP4832B). The standard curve ranged from 2 to 128 ng/mL, and standards were prepared in 0.5 M phosphate buffered saline containing gelatin. The antibody used was AFP 753180 (0.2 mL per tube of 1:40,000 dilution in 0.1% Normal Rabbit Serum in 0.5M phosphate buffered saline). Bovine prolactin (bPRL; AFP4832B) was iodinated using the Chloramine T procedure (6 µg Chloramine T per µg prolactin to be iodinated). The working solution provided 12 000 counts per minute in 0.1 mL 0.05M phosphate buffered saline containing gelatin. The bound and free fractions were separated using a sheep-anti-rabbit double antibody and 5% polyethylene glycol. Bovine Growth Hormone had 0.06% cross reactivity, while bovine follicle stimulating hormone, luteinizing hormone, and thyroid stimulating hormone all showed <0.0001% cross-reactivity with the antiserum (as provided by the NHPP). The intra-assay coefficient of variation was 8.5 and 4.4% for bull and Holstein calf plasma with mean bPRL concentrations of 49.4 and 22.1 ng/mL, respectively. 5.3.4. Statistical analysis. 5.3.4.1. Experiment 1 – pilot ergot alkaloid feeding study. Percent pellet consumption data were analyzed using repeated measures by the SAS mixed procedure (SAS Inc. Cary, NC USA). The main effects of treatment (i.e., Tx - control, low, or high) and day of study (i.e., Day -7 through 13) or experimental period (i.e., pre-treatment, treatment, and post-treatment), as well as associated interactions (i.e., Tx*Day or Tx*experimental period), were included in the model. 5.3.4.2. Experiment 2 – long-term ergot alkaloid feeding study. Statistical Analysis Software (SAS) version 9.4 with Enterprise Guide 6.1 (SAS Institute, Cary NC USA) was used for all analyses. The repeated measures mixed procedure was used to test for the effect of treatment (i.e., two ergot concentrations), experimental period (i.e., pre-treatment, treatment, post-treatment) or week of experiment (i.e., -12 through 18), and associated interactions (treatment*experimental period, i.e., Tx*EP, or treatment*week, i.e., Tx*Week). The data are presented as arithmetic means ± standard error of mean of each treatment group and of each experimental period. Variables analyzed included rectal temperature, prolactin concentration, body weight, scrotal circumference, CASA parameters (sperm concentration, ejaculate volume, sperm production, total motility, and progressive motility), flow cytometric comparisons) and morphology (percent normal, percent of each head, midpiece, principle piece, and acrosome defects, and percent proximal droplets, detached normal heads, detached abnormal heads, and percent live staining). Random factors included in the model were ambient temperature outside

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(“Temp_out”), ambient temperature inside the barn (“Temp_in”), and date of semen/blood collection (i.e., Tuesday or Thursday; “Date_of_Collection”). The best fit model for the data was selected based on the smallest Akaike information criteria (AICc) value from the ten tested covariance structures (simple, compound symmetry, heterogeneous compound symmetry, Toeplitz, banded Toeplitz, Huynh-Feldt, autoregressive, heterogeneous autoregressive, ante- dependence, and unstructured). Final analysis of the data (Type 3 Test of Fixed Effects) included main effects (Tx = Treatment, EP = Experimental Period and Week) or interaction terms (Tx*EP or Tx*Week). Statistical significance was considered p≤0.05 (=0.05). Multiple comparisons were conducted where applicable using the differences of least square means. The syntax used for the final analysis in SAS is given in Appendix L. 5.4. RESULTS 5.4.1. Experiment 1 – Pilot ergot alkaloid feeding study. Data for percentage pellets consumed for each treatment group throughout the study and during the pre-treatment, treatment, and post-treatment periods of the pilot study are presented in Figure. 5.2. There were significant interactions for Treatment*Day (p<0.02) with a substantial decrease in pellet consumption at the start of feeding period in both treatment groups but not in the control (Figure 5.2A). Pellet consumption did not change between experimental periods for the control and 40 µg/kg body weight (BW) groups. In the 40 µg/kg BW ergot group, feed consumption decreased from 42 ± 8% in the pre-treatment to 39 ± 5% in the treatment period (Treatment*Experimental Period p<0.01; Figure 5.2B). Pellet consumption further decreased to 32 ± 9% in the post-treatment period, however pre- and post-treatment values did not differ. During the treatment and post-treatment periods, feed consumption was lower in both the 40 µg/kg and 40 µg/kg BW ergot groups when compared to the control group consumption (Figure 5.2B). For all data combined, pellet consumption (mean ± standard error) was 57 ± 2%. 5.4.2. Experiment 2: Long term ergot alkaloid feeding study. No clinical symptoms of ergot toxicity were observed in the animals throughout the treatment or post-treatment period. Average outside ambient temperature during the pre-treatment, treatment, and post-treatment

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FIGURE 5.2. Pellet consumption (percentage of pellets offered that was consumed; mean ± standard error) in adult Angus bulls (n=14). Bulls were offered feed daily in individual pens. Pellet consumption was calculated daily. (A) Percentage of feed consumed before (pre-treatment period; days -7 through -1) during (treatment period; 0 through 9) and after (post-treatment period; 10 through 13) exposure to no ergot (control group, n=5), 40 µg ergot alkaloids/kg body weight (n=4), (targeted intake if all ergotized pellets were consumed), and 80 µg ergot alkaloids/kg body weight (n=5). (B) Percentage of feed consumed by experimental period (pre- treatment, treatment, and post-treatment). Data are presented as mean ± SEM. Values with uncommon alphabets in Fig. B are different at p≤ (=0.05). The asterisk (*) in Fig. A indicates consumption on day 0 and day 1 are different (p<0.01) for both ergot treatments.

179 periods was -13 (-31 to 1), 0.5 (-18 to 19), and 21 (13 to 28) °C. Experimental design allowed use of pre-treatment data as base-line data for detecting effect of treatment in low- and high ergot groups. 5.4.2.1. Prolactin, body weight, rectal temperature, and scrotal circumference. Data gathered at 2-week intervals for various endpoints are presented in Figures 5.3 to 5.6 on the left panels while period-wise data (mean value for each period) are shown in right-side columns. Plasma prolactin concentrations decreased markedly (Week p<0.01) during the treatment weeks for both low and high ergot groups with a recovery during the post-treatment weeks (Figure 5.3A). Overall, prolactin concentrations (data combined between groups) in the pre- treatment, treatment, and post-treatment periods were 64.9 ± 5.6, 34.0 ± 3.4, and 96.6 ± 7.8 ng/mL, respectively (Experimental Period p<0.01; Figure 5.3B). Prolactin concentration was lower during the treatment period than both the pre- and post-treatment periods. Prolactin decreased from week -2 to week 0 and 2 and increased from week 8 to 10. Bulls gained weight throughout the study (Week and Experimental period P<0.01; Figure 5.3C, D) without any effect of ergot feeding. Average body weight in the pre-treatment, treatment, and post-treatment periods was 902 ± 13, 968 ± 11, and 1005 ± 10 kg, respectively. Rectal temperature was lower (Treatment p=0.04; Figure 5.3E) in the high ergot group (37.9 ± 0.07 °C) than the low ergot group (38.2 ± 0.07 °C). Rectal temperature was higher (Experimental Period p≤0.01, Figure 5.3F) in the treatment (38.2 ± 0.1 °C) and post-treatment (38.4 ± 0.1 °C) experimental periods versus the pre-treatment period (37.7 ± 0.1 °C); treatment and post-treatment period rectal temperatures did not differ. Overall, rectal temperature was 38.1 ± 0.05 °C (all data combined). Scrotal circumference varied during the study period (Week p<0.01; Figure 5.3G), decreasing from week 0 to weeks 16 and 18 (data combined between groups). Scrotal circumference increased by 0.8 cm from pre-treatment to treatment period in the low ergot group (Treatment*Experimental Period p=0.05; Figure 5.3H) but no change was recorded for the high ergot group. 5.4.2.2. Sperm volume, concentration, and motility parameters (CASA). Sperm concentration and sperm total motility remained unchanged (p≤0.11) throughout the study (Figure 4A, B, G, H). Ejaculate volume and the number of sperm per ejaculate was lower

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FIGURE 5.3. Plasma prolactin concentration (A, B), body weight (C, D), rectal temperature (E,F), and scrotal circumference (G, H) of adult Angus bulls in low ergot (n=8; 1113 µg/kg DMI) and high ergot (n=6; 2227 µg/kg DMI) groups before (12 weeks, i.e., 84 days), during (9

181 weeks, i.e., 61 days), and after (10 weeks, i.e., 68 days) daily ergot alkaloid exposure in their feed. Blood was collected for subsequent plasma prolactin analysis every two weeks. Body weight, rectal temperature, and scrotal circumference were measured every two weeks. Data are presented as mean ± SEM. Horizontal lines indicate group data were averaged by week and combined to compare experimental periods. Values with uncommon alphabets are different at p≤ (=0.05). Asterisks indicate differences between weeks (p≤0.05) but no weekly or period- wide effects were recorded.

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FIGURE 5.4. Sperm concentration (A, B) , ejaculate volume (C, D), sperm production (E, F), total motility (G, H), and progressive motility (I, J) of fresh of adult Angus bulls in low ergot (n=8; 1113 µg/kg DMI) and high ergot (n=6; 2227 µg/kg DMI) groups before (12 weeks, i.e., 84 days), during (9 weeks, i.e., 61 days), and after (10 weeks, i.e., 68 days) daily ergot alkaloid exposure in their feed. Sperm endpoints were assessed every two weeks by computer assisted semen analysis (CASA). Data are presented as mean ± SEM. Group data were analyzed by repeated measures via the mixed procedure in SAS. Analysis by treatment (Tx), week of treatment, and the interaction (Tx*Week) is given in panels A, C, E, G, and I. Analysis by Tx, experimental period (EP), and the interaction (Tx*EP) is given in panels B, D, F, H, and J. Values with uncommon alphabets are different at p≤ (=0.05).

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(Treatment p=0.02; Figure 5.4 C, E, F) in the low ergot group compared to the high ergot group but no weekly or period-wide effects were recorded (Appendix M). Bulls in the low ergot treatment produced an average of 8.4 ± 0.6, 7.8 ± 0.6, and 6.9 ± 0.6 mL of semen during the pre- treatment, treatment, and post-treatment periods, respectively (Appendix M). These ejaculates contained 5.6 ± 0.8, 4.7 ± 0.7, and 4.2 ± 0.5 x109 sperm, respectively. Bulls in the high ergot treatment produced 11.2 ± 0.9, 13.7 ± 1.7, and 12.3 ± 1.5 mL of semen in the pre-treatment, treatment, and post-treatment periods, respectively. These ejaculates contained 8.9 ± 1.0, 8.5 ± 1.3, and 8.8 ± 1.4 x109 sperm, respectively. Overall, bulls produced an average of 9.7 ± 0.4 mL of semen containing 6.53 ± 0.3 x109 sperm (all data combined). During the treatment, progressive motility decreased from 64.0 ± 2.3% (pre-treatment period) to 56.9 ± 2.4% (treatment period) in the high ergot treatment (Treatment*Experimental Period p=0.05; Figure 5.4J; Appendix M) while no change was recorded for the low ergot group during these periods (57.0 ± 1.9% versus 58.0 ± 2.1%, respectively). The post-treatment period values for progressive motility for the high and low ergot groups were 60.1 ± 2.1% and 60.0 ± 2.1%, respectively, and did not differ from their respective pre-treatment or treatment period values. Week-wise analysis for progressive motility did not detect any differences (p≥0.11; Figure 5.4I). 5.4.2.3. Mitochondrial membrane potential and acrosome intactness (flow cytometry). Plasma membrane integrity (i.e., absence of PI fluorescence), mitochondrial membrane potential (i.e., fluorescence intensity of MitoTracker DR) and acrosome intactness (absence of FITC-PNA fluorescence) were assessed by flow-cytometry. Sperm population with very low PI staining (i.e., intact plasma membrane) were considered live sperm and their proportions (in relation to total sperm events) were further categorized into intact-acrosome versus compromised-acrosome sperm or into those having low (LMMP), medium (MMMP) versus high (HMMP) mitochondria membrane potential. Combined among treatment groups, percentage of live sperm with HMMP decreased (Experimental Period p<0.01; Figure 5.5B) from 16.5 ± 0.7% in the pre-treatment period to 15.1 ± 0.6% in treatment period and to 12.4 ± 1.5% in the post-treatment period. Likewise, percentage of live sperm with MMMP decreased (Experimental Period p<0.01; Figure 5.5D) between the pre-treatment (29.2 ± 1.3%) and the treatment (25.5 ± 1.1%) periods with a subsequent recovery between the treatment and the post-treatment (32.5 ± 1.7%) period (data

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Ergot Treatments

FIGURE 5.5. Sperm populations with intact plasma membrane and different levels of mitochondrial membrane potentials and acrosome integrity of fresh adult Angus bull semen

186 semen collection by electroejaculation. Data for mitochondrial membrane potential are missing for weeks 10 and 12 due to machine errors. Sperm with intact plasma membrane and high mitochondrial membrane potential (A,B), intact plasma membrane and medium mitochondrial membrane potential (C, D), and intact plasma membrane with low mitochondrial membrane potential (E, F), intact plasma membrane and intact acrosomes (G, H) and intact plasma membrane and compromised acrosomes (I, J) were determined by flow cytometry every two weeks following semen collection by electroejaculation. Data are presented as mean ± SEM. Bulls were randomized into two ergot treatment groups, low (1113 µg/kg; n=8) and high (2227 µg/kg; n=6). Bulls were group fed ergot alkaloids in their total mixed ration daily for the treatment period. Group data were analyzed by repeated measures via the mixed procedure in SAS. Analysis by treatment (Tx), week of exposure, and their interaction is given in panels A, C, E, G, and I. Analysis by Tx, experimental period (EP), and their interaction (Tx*EP) is given in panels B, D, F, H, and J. Horizontal lines indicate group data were averaged by week and combined to compare experimental periods. Uncommon alphabets are different at p≤ ( =0.05). Asterisks indicate differences between weeks (p≤0.05).

187 combined between treatment groups). Live sperm with LMMP, MMMP, and HMMP also varied by week of experiment (Week p<0.01; Figure 5.5 A, C). The percentage of live sperm with HMMP decreased from 16.9 ± 1.4 to 11.6 ± 0.7% from week 0 to 2, respectively. Similarly, the percentage of live sperm with MMMP decreased from 30.1 ± 2.9 to 21.8 ± 1.3% from week 0 to before (six weeks, i.e., 42 days), during (9 weeks, i.e., 61 days), and after (10 weeks, i.e., 68 days) ergot treatment. Flow cytometric analysis was conducted every two weeks following 2, respectively. Conversely, the percentage of live sperm with LMMP increased from 12.1 ± 1.2% to 33.9 ± 1.5% from week 0 to 2, respectively. The proportion of live sperm with intact acrosomes varied during the study weeks (Week p<0.01; Figure 5.5G). A substantial decrease was reported in the post-treatment period between weeks 10 (70.4 ± 1.4%) and 14 (46.1 ± 3.4%). As expected, the proportion of live sperm with compromised acrosomes was low during the study period (0.88 ± 0.05%). A progressive decrease (Experimental Period p<0.03) in proportion of live sperm with compromised acrosomes was noticed (Figure 5.5I). 5.4.2.4. Sperm morphology (eosin-nigrosin stains). Experimental period data for sperm morphological defects are presented in Figure. 5.6 and additional details are provided in Appendix M. Averaged among the treatment groups, the percentage of live sperm (i.e., sperm which excluded eosin-nigrosin stain) was lower (Experimental Period p<0.01; Figure 5.6F) during the treatment period (64.8 ± 1.7%) versus the pre-treatment (71.5 ± 1.6%) and post- treatment (71.5 ± 1.3%) periods. No effects of ergot on percentage of sperm showing normal morphology were detected (main effects p≥0.12; interactions p≥0.64; Figure 5.6 A, B). Similarly, no main effects (Treatment p≥0.09, Experimental Period p≥0.14) or interactions (p≥0.27) were detected for head defects, midpiece defects, principle piece defects, proximal droplets, detached normal heads, or detached abnormal heads. The percentage of acrosome defects was higher (Treatment p=0.01) in the low ergot group (1.1 ± 0.2%) compared to the high ergot treatment (0.3 ± 0.1%). 5.5. DISCUSSION This study evaluated the effects of feeding two levels of permissible ergot alkaloid concentrations (1113 µg/kg and 2227 µg/kg of dry matter intake) to adult beef bulls over one spermatogenic cycle (i.e., 61 days) on weight gain, scrotal circumference, thermotolerance, plasma prolactin concentration, and characteristics of fresh semen. The pilot experiment

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FIGURE 5.6. Sperm morphology determined by eosin-nigrosin staining and differential microscopic counting of fresh adult Angus bull semen before (12 weeks, i.e., 84 days), during (9 weeks, i.e., 61 days), and after (10 weeks, i.e., 68 days) ergot treatment. Eosin-nigrosin slides were prepared every two weeks following semen collection by electroejaculation. Percentage of live sperm (A, B), percentage of morphologically normal sperm (C, D), and percentage of sperm having knobbed acrosomes (E, F) was determined every two weeks by differential counting of eosin-nigrosin stained sperm smears following semen collection by electroejaculation. Data are presented as mean ± SEM. Bulls were randomized into two ergot treatment groups, low (1113 µg/kg; n=8) and high (2227 µg/kg; n=6). Bulls were group fed ergot alkaloids in their total

189 mixed ration daily for the treatment period. Group data were analyzed by repeated measures via the mixed procedure in SAS. Analysis by treatment (Tx), week of exposure, and the interaction (Tx*Week) is given in panels A, C, E. Analysis by Tx, experimental period (EP), and the interaction (Tx*EP) is given in panels B, D, F. Horizontal lines indicate group data were averaged by week and combined to compare experimental periods. Uncommon alphabets are different at p≤ (=0.05).

190 informed that individual feeding was not consistent and feasible under our farm conditions, therefore group feeding was used for the long-term study. It was speculated that other factors, including bull temperament, contributed to inconsistent individual feeding. Although individual feeding is ideal for concentration-response experiments, safety concerns from working with the bulls and relevance of group feeding to on-farm exposures drove the compromise to group- feeding. In addition, individual bulls served as their own controls for the long-term study (the pre-treatment period) in a repeated measures design. As this study employed adult bulls, the impact of time on the various semen/sperm endpoints was assumed to minimal. The authors chose to prioritize including multiple ergot alkaloid concentrations to be tested as opposed to further decreasing sample size to include an additional control treatment. Despite the limitations in the present study, some important observations were made that contribute to a better understanding of the effects of ergot alkaloids on adult breeding bulls. In the long-term study, plasma prolactin concentrations were reduced by 52.4% of the pre-treatment values during the feeding period in both groups with immediate recovery after withdrawal of ergot. Bulls continue to gain weight, did not demonstrate clinical symptoms of ergotism, and had variations in rectal temperature that were within the normal physiological range for beef cattle. Changes recorded in semen and sperm characteristics included: a decrease of 7% in percentage of progressively motile sperm in ejaculates from bulls exposed to the higher ergot concentration with subsequent recovery; an average decrease of 7% in live sperm in both high and low ergot groups during the treatment period (that returned to baseline value in the post-treatment period); decreased percentage of live sperm with high- and medium mitochondrial potential during the ergot feeding period in both group; and a small transient increase in live sperm with intact acrosomes immediately after end of ergot feeding. Scrotal circumference increased between pre-treatment and treatment periods in the low ergot group but not in the high ergot group and a progressive decrease from beginning of ergot feeding to end of experiment was noticed; further validation is required to determine if the observed effect resulted from ergot exposure. Overall, observed changes in semen characteristics were minor in nature and would have not resulted in failure in the breeding soundness evaluation of any bull. The proposed hypothesis that a concentration- response relationship between ergot in feed and reductions in plasma prolactin concentrations, sperm motility, sperm function, and increase in sperm abnormalities was partially supported. An important caveat to mention is that sperm characteristics under study in the long-term experiment

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(CASA parameters, morphology, flow cytometric assessments), although correlated with fertility, are not direct indicators of bull fertility. Whether observed changes have any significant effect on pregnancy rates requires further testing by fertility trials. The pilot study indicated that moderate feed refusal occurred in bulls individually offered 40 to 80 µg/kg BW ergot alkaloids in their feed. This was clearly evident on the second day of the treatment period, in which feed consumption decreased substantially in the two ergot treatment groups, but not in the control group. Feed refusal has been documented previously in ergot-exposed pigs and dairy cattle in Australia fed diets infected with Claviceps africana (1-40 mg dihydroergosine/kg feed) (Blaney et al. 2000b). In addition, “summer slump” is a syndrome of fescue toxicosis characterized by feed refusal in warmer temperatures as livestock experience impaired thermoregulatory ability (Evans, Rottinghaus, and Casteel 2004b). It was speculated that poor palatability contributed to the feed refusal observed. It was also considered that other factors, including intractable bull temperament (to frequent interaction with researchers), separation from pen-mates, and movement from home pen, contributed to the variable consumption across all groups in our study. This includes the variable pellet consumption during the pre-treatment period. Although individual feeding is ideal for calculating ergot alkaloid dose received by animals in scientific studies, it was found that individual feeding was neither consistent nor successful, leading to the choice of group feeding for the long-term study. Plasma prolactin concentration decreased substantially during the treatment period in both groups. Previous studies from the authors’ laboratory found no effect of C. purpurea ergot alkaloids on circulating prolactin in early lactational and periparturient cows (Grusie et al. 2018b; Cowan et al. 2018, 2019); however, it was speculated other factors (such as temperature, photoperiod, suckling, etc.) contributed to the observed results. Similarly, no changes in prolactin in bulls given 40 µg/kg ergotamine tartrate daily in their ration for 224 days (Schuenemann et al. 2005b). Conversely, studies of tall fescue ergot alkaloids exposure in bulls consistently report decreased circulating prolactin (Evans et al. 1988; Schuenemann et al. 2005a; Looper et al. 2009; Stowe et al. 2013; Pratt et al. 2015b; Burnett, Bridges, and Pratt 2017; Burnett et al. 2018). Dopamine suppresses prolactin secretion from the anterior pituitary gland by binding to D2 receptors on pituitary lactotrophs (Ben-Jonathan and Hnasko 2001). Ergot alkaloids, as dopamine agonists, also bind to D2 receptors to inappropriately suppress prolactin secretion (Hökfelt and Fuxe 1972; Anlezark, Pycock, and Meldrum 1976; Sibley and Creese

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1983). It is interesting to note that the plasma prolactin concentrations reported in the present study during the treatment period are consistent with other ergot-related decreases in serum prolactin, in which fescue alkaloid exposed bulls had <34 ng/mL (Schuenemann et al. 2005a; Looper et al. 2009; Stowe et al. 2013; Pratt et al. 2015b). There is some suggestion that ergot alkaloid toxicity in male reproduction is prolactin- mediated (Pratt and Andrae 2015). The role of prolactin in male reproductive physiology is broad (Bartke 2004). In rodents, prolactin acts in concert with gonadotropins to stimulate testicular growth and function (Bartke 1971; Hafiez, Lloyd, and Bartke 1972; Bartke, Croft, and Dalterio 1975; Zipf, Payne, and Kelch 1978; Bartke 1980; Dombrowicz et al. 1992). Further, prolactin has been detected in bull seminal fluid and on epididymal epithelial cells and germ cells (Pratt et al. 2015a). The prolactin receptors are located on most cell types of the testes: porcine Sertoli cells (Guillaumot, Tabone, and Benahmed 1996), human Leydig cells, and differentiating germ cells (Hair et al. 2002). In rams, prolactin regulates testosterone secretion (Sanford and Dickson 2008; Sanford and Baker 2010). However, the exact role of prolactin in bull reproduction is not clear. It is unclear if decreased prolactin concentrations in the present study contributed to decreased sperm production in the low ergot treatment. Further, it was not determined if suppressed prolactin disturbed the microenvironment within the bull testes, as such investigation was outside the scope of the present study. Bulls gained weight throughout the study, indicating that the concentrations offered in feed were not sufficient to cause feed refusal. Other studies have suggested that ergot alkaloids decrease dry matter or nutrient intake, corresponding to decreased weight gain (Aldrich et al. 1993; Matthews et al. 2005). A potential mechanism for this is reduced blood flow to the forestomach epithelium, leading to decreased absorption of nutrients and volatile fatty acids (Foote et al. 2013). Decreased weight gain has been reported in some studies of ergot-exposed bulls (Pratt et al. 2015b), but not others (Evans et al. 1988; Stowe et al. 2013; Burnett et al. 2018). It is unclear if ergot exposure affected overall growth or nutrient absorption in our bulls, as we did not have a reference no-treatment group. However, it is promising that bulls in this study apparently suffered no loss of productivity. Rectal temperature varied by experimental period and by treatment group. All temperatures recorded, however, were within the normal physiological range for cattle (Robertshaw 2004). No other symptoms of heat intolerance were observed. Our observations

193 could be explained partially by a substantial portion of the ergot exposure period being conducted in cooler temperatures during March, April, and part of May. As a result of their vasoconstrictive activity, ergot alkaloids reduce evaporative heat loss from the skin by decreasing peripheral blood flow (Rhodes et al. 1991; Aldrich et al. 1993). Clinical hyperthermia (rectal temperatures >40 °C) resulting from ergot exposure has been recorded following exposure of steers to enforced sunlight (Bourke 2003). Further, heat intolerance is a commonly cited symptom of fescue toxicosis in cattle (Spiers et al. 2012). Some studies of bulls grazing toxic tall fescue pastures report increased rectal temperature following prolonged exposure (Schuenemann et al. 2005a, b). However, rectal temperature is not always altered in ergot exposed bulls (Evans et al. 1988). Bulls in the present study had shelter/shade access in their pens, which would allow them to recover from any heat stress. The ambient temperatures in this study (-18 to 19 °C during the ergot feeding period) were unlikely to induce heat stress. Scrotal circumference increased during the treatment period for the low ergot group. Some studies of ergot alkaloids reported decreased scrotal circumference in bulls (Jones et al. 2004; Stowe et al. 2013), while others report no effect (Evans et al. 1988; Schuenemann et al. 2005a, b; Looper et al. 2009; Burnett et al. 2018). One study reported decreased scrotal circumference in yearling bulls fed 800 µg ergovaline/kg feed for 126 days, but did not find any accompanying decreases in semen quality (Stowe et al. 2013). Increased scrotal circumference is a positive factor for bull fertility and age of puberty in female offspring (Brinks, McInerney, and Chenoweth 1978; Smith, Brinks, and Richardson 1989), given bulls meet a minimum standard of scrotal circumference (Barth 2013). It is unclear why changes were not detected in the high ergot treatment and whether the observed changes were incidental. A concentration-dependent decrease in progressive motility was observed in the present study. In contrast, no effect was detected for sperm concentration, ejaculate volume, sperm production, or total sperm motility. In vitro studies have indicated that ergot alkaloids reduce sperm motility (Wang et al. 2009; Page et al. 2018). In contrast to our studies, however, the in vitro studies reported a decrease in total motility as well as progressive motility. This result may be due to in vitro versus in vivo conditions. Wang et al. (2009) concluded that ergot alkaloids (specifically ergotamine and dihydroergotamine) acted directly on sperm via alpha-adrenergic receptors to decrease sperm motility. In vitro studies suggest ergot alkaloids have a direct toxic effect on sperm.

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Results of the present study are in partial agreement with in vivo studies. In bulls exposed to endophyte-infected tall fescue ergot alkaloids (600 µg/kg) for 121-days, a decrease in percent motile sperm and decrease in progressive motility (19%) in yearling bulls was reported (Looper et al. 2009). However, bulls supplemented with 40 µg/kg BW ergotamine tartrate in their diet for 224 days had no decrease in progressive motility estimated by light microscopy (Schuenemann et al. 2005b). Similarly, yearling bulls exposed to 800 µg/kg daily for 126 days had no changes in any CASA-measured parameters (Stowe et al. 2013). Another study reported lower sperm concentrations in bulls that grazed toxic tall fescue pasture for 155 days; however, no ergot alkaloid concentration was given (Pratt et al. 2015b). This study also reported decreased post- thaw motility and progressive motility fescue-exposed bulls. Burnett, Bridges, and Pratt (2017) found decreased post-thaw progressively motile sperm in bulls that grazed endophyte-infected tall fescue for 112 days. Further, authors of this study found prolonged deleterious impacts on post-thaw sperm motility up to 56 days following removal of bulls from the toxic fescue pasture. Cryopreservation and post-thaw analysis were outside the objectives of this study, but these analyses were conducted and the data will be presented in different study. A study on yearling and two-year old bulls found no negative impact on sperm concentration, total motility, or progressive motility after grazing toxic tall fescue for 56 days (no ergot alkaloid concentration reported; Burnett et al. 2018). However, this study had conflicting results on pregnancy rates in heifers and cows following timed artificial insemination. Considered together with the data of the present study, ergot alkaloids in feed produce subtle alterations in sperm production and motility. Ergot alkaloid concentrations >600 µg/kg feed are inconsistently associated with changes in sperm motility, concentration, and morphology. Feed ergot alkaloid concentration data must be consistently reported to better establish concentration-effect data. Flow cytometry data from the present study indicated transient changes in mitochondrial membrane potential and acrosomal integrity due to ergot treatment. Ergot alkaloids have been found to be vasoconstrictive in the testicular artery of yearling bulls (Aiken et al. 2015), thus the change in mitochondrial membrane potential could represent a secondary toxic change due to vasoconstriction or impaired thermoregulation. Dysfunction with testicular thermoregulation affects semen quality by increasing sperm with morphological defects (Kastelic et al. 1996; Barth and Bowman 1994; Rahman et al. 2011) and decreasing motility (Brito et al. 2004; Rahman et al. 2011). Reactive oxygen species, produced during tissue vasoconstriction and as a

195 by-product of oxidative metabolism, damage sperm mitochondria (Guthrie and Welch 2012). Mitochondrial membrane potential is correlated with sperm motility, as mitochondrial respiration produces adenosine triphosphate for movement of the sperm flagellum (Ruiz-Pesini et al. 2000; Turner 2003; Paoli et al. 2011; Amaral et al. 2013). As such, mitochondrial membrane potential is correlated with fertility (Troiano et al. 1998; Mazur et al. 2000; Kasai et al. 2002; Paoli et al. 2011). Based on the decreased in sperm with medium to high mitochondrial integrity, ergot alkaloids may disrupt energy homeostasis of sperm. However, further studies are required to corroborate the observations of the present study and further understand the underlying mechanisms, such as studies utilizing flow cytometric probes to measure reactive oxygen species production. As changes in total motility were not detected in the present study, it cannot be stated for certain that ergot alkaloids act in this manner or damaged sufficient mitochondria to affect fertility. Acrosome integrity was largely unaffected by ergot treatment; however, the proportion of live sperm with intact acrosomes decreased during the post-treatment period. This observation was not seen with a concomitant increase in live sperm with compromised acrosomes in the present study, suggesting that more sperm were dead. Further studies are required to confirm our findings and to ascertain whether post-treatment acrosomal changes affect fertility in ergot- exposed bulls. The data for this study indicated no effect of ergot treatment on abnormal sperm morphology. The low ergot treatment had higher knobbed acrosomes than the high ergot treatment, but this was regardless of experimental period. The initial hypothesis was that ergot alkaloid exposure would decrease the percentage of morphologically normal sperm and increase the percentage of morphological defects. Bulls may show increased incidence of certain morphological defects in the weeks following environmental stress or impaired thermoregulation (Barth and Bowman 1994). In addition, one study reported decreased morphologically normal sperm in ergot alkaloid exposed yearling bulls (Pratt et al. 2015b). In contrast, another report suggested no change in morphologically normal sperm following 56-day exposure to fescue ergot alkaloids (Burnett et al. 2018). Two earlier studies also reported no change in sperm morphology as assessed by eosin-nigrosin stains (Schuenemann et al. 2005a,b). Based on the results of the present study, it would not be possible to determine if bulls were exposed to ergot alkaloids based on the spermiogram, or even consecutive spermiograms.

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In contrast, other toxins consumed by bulls produce characteristic morphological defects, e.g., gossypol toxicity causes midpiece defects (Chenoweth et al. 2000). Taken together with the sperm concentration and motility data, ergot exposed adult bulls would be unlikely to fail a standardized breeding soundness evaluation based on morphology following prolonged exposure to ergot alkaloids in their feed. Similarly, an earlier report also concluded that based on lack of changes in motility and morphology, ergot-exposed bulls were unlikely to fail a breeding soundness evaluation compared to non-exposed bulls (Stowe et al. 2013). In addition to the above suggestion that normal sperm production and concentration are unaffected, it appears that sperm maturation in the epididymis is also unaffected by ergot exposure. If the epididymal environment was affected, one would expect to see an increased incidence of distal midpiece reflexes and proximal droplets in ejaculates (Barth 2013). Testosterone also affects the epididymal environment; although not measured in the present study, other work f ound no effect of ergot on circulating testosterone (Schuenemann et al. 2005a,b; Looper et al. 2009; Pratt et al. 2015b). Lastly, the percentage of live sperm (based on eosin-nigrosin staining data) decreased somewhat, however this was not consistent with the total motility data from CASA. Objective methods of characterizing sperm with higher numbers counted are likely more accurate, however this observation should be investigated further. Based on the results of this study, feed containing up to 2227 µg/kg total ergot alkaloids may affect progressive motility and mitochondrial membrane potential of sperm but does not interfere with other sperm characteristics (i.e., total motility, sperm concentration, acrosome intactness, or normal morphology). Plasma prolactin concentrations decreased by 50% during 9- week exposure followed by recovery within 2 weeks after exposure. The overall impact of ergot alkaloid exposure on breeding soundness of adult bulls is mild. Changes in sperm motility and morphology would likely be undetectable with a standard breeding soundness evaluation. Transient changes in sperm mitochondrial membrane could indicate interference of ergot alkaloids with sperm energy homeostasis. Further studies are necessary to confirm these observations and to assess the fertilization capacity of sperm from ergot-exposed bulls. In terms of spermatogenesis, adult bulls can tolerate ergot alkaloids in their feed at current Canadian permissible concentrations (i.e., <3000 µg/kg of dry matter intake); however, a major effect on plasma prolactin (this study) and vasoconstrictive effects in cattle (recorded in previous studies) warrants caution against such exposure.

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6. CHAPTER 6 – GENERAL DISCUSSION

The research described in this thesis was conducted to meet the growing need for scientific evidence with Canadian relevance on ergot alkaloid exposure and effect in cattle in the face of rising mycotoxin contamination. Long considered an “ancient” disease, the recent appearance of ergot contamination and ergotism in livestock took many by surprise. Scientific information was highly sought after by regulators and producers, as ergot mycotoxin research in cattle was largely unavailable in Canada at the time. The research described in this thesis has provided answers to the numerous questions related to ergot alkaloids. The major indicators and conclusions associated with the research described herein are presented in Table 6.1. It was confirmed that the vascular system is affected in cattle at moderate concentrations in feed; however, vascular changes were not persistent once the alkaloids were removed from the feed. In an attempt to characterize the pharmacokinetic behaviour of the ergot alkaloids, which is largely unavailable in the scientific literature, it was found that ergot alkaloids cannot be detected in blood following exposure in feed. This led to the conclusion that vascular effects occur at the sub-µg/kg concentration. There is low oral bioavailability of the alkaloids and increased analytical sensitivity (approximately an order of magnitude greater) is necessary to quantify the alkaloids in bovine plasma. Lastly, we examined the effects of prolonged exposure in adult bulls, as producers suspected that ergot exposure decreases bull breeding capacity and semen quality. The results of this study found that ergot exposure had limited effect on major parameters examined in breeding soundness evaluations. This lead the conclusion that ergot exposure does not affect semen quality of adult bulls at current permissible Canadian concentrations (up to 3000 µg/kg) for livestock feed. However, reductions in plasma prolactin in both treatment groups of bulls were observed.

6.1 Vascular changes. The data described in this thesis indicates that vascular, rather than endocrine or reproductive, changes are produced at lower concentrations of ergot alkaloids in cattle feed. In fact, changes in the peripheral vascular system of cattle appear to be one of the most sensitive indicators of ergot alkaloid exposure. Constriction and reduced blood flow in the various arteries have been reported in studies of fescue toxicosis in cattle and other livestock (Aiken et al. 2007, 2009, 2011, 2016; McDowell et al. 2013; Aiken and Flythe 2014), in addition to the work described herein (Cowan et al. 2018, 2019). There are several conclusions that c

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TABLE 6.1. Major indicators and effects of ergot alkaloid exposure in beef cattle. Endpoint Indicator of Approx. no effect Diagnostic Individual Problems exposure or concentration (total usefulness or herd toxicity ergot alkaloids unless indicated otherwise) Feed analysis Both N/A High Both Unclear guidelines

Prolactin Neither (C. >2115 µg/kg feed Limited Herd Animal variability concentration purpurea) 390 µg ERV/kg

1 a 99 Exposure feed

(fescue) Vascular effects Exposure 201 µg/kg feed Limited Herd Animal variability Lack of field application (i.e., group and time averaged data) Gangrene Toxicity 473 µg/kg feedb High Both Animals have passed ‘point of no return’ Plasma analysis Neither 29252 µg/kg feed Limited Both Concentrations too low (250 µg/kg body Lack of sensitivity weight) Low oral bioavailability Sperm analysis Neither (C. 1113c-2227 µg/kg Limited Individ. Not sensitive to ergot exposure purpurea) feed Flow cytometry lacks field Exposure 600 µg ERV/kg applicability (fescue) feedd Contradictions within literature

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Feed refusal Both 40 µg/kg body Moderate Both May be difficult to assess in Both (C. weight large herds africana) 400-3000 µg/kg feede Milk production Exposure No data avail. in High Both More applicable to dairy cattle beef cows Dairy: 400-3000 µg/kg feede Performance No (C. 2227 µg/kg feed Limited Both Not observed in the present purpurea) thesis Changes in rectal No 2227 µg/kg feed Limited Individ. Hyperthemic ergotism temperature Yes 1200 mg/kg feedf High uncommon in Canada (useful in more temperate regions, e.g.

Australiaf) 2

0 a b c d

0 Aiken et al. 2007; Craig, Klotz, and Duringer 2015; progressive motility only; Looper et al. 2009 (decreased motility and

morphology); eBlaney et al. 2000b. Reported for dairy cattle and C. africana (sorghum ergot); fBourke 2003. Enforced sunlight in ergot (C. purpurea) exposed cattle. This study reported milligram per kg concentrations. Abbreviations: ERV, ergovaline; N/A, not applicable

200 be drawn from this study. First, vascular effects occur well below the allowable value of 3000 µg/kg set by the Canadian Feed Inspection Agency for cattle feed. This calls into question the predictive value of the current guideline as a ‘safe’ concentration for ergot alkaloids in livestock feed. In addition, vascular changes are both concentration and time dependent, where higher concentrations fed on a short-term basis produce similar changes following long-term, low concentration exposure. The current regulations do not consider this important relationship between concentration and duration of exposure. Lastly, vascular changes in cattle return to pre- treatment values once ergot is removed from the feed. Although the vascular contractile effects of ergot alkaloids are persistent in vitro (Pesquiera et al. 2014), it appears that in vivo clearance mechanisms are sufficient once contaminated feed is removed. To be protective of cattle from ergotism, future standards for ergot alkaloids in livestock feed should be based on concentrations at which vascular changes occur.

6.2 Diagnostic considerations. An important caveat in interpretation of the data is that our results describe group averaged and time averaged data. Therefore, the diagnostic feasibility of ultrasonography on arteries may be limited. Overall, diagnosis of subclinical ergot exposure in beef cattle continues to be a challenge. We did not observe reduced animal performance in our studies, further suggesting that ergot toxicity may not manifest until the animal displays clinical disease. As determined in Chapter 4, simple analysis of blood samples for presence of ergot alkaloids is not possible at the present time. The plasma prolactin concentration does not appear to be a suitable bioindicator in cows, but potentially for bulls. This is likely due to high inter- animal variability in plasma prolactin concentration. The endocrinology of prolactin likely has many factors involved that were not necessarily controlled for in our studies were outside the scope of this thesis. These factors will be discussed below in section 6.4. As seen in Chapters 2 and 3, decreased plasma prolactin is not observed in all ergot alkaloid exposures. In contrast, prolactin concentration was decreased during the treatment period for bulls (Chapter 5). As such, standards and diagnoses should exercise caution in relying solely on plasma prolactin concentration. Plasma prolactin concentration could and should be included in these assessments, with the understanding the prolactin physiology is complex and may not always reflect ergot alkaloid exposure. In bulls, routine spermiograms or motility analyses of ejaculates are not sensitive to ergot exposure. Progressive motility was decreased in the high ergot treatment (Chapter 5); however, when considered with results of other studies of fescue toxicosis in bulls,

201 semen quality is not consistently altered by moderate and permissible concentrations of ergot in feed (600-2227 µg/kg). This leads to the question: how do you identify ergot exposure in cattle before toxicity occurs? At the present time, chemical analysis of suspect feed remains the only practical method to detecting ergot exposure under field conditions.

6.3 Effect of season. Seasonality is an important factor to consider with ergot research that was not tested in this thesis. The research described in Chapters 2 and 3 was conducted during the relatively mild Canadian summers. In Chapter 4, the study spanned most of the calendar year, but ergot exposure occurred during the spring and early summer (March through May). It would be important to repeat these studies during the comparatively harsh climate of Canadian winters, where animals are more likely to display more severe symptoms. These include end-stage gangrenous ergotism, due to increased risk of frostbite and general physiological peripheral constriction mechanisms to conserve body heat. There is clear seasonality of gangrenous ergotism – animals are less likely to be clinically affected during the summer (unless extreme concentrations were consumed; Craig, Klotz and Duringer 2015). Physiological and subclinical changes occur above ~200 µg/kg total ergot alkaloids in the summer, but the question remains whether this concentration is truly a no-adverse effect concentration during the winter. In one case study, a feed concentration of 473 µg/kg was associated with tail sloughing in cattle when temperatures were -20°C (Craig, Klotz, and Duringer 2015). In the same report, other cases with higher concentrations in feed (>11 500 µg/kg) and warmer temperatures (-1 to -4 °C) were associated with gangrenous symptoms. Regulations for ergot in livestock feed need to consider the season in which animals are exposed. In addition, it would be important to test if recovery from ergot alkaloid exposure differs between the summer and winter months.

6.4 Prolactin. In early lactational and periparturient beef cows, we were unable to demonstrate decreased plasma prolactin concentration as a bioindicator of ergot alkaloid exposure. In contrast, we observed ergot-mediated prolactin suppression in adult bulls. While there is substantial evidence in laboratory and livestock species to support ergot alkaloids as prolactin suppressors (Karg, Schams and Rheinhardt 1972; Shaar and Clemens 1972; Floss, Cassady, and Robbers 1973; Smith et al. 1974; Nasr and Pearson 1975; Hurley et al. 1980; Porter and Thompson 1992; Stuedemann and Thompson 1993; Paterson et al. 1995; Browning et al. 1997), our studies in cattle had contradictory results. The reasons for this observation could be multi-

202 fold. There could be a certain degree of compensatory action at the level of the hypothalamus and pituitary when moderate concentrations of ergot alkaloids are encountered in feed. In addition, other physiological factors that affect prolactin physiology were undoubtedly present in these studies. Such factors include ambient temperature, circadian rhythm, photoperiod, stress, and those exclusive to female mammals (i.e., suckling, lactation, and stage of the estrus cycle) (Freeman et al. 2000; Auchtung et al. 2003). All these factors could not be controlled for in the studies described within this dissertation. In cows, other hormones such as growth hormone appear to play an important role in lactation. Thus, interference with prolactin secretion may not be as severe as in other livestock, i.e., horses (Evans et al. 2002, 2011) and swine (Blaney et al. 2000a,b; Oresanya et al. 2003). It is possible that the other physiological factors mentioned above were able to effectively mask the effect of ergot alkaloids on prolactin secretion in beef cows. Sex differences in prolactin secretion also appear to be important, as both concentrations of ergot alkaloids tested (1113 and 2227 µg/kg) suppressed prolactin in adult bulls. As there is a difference in species sensitivity and apparent sex-differences to ergot-induced changes in prolactin dynamics, feed guidelines should reflect this difference.

6.5 Semen characteristics. Results of the bull studies indicate that reproduction is generally not affected in otherwise clinically normal adult bulls. Fescue toxicosis studies in bulls and the subsequent effects are contradictory. Some studies report reduced motility, decreased sperm concentration, and increased percentages of abnormal sperm (Looper et al. 2009; Pratt et al. 2015b), whereas others report no effect (Schuenemann et al. 2005a,b; Stowe et al. 2013). The results of the study described in this thesis suggest the latter. Differences among these studies may be associated with alkaloid profile in the feed, duration of exposure, concentration in the feed, or ambient temperature. When considered in association with the results of the cow studies, it is apparent that peripheral vascular changes should be expected in cattle exposed to moderate concentrations of ergot alkaloids in their feed. A study from Aiken et al. (2015) indicated that both the caudal testicular arteries were constricted in yearling bulls exposed to fescue alkaloids. Constriction and lack of blood flow to the testicular artery represents a putative toxic pathway. The results of Chapter 5 also indicate that changes in CASA parameters and spermiograms would not reflect ergot alkaloid exposure and thus may be missed by traditional breeding soundness evaluations or examination by clinicians. Feed testing of breeding bulls is required to assess exposure and potential disease. Although further studies of in vitro fertilization using

203 semen of ergot exposed bulls should be conducted, we can say with reasonable confidence that C. purpurea ergot alkaloids do not affect breeding potential of adult bulls at total ergot alkaloid concentrations up to 2227 µg/kg in the feed. Sperm characteristics are not a sensitive indicator of ergot exposure. Consequently, it has limited clinical relevance for diagnostic purposes.

6.6. Detection of ergot alkaloids in plasma. Our attempts to develop a method to detect ergot alkaloids in plasma following feed exposure led us to numerous conclusions. Firstly, we can confirm that bioavailability of the ergot alkaloids is low in livestock. Oral bioavailability of various ergot alkaloids has been reported to be <2% in numerous human studies (Ala-Hurula et al. 1979a,b; Ekbom, Paalzow, Waldenlind 1981; Waldenlind et al. 1982; Ibraheem, Paalzow, Tfelt-Hansen 1983) and was postulated for livestock. The low oral bioavailability of the ergot alkaloids speaks to the potency of the compounds but also suggests that bioactive metabolites may contribute to the pharmacological effect. Second, further analytical sensitivity is required to detect ergot alkaloids in plasma. Following intravenous dosages in livestock, the concentrations detected are in the low µg/kg range. Instrumentation with robust quantification at the low µg/kg and high ng/kg concentration would be necessary in order to correlate subclinical or clinical changes with the plasma concentration. The collection of blood or other tissues for ergot analysis would not be clinically useful. Rapid elimination from tissues further compromises detection probability. Human studies report that the plasma concentrations of ergot alkaloids do not correlate well with clinical symptoms (Tfelt-Hansen, Eickhoff, Olesen 1980; Perrin 1985; Tfelt- Hansen 1988; Bigal and Tepper 2003). One other peripherally related observation is that feeding cows a one-time concentration of up to 29 000 µg/kg in feed did not produce any signs of nervous ergotism, nor did it impact lactation. The observation supports the time and concentration interaction stated previously. Nervous ergotism is rare in livestock and the feed concentration or dose threshold at which effect occur is ill-defined. The identification of the threshold is further compromised by the variability in ergot alkaloid composition in feed types (which will be discussed below). What is unclear is why we were unable to detect ergot alkaloids in plasma when other studies in humans, goats, and horses have done so. The methods for sample extraction and clean-up will need to be refined. Pharmacokinetic information is still sorely needed for livestock to understand tissue withdrawal times. As the method developed had good linearity, accuracy, and precision, in vitro pharmacokinetic studies could be undertaken to start to fill in some of the gaps in the literature. Lastly, the contribution of active metabolites to

204 ergot toxicity in livestock is unknown. The physiological activity of -inine epimers of ergopeptine alkaloids is reportedly low (Berde and Stürmer 1978; Pierri et al. 1982; Merkel et al. 2012); however, the contradiction between plasma concentration and pharmacological effect suggests that active metabolites are involved in clinical manifestations. Like the parent compounds, pharmacokinetic information on active metabolites is largely unavailable.

6.7 Comparison with fescue toxicosis. Most livestock research related to ergot alkaloids is associated with exposure to ergot alkaloids from endophyte-infected tall fescue, i.e., fescue toxicosis. To the authors’ knowledge, fescue toxicosis is not reported in Canada. In contrast, fescue toxicosis is a syndrome of livestock in the United States due to the widespread use of tall fescue as forage. As indicated in Chapter 1, ergopeptine alkaloids from endophyte infected tall fescue are similar to those found in C. purpurea sclerotia. Ergotamine is considered the most potent of the ergopeptine alkaloids in C. purpurea sclerotia (Craig, Klotz and Duringer 2015), such that other ergopeptine alkaloids have been described in terms of ‘ergotamine equivalence’ concentrations. The primary alkaloid of concern in tall fescue – ergovaline – however, is not detected in C. purpurea sclerotia. The differences in results of studies of fescue alkaloid exposed livestock and C. purpurea exposed livestock may be related to differences in potencies of the ergot alkaloids and the relative composition of the alkaloids. For example, fescue toxicosis studies have reported more drastic constriction in the caudal artery than in our studies; these studies reported 35-42% decreases in luminal area at concentrations ranging from 390 to 850 µg ergovaline per kg feed (Aiken et al. 2007, 2009). These studies also reported reductions in serum prolactin concentration in the cows and bulls. Studies of fescue toxicity often cite depressed prolactin as a measure of effectiveness of treatments in inducing symptoms (Stowe et al. 2013). In addition, the detection of negative impacts on sperm motility and male endpoints (Looper et al. 2009) may be explained by differing potencies of fescue and C. purpurea. Ergovaline has been shown to have similar in vitro contractile potency as ergotamine (Klotz et a. 2007, 2010; Foote et al. 2011, 2012; Pesquiera et al. 2014). In addition to potency, relative ergot alkaloid abundance in fescue and C. purpurea contaminated feed undoubtedly vary. Our studies indicate that ergotamine, although perhaps the most potent, is not the most abundant alkaloid. In contaminated feeds in our studies, ergocristine was the most abundant alkaloid. Its concentration ranged from approximately 40 to 70% of the total alkaloid content of the feed. This is consistent with a survey of feedstuffs described by Grusie et al. 2018a. It is unclear what proportion of

205 ergocristine, ergocornine, ergosine, and ergocryptine, etc., comprises contaminated tall fescue seed, however fescue studies generally report only ergovaline and its epimer ergovalinine (Aiken et al. 2007, 2009). The results of an in vitro study suggested that tall fescue seed extract contained mostly ergovaline, as in vitro contractility produced by the extract and pure ergovaline did not vary greatly (Foote et al. 2012). Overall, it appears that ergovaline is both the most potent and most abundant alkaloid in endophyte infected tall fescue. Although toxicity of the alkaloids is assumed to be additive, relative potency of the alkaloids is important (as identified by Craig, Klotz, and Duringer 2015). This brings up an important consideration for regulations – should the alkaloids in feed be reported relative to ergotamine concentration or simply as total alkaloid concentration? Overall, the differences between C. purpurea and endophyte infected tall fescue mean that direct comparisons between studies should be made with a certain degree of caution.

6.8 Establishing a no-effect concentration and impact on regulations. The results of Chapters 2 and 3 indicate that vascular effects do not occur below 200 µg/kg total alkaloids in feed. We suggest that this be considered a no-effect concentration for beef cattle dependent on vascular changes and long-term duration of exposure. Chapter 5 indicated that bulls could tolerate from 1113 to 2227 µg/kg total alkaloids in feed. Cattle can tolerate higher concentrations when exposed for a limited period of time, as indicated in Chapter 4, in which cattle experienced no untoward effects from being fed a one-time amount of up to 29 000 µg/kg. If this concentration were fed for a longer period of time, we would expect clinical disease. If producers submitted feed samples with results near 200 µg/kg, dilution of samples in clean grain may be recommended. As indicated before, this concentration cannot be an acceptable standard during the winter season unless research indicates the contrary.

6.9 Food safety. Ergot alkaloids do not pose a major food safety concern. Vascular effects do not persist once animals are taken off contaminated feed. Elimination half -life values have been reported to be between minutes to a few hours for some ergot alkaloids (Ibraheem, Paalzow, Tfelt-Hansen 1983; Tfelt-Hansen and Johnson 1993; Jaussaud et al. 1998; Bony et al. 2001), therefore we do not suspect that the compounds would be detected in tissues based on our observations in Chapter 4. Ergopeptine alkaloids, being relatively lipophilic compounds, may be expected to be detected in the milk of lactating animals. However, ergovaline is below detectable concentrations in the milk of dairy cows (Fink-Gremmels 2008), therefore milk residues are not

206 a concern. There is no risk to the public consuming animal products from ergot-exposed livestock. 6.10 Future directions. In livestock, there are numerous avenues to further examine ergot exposure and toxicity. Feeding trials in colder climactic conditions and the differential effects on the peripheral vascular system would be an important contribution to the ergot literature as this data is largely unavailable. Pharmacokinetic studies and sensitive methods to detect ergot alkaloids in bovine plasma are still needed. Further studies to understand the metabolite profile of ergot alkaloids, i.e., their -inine epimers, would be important to ascertain if these compounds contribute to pharmacologic effect in animals. Similarly, comparisons of the potencies of the individual alkaloids and in producing vascular effects would be valuable contributions to the literature. These data could be presented as in the report from Craig, Klotz, and Duringer (2015), in which the concentration of each ergot alkaloid was expressed as a fraction or equivalence concentration of ergotamine. This may be considered a more suitable approach to express livestock feed guidelines for ergot. Further investigation of prolactin as a potential biomarker for C. purpurea exposed cattle would be interesting, due to the apparent contradiction between data from cows and bulls described herein. Development of diagnostic methods to detect ergot alkaloid exposure in cattle, such as infrared thermographic imaging of hooves, tail tips, ear tips, etc., would be important for livestock clinicians. Mitigation strategies, such as the inclusion of mycotoxin binding agents in feed, are becoming increasingly necessary due to widespread feed contamination. Studies with binders could use the vascular data described herein to study attenuation of ergot alkaloid induced changes. Testicular histopathology studies could be conducted to evaluate the effect of ergot alkaloids on spermatogenesis. 6.11 Conclusion. The research described in this thesis has contributed to an improved understanding of C. purpurea ergot alkaloid exposure to cattle in Canada and globally. Certainly, there are numerous areas of ergot exposure and toxicity that remain to be investigated, some of which have been highlighted in the above discussion. The information presented will inform clinicians and producers on the characteristics of ergot poisoning (i.e., not detectable in blood, vascular changes first, bull breeding soundness unaffected, feed testing required), will enable regulators to refine current guidance values for ergot alkaloids in livestock feed, and will contribute to the global understanding on ergot alkaloid mycotoxins and their effects on livestock.

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8. APPENDICES Appendix A. Syntax for statistical analysis in SAS Proc mixed in Chapter 2. The model was tested with nine covariance matrices to determine the best fit using the lowest AICC score. Word “simple” in the following synt (i.e., “type=”) was replaced with “cs”, “csh”, “toep”, “toep(1)”, hf, “ar(1)”, “arh(1)”, and “ante(1)” to run compound symmetry, heterogeneous compound symmetry, Toeplitz, banded Toeplitz, Huynh-Feldt, autoregressive, heterogeneous autoregressive, and ante-dependence covariance matrices, respectively. ID = unique identifier for each of the 16 cows in the study; Group = Control, Low, Medium or High; Day = days of data collection (-4 to 9 or 14); Period = Before, During or After; Rep = Replicate 1 or Replicate 2; Diam = Recorded diameter of the blood vessel

ODS graphics on; Proc mixed data=WORK.ACUTEERGOT cl covtest; class ID Group Day Period Rep; model DIAM = Group|Period / DDFM=kr htype=3; random intercept/subject=Rep; repeated Day/subject=ID (Group) type=simple; run; ODS graphics off; Once the best fit covariance matrix was selected, the final analysis was run. ODS graphics on; Proc mixed data=WORK.ACUTEERGOT cl covtest plots=residualpanel; class ID Group Day Period Rep; model DIAM = Group|Period / DDFM=kr htype=3; random intercept/subject=Rep; repeated Day/subject=ID (Group) type=cs r rcorr; lsmeans Group|Period / pdiff; run; ODS graphics of

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Appendix B. Tabular presentation of measured (2.1) and calculated (2.2) hemodynamic variables from Chapter 2. B.1. Hemodynamic variables of three arteries measured by Doppler ultrasonography in beef cows (n=4 per treatment group) fed increasing concentrations of ergot for one week. Arteries were imaged daily and data were compared by repeated measures mixed procedure. Data are presented as mean ± SEM for a given time period. Pair-wise comparisons were performed if the ANOVA p-value was ≤0.05 for the treatment (control, low, medium and high), experimental period (pre-treatment, treatment, post-treatment) or treatment*experimental period interaction term.

Ergot Treatment Group Control Low Medium High Variable (132 µg/kg DM) (529 µg/kg DM) (2115 µg/kg DM) Caudal artery Diameter (mm) Pre-treatment 2.9 ± 0.1 3.3 ± 0.1 3.1 ± 0.1x 2.9 ± 0.1x Treatment 2.9 ± 0.1a 3.1 ± 0.1a 2.8 ± 0.1ay 2.4 ± 0.1by Post-treatment 3.0 ± 0.1 3.1 ± 0.1 2.9 ± 0.1x 2.7 ± 0.1x Peak systolic velocity (m/s)

243 Pre-treatment 0.68 ± 0.03 0.55 ± 0.03x 0.76 ± 0.05 0.75 ± 0.05 Treatment 0.71 ± 0.03 0.67 ± 0.02ay 0.71 ± 0.03 0.75 ± 0.04 Post-treatment 0.64 ± 0.03 0.74 ± 0.05ay 0.71 ± 0.04 0.74 ± 0.04 Pulsatility index Pre-treatment 1.33 ± 0.05 1.20 ± 0.05x 1.41 ± 0.07 1.24 ± 0.05x Treatment 1.36 ± 0.04 1.47 ± 0.05y 1.47 ± 0.05 1.37 ± 0.05 Post-treatment 1.28 ± 0.05a 1.61 ± 0.05by 1.52 ± 0.08bx 1.50 ± 0.07by Resistivity index Pre-treatment 0.78 ± 0.02 0.77 ± 0.01 0.77 ± 0.02 0.74 ± 0.02 Treatment 0.78 ± 0.01 0.82 ± 0.01 0.83 ± 0.01 0.77 ± 0.01 Post-treatment 0.79 ± 0.01 0.84 ± 0.01 0.83 ± 0.01 0.83 ± 0.02 Mean arterial velocity (m/s) Pre-treatment 0.40 ± 0.02 0.36 ± 0.02 0.43± 0.03 0.45 ± 0.03

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Treatment 0.41 ± 0.02 0.37 ± 0.01 0.40 ± 0.01 0.43 ± 0.02 Post-treatment 0.40 ± 0.02 0.39 ± 0.03 0.39 ± 0.02 0.42 ± 0.02 End diastolic velocity (m/s) Pre-treatment 0.14 ± 0.02 0.13 ± 0.02 0.15 ± 0.02 0.20 ± 0.03 Treatment 0.15 ± 0.01 0.12 ± 0.01 0.12 ± 0.01 0.18 ± 0.01 Post-treatment 0.13 ± 0.01 0.12 ± 0.02 0.12 ± 0.01 0.12 ± 0.02 Pulse rate (bpm) Pre-treatment 59 ± 2 55 ± 1 59 ± 2 62 ± 2 Treatment 58 ± 1 52 ± 1 56 ± 1 58 ± 2 Post-treatment 56 ± 2 52 ± 2 55 ± 2 59 ± 2

Median sacral artery Diameter (mm) Pre-treatment 3.5 ± 0.1a 4.0 ± 0.1bx 2.9 ± 0.1bx 3.3 ± 0.1ax Treatment 3.7 ± 0.1a 3.6 ± 0.1ay 3.0 ± 0.1bx 3.1 ± 0.1bx Post-treatment 3.7 ± 0.1 3.8 ± 0.1x 3.3 ± 0.1ay 3.3 ± 0.1x 244 Peak systolic velocity (m/s)

Pre-treatment 1.16 ± 0.06 1.24 ± 0.10 1.25 ± 0.06 1.17 ± 0.06 Treatment 1.30 ± 0.05 1.33 ± 0.05 1.28 ± 0.05 1.32 ± 0.07 Post-treatment 1.22 ± 0.08 1.28 ± 0.07 1.28 ± 0.09 1.40 ± 0.08 Pulsatility index Pre-treatment 1.51 ± 0.05 1.66 ± 0.08 1.61 ± 0.07 1.66 ± 0.11 Treatment 1.71 ± 0.06 1.80 ± 0.06 1.75 ± 0.05 1.61 ± 0.07 Post-treatment 1.69 ± 0.08 1.80 ± 0.10 1.78 ± 0.07 1.79 ± 0.11 Resistivity index Pre-treatment 0.78 ± 0.01 0.80 ± 0.02 0.79 ± 0.02 0.76 ± 0.02 Treatment 0.80 ± 0.01 0.82 ± 0.01 0.84 ± 0.01 0.76 ± 0.02 Post-treatment 0.80 ± 0.02 0.81 ± 0.02 0.83 ± 0.01 0.80 ± 0.02 Mean arterial velocity (m/s) Pre-treatment 0.61 ± 0.03 0.59 ± 0.05 0.64 ± 0.04 0.55 ± 0.02 Treatment 0.63 ± 0.02 0.62 ± 0.03 0.62 ± 0.02 0.63 ± 0.03 Post-treatment 0.60 ± 0.04 0.59 ± 0.03 0.60 ± 0.04 0.63 ± 0.03

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End diastolic velocity (m/s) Pre-treatment 0.25 ± 0.02 0.25 ± 0.03 0.26 ± 0.02 0.26 ± 0.02 Treatment 0.25 ± 0.02 0.24 ± 0.02 0.20 ± 0.01 0.31 ± 0.02 Post-treatment 0.24 ± 0.03 0.23 ± 0.02 0.21 ± 0.01 0.28 ± 0.03 Pulse rate (bpm) Pre-treatment 62 ± 3 54 ± 1 60 ± 2 63 ± 2 Treatment 59 ± 1 53 ± 1 58 ± 2 59 ± 2 Post-treatment 58 ± 2 53 ± 2 57 ± 2 58 ± 3

Internal iliac artery Diameter (mm) Pre-treatment 7.2 ± 0.2 7.7 ± 0.3 7.5 ± 0.2 7.4 ± 0.3 Treatment 7.2 ± 0.2 8.0 ± 0.2 7.4 ± 0.1 7.3 ± 0.2 Post-treatment 6.9 ± 0.2 7.7 ± 0.3 7.0 ± 0.2 7.2 ± 0.3 245 Peak systolic velocity (m/s)

Pre-treatment 0.97 ± 0.06 0.88 ± 0.05 0.94 ± 0.04 0.98 ± 0.05 Treatment 0.93 ± 0.04 0.94 ± 0.03 0.95 ± 0.03 0.99 ± 0.04 Post-treatment 0.98 ± 0.05 1.07 ± 0.06 0.95 ± 0.04 1.04 ± 0.07 Pulsatility index Pre-treatment 1.58 ± 0.10 1.56 ± 0.06 1.58 ± 0.04 1.61 ± 0.05 Treatment 1.50 ± 0.05 1.68 ± 0.05 1.53 ± 0.04 1.59 ± 0.04 Post-treatment 1.66 ± 0.08 1.79 ± 0.08 1.65 ± 0.07 1.62 ± 0.08 Resistivity index Pre-treatment 0.79 ± 0.01 0.76 ± 0.01 0.79 ± 0.01 0.77 ± 0.01 Treatment 0.78 ± 0.01 0.80 ± 0.01 0.80 ± 0.01 0.77 ± 0.01 Post-treatment 0.80 ± 0.01 0.82 ± 0.01 0.80 ± 0.01 0.78 ± 0.02 Mean arterial velocity (m/s) Pre-treatment 0.50 ± 0.03 0.43 ± 0.02 0.47 ± 0.02 0.47 ± 0.02 Treatment 0.49 ± 0.02 0.45 ± 0.01 0.51 ± 0.02 0.49 ± 0.02 Post-treatment 0.49 ± 0.02 0.50 ± 0.02 0.47 ± 0.02 0.51 ± 0.03 End diastolic velocity (m/s) Pre-treatment 0.20 ± 0.01 0.21 ± 0.01 0.19 ± 0.01 0.22 ± 0.01

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Treatment 0.19 ± 0.005 0.19 ± 0.01 0.19 ± 0.01 0.22 ± 0.01 Post-treatment 0.19 ± 0.01 0.19 ± 0.01 0.19 ± 0.01 0.22 ± 0.01 Pulse rate (bpm) Pre-treatment 61 ± 2xy 55 ± 2 60 ± 2 62 ± 2x Treatment 62 ± 1y 54 ± 2 59 ± 2 59 ± 2ay Post-treatment 60 ± 2x 55 ± 2 57 ± 2 60 ± 2x Repeated measures analysis of variance (SAS mixed proc) Superscripts xy indicate differences in columns (among periods within a treatment) and superscripts ab indicate differences in rows (among treatments for a given period). Values with uncommon alphabets are different at p≤0.05

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B.2. Calculated hemodynamic parameters of three arteries measured by Doppler ultrasonography in beef cows (n=4 per treatment group) fed increasing concentrations of ergot for one week. Arteries were imaged daily and data were compared by repeated measures mixed procedure. Data are presented as mean ± SEM for a given time period. Pair-wise comparisons were performed if the ANOVA p-value was ≤0.05 for the treatment (control, low, medium and high), period (pre-treatment, treatment, post-treatment) or treatment*period interaction term. Ergot Treatment Group Control Low Medium High (132 µg/kg DM) (529 µg/kg DM) (2115 µg/kg DM)

Caudal artery Volume per pulse (mL) Pre-treatment 2.2 ± 0.3x 2.3 ± 0.4 3.0 ± 0.3x 2.9 ± 0.5x Treatment 2.6 ± 0.3ay 2.6 ± 0.2 2.0 ± 0.2ay 1.8 ± 0.2ay Post-treatment 1.9 ± 0.2x 2.6 ± 0.3 2.1 ± 0.3x 1.9 ± 0.2x Blood flow (mL/min) 24 Pre-treatment 133 ± 22xy 125 ± 25 177 ± 20x 169 ± 26x 7 ay a by by Treatment 151 ± 14 138 ± 11 108 ± 8 100 ± 11 Post-treatment 104 ± 13x 137 ± 20 112 ± 15ay 109 ± 13ay

Median sacral artery Volume per pulse (mL) Pre-treatment 6.1 ± 0.6 8.5 ± 0.9 4.3 ± 0.3 4.5 ± 0.4 Treatment 7.1 ± 0.5 7.5 ± 0.6 4.7 ± 0.3 5.3 ± 0.5 Post-treatment 6.7 ± 0.6 7.4 ± 0.6 5.9 ± 0.8 6.0 ± 0.7 Blood flow (mL/min) Pre-treatment 364 ± 35 471 ± 52 259 ± 20 282 ± 23 Treatment 408 ± 24 404 ± 38 264 ± 18 296 ± 21 Post-treatment 387 ± 30 402 ± 39 325 ± 38 340 ± 36

Internal iliac artery Volume per pulse (mL)

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Pre-treatment 21.2 ± 2.4 22.3 ± 1.8 21.3 ± 1.4 20.2 ± 1.6 Treatment 20.5 ± 1.6 25.8 ± 1.3 22.7 ± 1.0 21.7 ± 1.4 Post-treatment 19.5 ± 1.9 25.6 ± 1.9 19.5 ± 1.2 21.8 ± 1.9 Blood flow (mL/min) Pre-treatment 1254 ± 116 1214 ± 94 1273 ± 91 1242 ± 86 Treatment 1240 ± 81 1365 ± 54 1310 ± 53 1256 ± 86 Post-treatment 1125 ± 86 1398 ± 103 1108 ± 71 1292 ± 130 Repeated measures analysis of variance (SAS mixed proc) Superscripts xy indicate differences in columns (among periods within a treatment) and superscripts ab indicate differences in rows (among treatments for a given period). Values with uncommon alphabets are different at p≤0.05

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Appendix C. Tabular presentation of measured and calculated hemodynamic variables from Chapter 3. C.1. Hemodynamic endpoints (mean ± SEM) of caudal artery and internal iliac artery measured by Doppler ultrasonography in beef cows (n=32) during the pre-treatment (2 weeks), treatment (9 weeks), and post-treatment (3 weeks) experimental periods to increasing concentrations of ergot alkaloids in their feed in Control, Low, Medium and High groups. Arteries were imaged weekly and data were compared by repeated measures mixed procedure.

Ergot Treatment Control Low Medium High (5 µg/kg DM*) (48 µg/kg DM) (201 µg/kg DM) (822 µg/kg DM) n 9 9 8 6 CAUDAL ARTERY Diameter (mm) p-values: Tx=0.287, EP<0.001, Tx*EP<0.001 Pre-treatment 3.8 ± 0.1a 3.7 ± 0.1a 3.9 ± 0.1ab 4.2 ± 0.1a Treatment 3.7 ± 0.04a 3.8 ± 0.05b 3.8 ± 0.04b 3.6 ± 0.1b Post-treatment 3.6 ± 0.05ap 3.8 ± 0.1bp 4.1 ± 0.1apq 4.2 ± 0.1aq

24 Bloodflow (mL/min) p-values: Tx=0.139, EP=0.003, Tx*EP<0.001

9 f abf ag ag

Pre-treatment 299 ± 23 284 ± 26 391 ± 41 415 ± 39 Treatment 288 ± 15 308 ± 16b 279 ± 14b 261 ± 14b Post-treatment 292 ± 22 236 ± 14a 347 ± 40ab 315 ± 28ab

Blood volume per pulse (mL) p-values: Tx=0.394, EP=0.141, Tx*EP<0.001 Pre-treatment 3.2 ± 0.2f 3.1 ± 0.3afg 4.4 ± 0.5ag 4.2 ± 0.4ag Treatment 3.8 ± 0.2x 4.2 ± 0.2by 3.9 ± 0.2bxy 3.0 ± 0.2bx Post-treatment 4.5 ± 0.3 3.7 ± 0.2ab 5.1 ± 0.5ab 4.3 ± 0.4ab

Mean velocity (m/s) p-values: Tx=0.889, EP=0.018 Tx*EP=0.027 Pre-treatment 0.44 ± 0.03 0.44 ± 0.04 0.53 ± 0.04a 0.49 ± 0.04a Treatment 0.43 ± 0.02 0.45 ± 0. 02 0.40 ± 0.02b 0.43 ± 0.02b

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Post-treatment 0.48 ± 0.03 0.36 ± 0.03 0.42 ± 0.03b 0.36 ± 0.02b

Peak systolic velocity (m/s) p-values: Tx=0.936, EP=0.164, Tx*EP<0.001 Pre-treatment 0.71 ± 0.04a 0.74 ± 0.05ab 0.84 ± 0.06a 0.80 ± 0.05a Treatment 0.80 ± 0.02ab 0.82 ± 0.03b 0.76 ± 0.03b 0.79 ± 0.02a Post-treatment 0.90 ± 0.04bp 0.75 ± 0.05apq 0.81 ± 0.04bpq 0.72 ± 0.03bq

End diastolic velocity (m/s) p-values: Tx=0.385, EP=0.028, Tx*EP=0.004 Pre-treatment 0.22 ± 0.03f 0.19 ± 0.02abf 0.28 ± 0.03ag 0.24 ± 0.02afg Treatment 0.20 ± 0.01 0.20 ± 0.01b 0.20 ± 0.01b 0.20 ± 0.01ab Post-treatment 0.25 ± 0.02p 0.15 ± 0.01aq 0.19 ± 0.02bpq 0.17 ± 0.01bpq

Pulse rate (bpm) p-values: Tx=0.008, EP=0.001, Tx*EP=0.371 Pre-treatment 94 ± 3 90 ± 3 91 ± 3 99 ± 4

2 Treatment 78 ± 2 74 ± 1 73 ± 1 86 ± 2 50

Post-treatment 66 ± 2 64 ± 2 69 ± 2 75 ± 3

Pulsatility index p-values: Tx=0.348, EP=0.004, Tx*EP=0.273 Pre-treatment 1.17 ± 0.06 1.27 ± 0.06 1.12 ± 0.07 1.19 ± 0.07 Treatment 1.46 ± 0.04 1.48 ± 0.04 1.49 ± 0.04 1.44 ± 0.05 Post-treatment 1.46 ± 0.07 1.70 ± 0.06 1.59 ± 0.08 1.55 ± 0.09

Resistivity index p-values: Tx=0.468, EP=0.145, Tx*EP=0.051 Pre-treatment 0.70 ± 0.03fg 0.74 ± 0.02f 0.67 ± 0.03ag 0.70 ± 0.02fg Treatment 0.75 ± 0.01 0.76 ± 0.01 0.75 ± 0.01b 0.75 ± 0.01 Post-treatment 0.73 ± 0.02 0.80 ± 0.01 0.77 ± 0.02b 0.76 ± 0.02

INTERNAL ILIAC ARTERY Diameter (mm) p-values: Tx=0.960, EP=0.503, Tx*EP=0.004 Pre-treatment 7.5 ± 0.4 7.3 ± 0.3 7.8 ± 0.3a 7.4 ± 0.2 Treatment 7.3 ± 0.1 7.1 ± 0.1 6.8 ± 0.2b 7.1 ± 0.1

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Post-treatment 7.2 ± 0.2 7.0 ± 0.2 7.3 ± 0.2ab 7.4 ± 0.3

Blood flow (mL/min) p-values: Tx=0.485, EP=0.104, Tx*EP=0.001 Pre-treatment 2417 ± 260f 2531 ± 363f 2805 ± 376ag 2148 ± 176abf Treatment 1773 ± 111 1808 ± 105 1696 ± 117b 1827 ± 117b Post-treatment 1380 ± 97 1325 ± 97 1409 ± 90b 1345 ± 105a

Blood volume per pulse p-values: Tx=0.687, EP=0.027, Tx*EP=0.089 (mL) Pre-treatment 26.7 ± 3.1 29.7 ± 4.8 31.7 ± 4.6 22.1 ± 1.5 Treatment 24.0 ± 1.4 24.7 ± 1.2 24.1 ± 1.5 23.4 ± 1.2 Post-treatment 22.8 ± 1.7 22.3 ± 1.6 21.6 ± 1.3 19.1 ± 1.4

Mean velocity (m/s) p-values: Tx=0.076, EP<0.001, Tx*EP=0.042 Pre-treatment 0.87 ± 0.04afg 0.92 ± 0.05af 0.91 ± 0.06ag 0.81 ± 0.04abfg 2 b a b b 51 Treatment 0.65 ± 0.02 0.71 ± 0.02 0.72 ± 0.02 0.74 ± 0.03

Post-treatment 0.55 ± 0.02cpq 0.55 ± 0.02bp 0.56 ± 0.03bq 0.51 ± 0.03apq

Peak systolic velocity (m/s) p-values: Tx=0.291, EP= 0.051, Tx*EP=0.010 Pre-treatment 1.46 ± 0.05 1.66 ± 0.08 1.54 ± 0.06ab 1.45 ± 0.06ab Treatment 1.41 ± 0.03x 1.56 ± 0.03xy 1.58 ± 0.04by 1.56 ± 0.06bxy Post-treatment 1.40 ± 0.05pq 1.49 ± 0.05p 1.39 ± 0.06apq 1.29 ± 0.06aq

End diastolic velocity (m/s) p-values: Tx=0.045, EP=0.001, Tx*EP=0.064 Pre-treatment 0.43 ± 0.05 0.38 ± 0.04 0.44 ± 0.04 0.33 ± 0.04 Treatment 0.28 ± 0.01 0.27 ± 0.01 0.32 ± 0.01 0.29 ± 0.02 Post-treatment 0.25 ± 0.02 0.19 ± 0.02 0.25 ± 0.02 0.19 ± 0.02

Pulse rate (bpm) p-values: Tx=0.003, EP<0.001, Tx*EP=0.045 Pre-treatment 94 ± 3a 87 ± 3a 91 ± 3a 96 ± 2a Treatment 74 ± 1bxy 72 ± 1ax 70 ± 1bxy 77 ± 2by

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Post-treatment 61 ± 2bpr 60 ± 2br 65 ± 1bpq 71 ± 3bq

Pulsatility index p-values: Tx=0.026, EP<0.001, Tx*EP=0.136 Pre-treatment 1.22 ± 0.09 1.40 ± 0.09 1.27 ± 0.07 1.43 ± 0.07 Treatment 1.85 ± 0.06 1.92 ± 0.06 1.81 ± 0.05 1.71 ± 0.06 Post-treatment 2.18 ± 0.09 2.48 ± 0.10 2.12 ± 0.08 2.19 ± 0.10

Resistivity index p-values: Tx=0.029, EP=0.002, Tx*EP=0.113 Pre-treatment 0.70 ± 0.03 0.76 ± 0.03 0.72 ± 0.02 0.77 ± 0.02 Treatment 0.80 ± 0.01 0.82 ± 0.01 0.79 ± 0.01 0.80 ± 0.01 Post-treatment 0.83 ± 0.01 0.87 ± 0.01 0.82 ± 0.01 0.85 ± 0.01

2 * DM = dry matter 52

Pair-wise comparisons were performed if the ANOVA p-value was ≤0.05 for the treatment (i.e., Tx; control, low, medium and high), experimental period (i.e., EP; pre-treatment, treatment, post-treatment) or treatment*experimental period interaction term (i.e., Tx*EP). Superscripts abc indicate differences in columns (among periods within a treatment). Superscripts fgh indicate differences in pre-treatment period rows; xyz indicate differences in treatment period rows; pqr indicate differences in post-treatment period rows (among treatments for a given period). Values with uncommon alphabets are different at p≤0.05

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Appendix D. Syntax for statistical analysis used in Chapter 3. ODS graphics on; Proc mixed covtest; class CowID Tx Week EP CalfMonth Pasture model Diameter = Tx|EP / DDFM=kr htype=3; random CalfMonth Temp_in Temp_out Pasture; repeated Week/subject=CowID(Tx) type=?? r rcorr; lsmeans Tx*EP / pdiff=all; run; ODS graphics off; quit; *Note: “??” in the syntax was replaced with the name of the covariance structure for model comparisons*

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Appendix E. Preparation of calibration curve and quality control samples (4.1) and quality control sample concentrations (4.2) for Chapter 4. E.1. Calibration curve for four ergot alkaloids in bovine plasma. Working standards were prepared by parallel dilution using a solution of 85/15 (%v/v) acetonitrile:water with 10 mM ammonium acetate. Samples were extracted by protein precipitation with acetonitrile. Working standard concentration Dilution Theoretical Final post-extraction standard (ng/mL) factor concentration (i.e., 20-fold dilution) ergocristine ergosine, ergocornine, ergocristine ergosine, ergocornine, ergocryptine ergocryptine 5000 2500 (stock #1) - 250 125 2500 1250 2 125 62.5 1250 625 4 62.5 31.3 625 312.5 8 31.3 15.6 312.5 156.3 16 15.6 7.8 156.3 78.1 (stock #2) 32 7.81 3.91 78.1 39.1 2 3.91 1.95 39.1 19.5 4 1.95 0.975

E.2. Partial validation Quality control (QC) sample concentration pre- and post-extraction with protein precipitation. Pre-extraction concentration (ng/mL) Post-extraction concentration (ng/mL) Quality ergocristine ergocornine, ergocristine ergocornine, control ergocryptine, ergocryptine, ergosine sample ergosine HQC 4000 2000 200 100 MQC 2400 1200 120 60 LQC 100 50 5 2.5 LLOQ 39 19.5 1.95 0.975

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Appendix F. Pharmacokinetic studies (Chapter 4) – additional information

F.1. Ergot alkaloid composition of treatment pellets used in two replicates of pharmacokinetic trials in cows. CV = coefficient of variation between the replicates Ergot alkaloid Replicate 1 Replicate 2 CV % Ergocornine 4065 1259 75 Ergocristine 12437 18983 29 Ergocryptine 4230 3115 21 Ergometrine 2155 76 132 Ergosine 1513 990 30 Ergotamine 4852 2860 37

TOTAL 29252 27283 5

F.2. Body weight and calculated body weight (BW) dose of total ergot alkaloids of beef cows (n=12) offered in a one-time high concentration pellet for a pharmacokinetic study. (Replicate 1). Cow # Body weight (kg) Weight of feed Body weight dose offered (kg) (µg/kg BW)* 60 540 4.66 250.1 73 515 4.4 247.6 5 474 4.1 250.8 94 552 4.76 250.3 156 658 5.68 250.2 153 531 4.6 251.4 9 711 6.1 248.8 186 590 5.1 250.7 187 681 5.9 251.2 57 729 6.28 249.9 Average ± SEM 598 ± 28 5.2 ± 0.2 250.1 ± 0.4 *based on total alkaloid concentration in pellets of 29000 µg/kg

Animals excluded from calculation due to feed error/non-consumption: 12* 470 8.90 549.9 87* 687 3.50 147.7

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F.3. Body weight and calculated body weight (BW) dose of total ergot alkaloids to lactating Hereford cows (n=9) offered in a one-time high concentration pellet for a pharmacokinetic study. (Replicate 2) Cow ID Body weight (kg) Weight of feed Body weight dose offered (kg) (µg/kg BW)* 29 685 6.3 250.2 72 640 5.9 250.2 99 723 6.6 250.2 111 746 6.8 250.2 55 782 7.2 250.2 1 701 6.4 250.2 181 821 7.5 250.2 198 699 6.4 250.2 90 567 5.2 250.2 Average ± SEM 707 ± 25 6.4 ± 0.2 250.2 *based on total ergot alkaloid concentration of 27300 µg/kg

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Appendix G. Preliminary matrix effects analysis for Chapter 4.

G.1. Preliminary quantification data from plasma and HPLC-grade water samples spiked with ergot alkaloids prior to extraction. Samples were extracted with protein precipitation. Analyte Quality Nominal Calculated plasma Calculated water control Concentration concentration (ng/mL) concentration (ng/mL) (ng/mL) Ergocornine LLOQ 0.977 0.92 ± 0.07 0.93 ± 0.07 LQC 2.5 2.43 ± 0.23 2.33 ± 0.09 MQC 60 56.2 ± 1.0 54.8 ± 0.78 HQC 100 90.7 ± 0.9 96.3 ± 1.9

Ergocristine LLOQ 1.95 1.82 ± 0.15 1.79 ± 0.18 LQC 5 4.67 ± 0.21 4.74 ± 0.16 MQC 120 114.4 ± 2.2 116.5 ± 5.86 HQC 200 184.8 ± 2.5 215.3 ± 5.0

Ergocryptine LLOQ 0.977 0.99 ± 0.08 0.95 ± 0.04 LQC 2.5 2.48 ± 0.14 2.41 ± 0.08 MQC 60 56.8 ± 1.1 56.4 ± 3.1 HQC 100 93.0 ± 1.4 103.1 ± 2.3

Ergosine LLOQ 0.977 1.1 ± 0.1 1.1 ± 0.03 LQC 2.5 2.5 ± 0.1 2.6 ± 0.1 MQC 60 56.8 ± 0.6 56.5 ± 2.9 HQC 100 92.4 ± 0.8 103.1 ± 2.3

The concentration data above were derived (by the analysis software) from chromatographic peak area. The peak area data from the above data in plasma represents the “area pre-spike” variable in the below equations. The peak area data from water represents the “area pure” variable. In order to complete the matrix effects portion of the method validation, I will need to extract a sample and spike it with ergot alkaloids after the extraction. Equations to be used to assess matrix (plasma) effects on quantitation of ergot alkaloids:

Extraction efficacy (%) = Areapre-spike x 100

Areapure

Extraction recovery (%) = Areapre-spike x 100

Areapost-spike

Matrix Factor (MF) = Areapost-spike

Areapure

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Appendix H. Solid phase extraction attempts for Chapter 4. Solid phase extraction was attempted in July and August 2016.

Ergot alkaloids were extracted from thawed plasma samples according to the method described by Salvador et al. with slight modifications. ThermoScientific Hypersep Retain CX columns were each preconditioned with 2 mL methanol followed by 2 mL of water. Plasma samples (3 mL) were spiked with 5% formic acid (0.2 mL formic acid per mL plasma). The sample was loaded onto the column and allowed to elute via gravity. We previously determined that use of a vacuum was too aggressive and may have pulled the alkaloids off the column. The column was rinsed with 2 mL of 5% formic acid (90% aqueous) followed by 2 mL methanol. The alkaloids were eluted in a new test tube with 1 mL of 97.5% methanol/2.5% ammonium hydroxide solution five times.

We attempted to quantify ergot alkaloids in the SPE samples using LC-MS and the existing method established by Prairie Diagnostic Services with a limited quantification curve. The quantification curve used in these preliminary analyses was three points: 0.1, 1.25, and 12.5 ng/mL. At the time, only four ergot alkaloids were being ran in PDS for routine analysis: ergosine, ergocornine, ergocryptine, and ergocristine. Unfortunately, there were issues with some of the standards in these runs thus reliable quantification could not be made. However, it was demonstrated that alkaloids could be detected in bovine plasma following consumption of highly contaminated feed.

H.1. Ergot alkaloid standard quantification information following solid phase extraction using ThermoScientific Hypersep Retain CX columns. Data in italics indicates sample failure. Ergot Alkaloid Nominal Analyte Peak Calculated Accuracy (%) Concentration Area (counts) Concentration (ng/mL) (ng/mL) Ergocornine 12.5 3.93E+02 12.5 100 1.25 4.13E+02 13.4 1070 0.1 1.16E+02 0.1 100

Ergocristine 12.5 1.24E+02 12.5 100 1.25 3.22E+02 55 4400 0.1 6.61E+01 0.1 100

Ergocryptine 12.5 9.92E+01 12.5 99.9 1.25 3.72E+01 1.42 114

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0.1 2.89E+01 < 0 N/A

Ergosine 12.5 4.45E+05 12.5 100 1.25 4.34E+04 1.28 103 0.1 5.79E+01 0.0715 71.5 The samples of two cows from replicate 1 (cow IDs 68 and 72 from summer 2016) were extracted for quantification. The chromatograms for the values listed below were not double- checked for having peaks.

H.2. Concentration of ergot alkaloids in cow plasma following a one-time high concentration exposure in feed. Samples were extracted by solid phase extraction. Ergot alkaloid concentration (ng/mL)

Time Ergocornine Ergocristine Ergocryptine Ergosine Total Ergot (min) Alkaloids 42 0 21.4 3.63 0.09 25.1 49 0 50.8 2.16 0.08 53.0 62 0 90.4 2.16 0.08 92.6 120 16.4 16 3.63 0.08 36.1 239 0.101 26.7 3.63 0.08 30.5 361 0 22.2 9.54 0.08 31.8 487 0 41.7 7.32 0.08 49.1 699 0 14.3 9.53 0.08 23.9 937 0 31.1 2.16 0.10 33.4 1161 21 0 18.4 0.12 39.5 1421 0 23.1 14 0.09 37.2 1892 0.65 24 5.11 0.10 29.9 2393 0 25.8 0 0.09 25.9 2949 4.4 12.5 2.16 0.08 19.1

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Appendix I. Breeding soundness evaluations for bulls used in ergot feeding trials.

I.9. Breeding soundness evaluation results of adult Angus bulls used in ergot feeding trials. Bulls will asterisks (*) were only used for the pilot study. Bulls evaluated in October were purchased later than the other bulls and were only used in the long-term feeding trial. Percentage of Morphological Defects ID Age SC Motil. Head Midp. Princip. LN LA PDr KAcr Normal BSE Class. Evaluation conducted April 27, 2017 257C 2 38.5 VG 2 2 0 0 0 2 0 94 Sat 134C 2 39 VG 1 1 0 0 0 1 0 98 Sat 15C 2 37 VG 0 2 0 0 0 0 0 98 Sat 203B 3 40 VG 0 11 1 0 1 0 1 86 Sat

37C 2 42 Good 1 5 0 0 1 2 0 89 Sat 2 60 551C 2 37 VG 2 10 0 3 0 2 0 83 Sat 824B* 3 40.5 VG 3 1 0 0 0 3 0 93 Sat 766C 2 38 VG 1 0 0 0 0 0 0 99 Sat 122C* 2 37 Good 4 7 0 1 3 3 0 85 Sat 814B 3 41 VG 0 1 1 0 1 0 0 97 Sat 967B 3 40 VG 1 16 3 0 0 0 0 81 Sat 79Z 5 44 VG 4 1 0 3 0 0 0 93 Sat 3B* 2 35 Good 14 6 0 1 1 1 0 79 Sat 326Z 5 38 VG 5 5 0 1 0 0 0 90 Sat

Evaluation conducted December 4, 2017 92Z 5 39.5 F 3 1 - 5 3 - - 88 Sat 817C 2 40.5 G 2 7 3 1 - - - 82♯ Sat 886C 2 40.5 G 4 5 2 14 3 1 - 72 Sat SC = scrotal circumference; PDr = proximal droplets; KAcr = knobbed acrosomes; Sat = satisfactory; VG = very good; F = fair ♯ 5% diadems recorded under ‘other’ category for defects

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Appendix J. Alternate feed analysis for ergot alkaloid concentration. Analysis of wheat screenings and pellet samples was conducted by the Missouri University Veterinary Medical Diagnostic Laboratory and Romer Labs.

J.1 Ergot alkaloid composition and concentration (µg/kg) of ergotized wheat screenings and ergotized pellets. Samples were Analyzed by high performance liquid chromatography (HPLC) by the Missouri University Veterinary Medical Diagnostic Laboratory (Columbia, MO, USA) Sample ID Ergot alkaloid Composite sample Composite Individual Individual Wheat screenings 1 Wheat screenings 2 1 sample 2 bag 1 (2) bag 2 (14) Ergosine 120 125 135 130 26355 21815 Ergotamine 470 580 435 315 90155 69680 Ergocornine 260 160 190 150 52275 41900 Ergocryptine 320 275 265 280 67400 56925 Ergocristine 1235 1600 1180 995 280920 227185

SUM 2405 2740 2205 1870 517105 417505

AVERAGE 2573 ± 237 2038 ± 237 467305 ± 70428 2 61

J.2. Ergot alkaloid and corresponding -inine epimer composition and concentration (µg/kg) of ergotized screenings and pellets used for a long-term ergot feeding trial in adult bulls. The ergot concentration in the pellets was targeted based off the results of the screenings. Samples were analyzed by LC-MS-MS by ROMER LABS Diagnostic GmBH – Europe (Tulln, Austria, EU). Ergot alkaloid Pellets Screenings -Ergocryptine 67 ± 12 91815 ± 9182 Ergocornine 149 ± 26.7 51190 ± 5119 Ergocristine 306 ± 55.2 254740 ± 25474 261

Ergometrine 125 ± 22.5 48400 ± 4840 Ergosine 55 ± 10 28895 ± 2890 Ergotamine 189 ± 33.9 72160 ± 7216

Parent compound sum 891 547200

-Ergocryptinine 57 ± 10.3 85490 ± 8549 Ergocorninine 70 ± 12.6 70635 ± 7064 Ergocristinine 101 ± 18.2 312150 ± 31215 Ergometrinine 30 ± 6.1 8305 ± 997 Ergosinine 21 ± 4.3 32435 ± 3244 Ergotaminine 53 ± 9.6 84355 ± 8436 Epimer total 332 593370

Parent compound and epimer sum 1223 1140570

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Appendix K. Flow cytometric rationale and endpoints. Sperm with intact plasma membranes remained unstained and were considered viable. Propidium iodide binds to DNA in the nucleus of sperm when plasma membrane integrity is lost (Glazer 2014), therefore sperm with high PI (red) fluorescence were considered nonviable (i.e., dead). The FITC-PNA stain was used to assess acrosome integrity. Peanut (Arachis hypogea) agglutinin (PNA) is a lectin that binds to the inner membrane of the acrosome when the membrane integrity is compromised (Glazar 2014), suggestive of acrosome reaction. Intact acrosomal membranes will not allow passage of FITC tagged PNA through the membrane and, therefore, will not fluoresce. Intact acrosomes on spermatozoa are required for proper fertilization to occur, thus high proportions of sperm in which acrosome reaction has already occured can have negative impacts on breeding. The midpiece of mammalian spermatozoa contain clusters of mitochondria that are responsible for cellular energy production and

motility. Spermatozoa with high viability and motility are considered to have high mitochondrial membrane potential (MMP). Sperm

2 63

motility is correlated with mitochondrial membrane potential, thus low viability, nonmotile sperm tend to have low MMP (Amara l et al. 2013). K.1. Definitions of quadrants used for triple-stain flow cytometric assessment of bull sperm. Quadrant Title Definition Comparison: propidium iodide versus FITC-PNA QB1 CPM + IACR compromised plasma membrane + intact acrosomes QB2 CPM + RACR compromised plasma membrane + acrosome reacted QB3 IPM + IACR intact plasma membrane + intact acrosome QB4 IPM + RACR intact plasma membrane + acrosome reacted

Comparison: propidium iodide versus mitotracker deep red Q1 CPM + L and MMMP compromised plasma membrane + low and medium mitochondrial membrane potential

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Q2 CPM + HMMP compromised plasma membrane + high mitochondrial membrane potential Q3 IPM + L and MMMP intact plasma membrane + other than high mitochondrial membrane potential (i.e., low and medium) Q4 IPM + HMMP intact plasma membrane + high mitochondrial membrane potential QA1 CPM + LMMP compromised plasma membrane + low mitochondrial membrane potential QA2 CPM + M and HMMP compromised plasma membrane + other than low mitochondrial membrane potential (i.e., medium and high) QA3 IPM + LMMP intact plasma membrane + low mitochondrial membrane potential QA4 IPM + M and HMMP intact plasma membrane + other than low mitochondrial membrane potential (i.e., medium and high) QA4-Q4 IPM + MMMP intact plasma membrane + medium mitochondrial membrane potential QA2-Q2 CPM + MMMP compromised plasma membrane + medium mitochondrial membrane potential

Formulae for total cell populations associated with each stain 2

64 QA1+QA3 Total LMMP Total sperm with low mitochondrial membrane potential (i.e., intact and compromised plasma membranes) Q2+Q4 Total HMMP Total sperm with high mitochondrial membrane potential (QA4+QA2)-(Q2+Q4) Total MMMP Total sperm with medium mitochondrial membrane potential QB1+QB2 Total CPM Total sperm with compromised plasma membranes 100-(QB1+QB2) Total IPM Total sperm with intact plasma membranes QB1+QB3 Total IACR Total intact acrosomes sperm 100-(QB1+QB3) Total RACR Total acrosome reacted sperm

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Appendix L. Syntax used for statistical analysis for Chapter 5. For comparisons by experimental period and treatment:

ODS graphics on; Proc mixed covtest; class BullID Tx Week EP Date_of_Collection; model Motility = Tx|EP / DDFM=kr htype=3; random Temp_in Temp_out Date_of_Collection; repeated Week/subject=BullID type=?? r rcorr; lsmeans Tx*EP / pdiff=all; run; ODS graphics off; quit;

For comparisons by week of experiment and treatment: Proc mixed covtest; class BullID Tx Week Date_of_Collection; model Motility = Tx|Week / DDFM=kr htype=3; random Temp_in Temp_out Date_of_Collection; repeated Week/subject=BullID type=?? r rcorr; lsmeans Tx*Week / pdiff=all; run; *Note: “??” in the syntax was replaced with the name of the covariance structure for model comparisons*

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Appendix M. Tabular results of experimental data from Chapter 5. M.1. Plasma prolactin concentration, weight, rectal temperature, and scrotal circumference (mean ± standard error) in adult Angus bulls (n=14) exposed to ergot alkaloids in their feed. The experiment consisted of a pre-treatment (12 weeks), treatment (10 weeks), and post- treatment (10 weeks) period. Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). Group data were analyzed by repeated measures in the SAS mixed procedure testing for main effects of treatment (Tx) and experimental period (EP) and their interaction (Tx*EP). Ergot Treatment Low (1113 µg/kg) High (2227 µg/kg) Plasma prolactin (ng/mL) P-values: Tx=0.90, EP<0.01, Tx*EP=0.48 Pre-treatment 65.9 ± 7.2 63.5 ± 8.9 Treatment 37.0 ± 4.5 29.9 ± 5.1 Post-treatment 106 ± 13 84.4 ± 5.1

Weight (lbs) P-values: Tx=0.73, EP<0.01, Tx*EP=0.53 Pre-treatment 1949 ± 45 2036 ± 24 Treatment 2116 ± 40 2154 ± 24 Post-treatment 2184 ± 35 2253 ± 21

Rectal temperature (°C) P-values: Tx=0.23, EP<0.01, Tx*EP=0.39 Pre-treatment 37.7 ± 0.1 37.6 ± 0.1 Treatment 38.4 ± 0.1 38.0 ± 0.1 Post-treatment 38.6 ± 0.1 38.2 ± 0.1

Scrotal circumference (cm) P-values: Tx=0.21, EP=0.07, Tx*EP=0.05 Pre-treatment 40.8 ± 0.3a 40.1 ± 0.2 Treatment 41.6 ± 0.4b 40.0 ± 0.3 Post-treatment 40.6 ± 0.4ab 39.2 ± 0.3 Tx = treatment; EP = experimental period; Tx*EP = interaction Each value represents mean ± SEM Superscripts “a,b” indicate differences within columns. Values with uncommon alphabets are different at P≤ (=0.05).

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M.2. Sperm production and motility parameters of adult Angus bulls (n=14) exposed to ergot alkaloids in their feed. Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). The experiment consisted of a pre-treatment (12 weeks, i.e., 84 days), treatment (9 weeks, i.e., 61 days), and post-treatment (10 weeks, i.e., 68 days) period. Bulls were group-fed ergot alkaloids in their total mixed ration daily for the treatment period. Semen was collected by electroejaculation every two weeks and samples were analyzed by a Computer Assisted Sperm Analyzer system on the same day of collection. Group data were analyzed by repeated measures in the SAS mixed procedure testing for main effects of treatment (Tx) and experimental period (EP) as well as the interaction (Tx*EP). Ergot Treatment Low (1113 µg/kg) High (2227 µg/kg) n: 8 6 Sperm concentration (x109/mL) P-values: Tx=0.52, EP=0.48, Tx*EP=0.36 Pre-treatment 0.64 ± 0.07 0.79 ± 0.07 Treatment 0.59 ± 0.08 0.63 ± 0.06 Post-treatment 0.56 ± 0.05 0.67 ± 0.09

Ejaculate volume (mL) P-values: Tx=0.11, EP=0.27, Tx*EP=0.22 Pre-treatment 8.4 ± 0.6 11.2 ± 0.9 Treatment 7.8 ± 0.6 13.7 ± 1.7 Post-treatment 6.9 ± 0.6 12.3 ± 1.5

Sperm production (x109)* P-values: Tx=0.01, EP=0.84, Tx*EP=0.90 Pre-treatment 5.6 ± 0.8 8.9 ± 1.0 Treatment 4.7 ± 0.7 8.5 ± 1.3 Post-treatment 4.2 ± 0.5 8.8 ± 1.4

Total sperm motility (%) P-values: Tx=0.61, EP=0.11, Tx*EP=0.12 Pre-treatment 63.1 ± 1.8 68.6 ± 2.2 Treatment 62.4 ± 2.0 61.2 ± 2.4 Post-treatment 65.4 ± 1.8 65.4 ± 2.0

Progressive sperm motility (%) P-values: Tx=0.55, EP=0.29, Tx*EP=0.05 Pre-treatment 57.0 ± 1.9 64.0 ± 2.3a Treatment 58.0 ± 2.1 56.9 ± 2.4b Post-treatment 60.0 ± 2.1 60.1 ± 2.1ab *Sperm production = sperm concentration x ejaculate volume Tx = treatment; EP = experimental period; Tx*EP = interaction Superscripts “ab” indicate differences within columns. Values with uncommon alphabets are different at P≤ (=0.05).

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M.3. Flow cytometric comparisons of sperm populations assessed for plasma membrane integrity and mitochondrial membrane potential of semen from adult Angus bulls (n=14) exposed to ergot alkaloids in their feed. Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). The experiment consisted of a pre-treatment (6 weeks), treatment (9 weeks), and post-treatment (10 weeks) period. Data are presented as percentage of total gated cells and mean ± standard error. Sperm that were negative for propidium iodide (PI) fluorescence were considered to have intact plasma membranes and are denoted as IPM. Conversely, cells that exhibited propidium iodide fluorescence were considered to have compromised plasma membranes (CPM). Mitochondrial membrane potential was assessed at three levels/categories based on Mitotracker Deep Red fluorescence. Flow cytometric comparison Ergot treatment Propidium Mitotracker DR Experimental period Low (1113 µg/kg) High (2227 µg/kg) iodide Negative High MMP P-values: Tx=0.74, EP<0.01, Tx*EP=0.83 (IPM) Pre-treatment 17.3 ± 0.9 15.5 ± 0.9 Treatment 16.0 ± 0.9 13.9 ± 0.9 Post-treatment 12.5 ± 2.0 12.3 ± 2.2

Medium MMP P-values: Tx=0.39, EP<0.01, Tx*EP=0.48 Pre-treatment 30.5 ± 1.8 27.4 ± 1.7 Treatment 27.8 ± 1.6 22.3 ± 1.2 Post-treatment 33.3 ± 2.3 31.5 ± 2.5

Low MMP P-values: Tx=0.30, EP=0.14, Tx*EP=0.56 Pre-treatment 14.2 ± 0.8 12.8 ± 1.0 Treatment 20.2 ± 1.8 17.8 ± 2.1 Post-treatment 12.5 ± 0.8 13.1 ± 1.5

Positive High MMP P-values: Tx=0.37, EP<0.01, Tx*EP=0.24 (CPM) Pre-treatment 11.2 ± 1.2 12.5 ± 0.8 Treatment 10.0 ± 0.8 12.7 ± 0.8 Post-treatment 15.6 ± 1.6 16.1 ± 2.3

Medium MMP P-values: Tx=0.01, EP=0.14, Tx*EP=0.40 Pre-treatment 19.8 ± 1.6 24.1 ± 2.0 Treatment 16.8 ± 1.4 22.9 ± 2.0 Post-treatment 20.2 ± 1.9 21.4 ± 2.4

Low MMP P-values: Tx=0.79, EP<0.01, Tx*EP=0.60 Pre-treatment 7.0 ± 0.5 7.7 ± 0.5 Treatment 9.1 ± 0.9 10.4 ± 1.0

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Post-treatment 6.0 ± 0.8 5.7 ± 0.7 IPM = intact plasma membrane; CPM = compromised plasma membrane; MMP = mitochondrial membrane potential; Tx=treatment; EP=experimental period Statistical significance was considered at P≤ (=0.05).

M.4. Flow cytometric comparisons of sperm populations assessed for plasma membrane integrity and acrosomal membrane integrity. Data are presented as percentage of total gated cells and mean ± standard error. Sperm that were negative for propidium iodide (PI) fluorescence were considered to have intact plasma membranes and are denoted as IPM. Conversely, cells that exhibited propidium iodide fluorescence were considered to have compromised plasma membranes (CPM). Sperm that had low-to- negative FITC-PNA fluorescence were considered acrosomal intact (I.e., intact acrosome or IACR) and cells with high fluorescence were considered to have ruptured/compromised acrosomes (i.e., RACR). Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). The experiment consisted of a pre-treatment (6 weeks), treatment (9 weeks), and post-treatment (10 weeks) period. Ergot Treatment Flow cytometric comparison Experimental Low (1113 µg/kg) High (2227 µg/kg) period Propidium FITC-PNA P-values: Tx=0.48, EP=0.23, Tx*EP=0.47 iodide Negative (IPM) Negative (IACR) Pre-treatment 60.8 ± 2.8 54.6 ± 3.1 Treatment 62.2 ± 2.3 53.4 ± 2.3 Post-treatment 62.0 ± 2.3 60.8 ± 3.0

Negative (IPM) Positive (RACR) P-values: Tx=0.29, EP<0.01, Tx*EP=0.16 Pre-treatment 1.4 ± 0.17 0.92 ± 0.12 Treatment 1.2 ± 0.11 1.22 ± 0.09 Post-treatment 0.37 ± 0.03 0.44 ± 0.05

Positive (CPM) Negative (IACR) P-values: Tx=0.09, EP<0.01, Tx*EP=0.15 Pre-treatment 19.2 ± 1.8 23.2 ± 1.8 Treatment 18.9 ± 1.2 22.4 ± 1.5 Post-treatment 17.9 ± 1.1 18.1 ± 1.5

Positive (CPM) Positive (RACR) P-values: Tx=0.85, EP=0.99, Tx*EP=0.53 Pre-treatment 18.7 ± 2.1 21.3 ± 2.4 Treatment 17.7 ± 1.5 23.0 ± 2.0 Post-treatment 19.8 ± 1.5 20.7 ± 1.8

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IPM = intact plasma membrane; CPM = compromised plasma membrane; IACR = intact acrosomes; RACR = reacted acrosomes; Tx = treatment; EP = experimental period Statistical significance was considered at P≤ (=0.05).

M.5. Total cell populations from assessments of plasma membrane integrity, mitochondrial membrane potential, and acrosomal membrane integrity in fresh semen from adult Angus bulls (n=14) exposed to ergot alkaloids in their feed. Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). The experiment consisted of a pre-treatment (6 weeks), treatment (9 weeks), and post-treatment (10 weeks) period. Bulls were group-fed ergot alkaloids in their total mixed ration daily for the treatment period. Semen was collected by electroejaculation every two weeks and samples were analyzed by flow cytometry. Data are presented as percentage of total percentage of gated cells and mean ± standard error. Flow cytometric Ergot Treatment Sperm population characteristics Low (1113 µg/kg) High (2227 µg/kg) % intact plasma membranes P-values: Tx=0.48, EP=0.29, Tx*EP=0.47 Pre-treatment 62.1 ± 2.8 55.6 ± 3.1 Treatment 63.5 ± 2.2 54.6 ± 2.3 Post-treatment 62.4 ± 2.3 61.2 ± 3.0

% compromised plasma membranes P-values: Tx=0.48, EP=0.29, Tx*EP=0.47 Pre-treatment 37.9 ± 2.8 44.4 ± 3.1 Treatment 36.6 ± 2.2 45.4 ± 2.3 Post-treatment 37.6 ± 2.3 38.8 ± 3.0

% high MMP P-values: Tx=0.91, EP=0.13, Tx*EP=0.99 Pre-treatment 28.4 ± 1.4 28.0 ± 0.8 Treatment 26.0 ± 1.0 26.6 ± 1.3 Post-treatment 28.1 ± 2.1 28.4 ± 2.3

% medium MMP P-values: Tx=0.49, EP<0.01, Tx*EP=0.51 Pre-treatment 50.3 ± 1.3 51.6 ± 0.9 Treatment 44.6 ± 1.7 45.2 ± 2.0 Post-treatment 53.5 ± 2.1 52.8 ± 1.9

% low MMP P-values: Tx=0.57, EP=0.15, Tx*EP=0.96 Pre-treatment 21.2 ± 0.7 20.4 ± 0.9 Treatment 29.3 ± 2.3 28.3 ± 2.7 Post-treatment 18.5 ± 1.0 18.8 ± 1.6

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% intact acrosomes P-values: Tx=0.89, EP=0.99, Tx*EP=0.53 Pre-treatment 80.0 ± 2.2 77.8 ± 2.5 Treatment 81.1 ± 1.6 75.8 ± 2.0 Post-treatment 79.9 ± 1.5 78.8 ± 1.8

% compromised acrosomes P-values: Tx=0.89, EP=0.99, Tx*EP=0.53 Pre-treatment 20.1 ± 2.2 22.2 ± 2.5 Treatment 18.9 ± 1.6 24.2 ± 2.0 Post-treatment 20.1 ± 1.5 21.2 ± 1.8 Tx = treatment; EP = experimental period; Tx*EP = interaction Statistical significance was considered at P≤ (=0.05).

M.6. Morphology of sperm collected from adult Angus bulls (n=14) exposed to ergot alkaloids in their feed. Bulls were randomly sorted into two treatment groups: low ergot (n=8) and high ergot (n=6). The experiment consisted of a pre-treatment (12 weeks), treatment (9 weeks), and post-treatment (10 weeks) period. Bulls were group-fed ergot alkaloids in their total mixed ration daily for the treatment period. Sperm smears were prepared using eosin- nigrosin stain and fresh semen every two weeks following semen collection. Data presented are percentage of total cells counted and mean ± standard error. Ergot treatment Morphological endpoint Low (1113 µg/kg) High (2227 µg/kg) % Normal P-values: Tx=0.58, EP=0.12, Tx*EP=0.64 Pre-treatment 71.8 ± 2.8 83.0 ± 2.1 Treatment 71.7 ± 3.1 82.3 ± 1.8 Post-treatment 74.7 ± 2.4 82.2 ± 1.8

% Head Defects P-values: Tx=0.67, EP=0.16, Tx*EP=0.86 Pre-treatment 4.1 ± 0.5 3.9 ± 0.6 Treatment 4.3 ± 0.6 4.7 ± 0.9 Post-treatment 5.3 ± 0.7 5.2 ± 0.9

% Midpiece Defects P-values: Tx=0.58, EP=0.53, Tx*EP=0.27 Pre-treatment 15.2 ± 2.4 7.2 ± 1.5 Treatment 13.0 ± 2.5 7.3 ± 0.9 Post-treatment 11.2 ± 2.1 6.3 ± 0.8

% Principle Piece Defects P-values: Tx=0.83, EP=0.87, Tx*EP=0.88 Pre-treatment 2.4 ± 0.7 1.2 ± 0.3 Treatment 2.7 ± 0.9 0.8 ± 0.2 Post-treatment 1.1 ± 0.3 0.5 ± 0.1

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% Proximal droplets P-values: Tx=0.09, EP=0.31, Tx*EP=0.33 Pre-treatment 0.8 ± 0.1 1.3 ± 0.2 Treatment 1.9 ± 1.0 1.3 ± 0.2 Post-treatment 1.0 ± 0.2 1.9 ± 0.3

% Acrosome defects P-values: Tx<0.01, EP=0.45, Tx*EP=0.64 Pre-treatment 1.1 ± 0.4 0.4 ± 0.1 Treatment 0.6 ± 0.2 0.3 ± 0.1 Post-treatment 1.5 ± 0.6 0.3 ± 0.1

% Normal detached heads P-values: Tx=0.94, EP=0.14, Tx*EP=0.67 Pre-treatment 4.0 ± 0.8 2.3 ± 0.4 Treatment 5.0 ± 1.0 2.5 ± 0.4 Post-treatment 4.1 ± 0.8 2.2 ± 0.3

% Abnormal detached P-values: Tx=0.48, EP=0.87, Tx*EP=0.90 heads Pre-treatment 0.7 ± 0.2 0.8 ± 0.3 Treatment 0.9 ± 0.2 1.0 ± 0.4 Post-treatment 1.0 ± 0.3 1.2 ± 0.4

% Live (dye exclusion) P-values: Tx=0.86, EP<0.01, Tx*EP=0.32 Pre-treatment 71.2 ± 2.0 71.8 ± 2.7 Treatment 66.5 ± 1.9 62.5 ± 2.9 Post-treatment 71.6 ± 1.7 71.4 ± 2.0 Tx = treatment; EP = experimental period; Tx*EP = interaction Statistical significance was considered at P≤ (=0.05).

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