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Studies on the mycotoxin zearalenone -- Barley zearalenone contamination survey and In Vitro effects… Wallace, Christa Jeanne 1991

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STUDIES ON THE MYCOTOXIN ZEARALENONE: BARLEY ZEARALENONE CONTAMINATION SURVEY AND IN VITRO EFFECTS OF ZEARALENONE ON OOCYTES AND PRE-IMPLANTATION EMBRYOS by CHRISTA JEANNE WALLACE B.Sc. Hon. (Agr.), University of B r i t i s h Columbia, 1988 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH A p r i l , 1991 Christa Jeanne Wallace, COLUMBIA 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PKM\,HP>\- ScACtOC^r The University of British Columbia Vancouver, Canada Date fivPEAL. SL*\ v W \ DE-6 (2/88) i i ABSTRACT The mycotoxin zearalenone i s known for i t s harmful e f f e c t s on l i v e s t o c k reproduction. Animal exposure occurs through feed sources colonized by Fusarium species which produce the mycotoxin. Since regular screening procedures for zearalenone are not conducted on Western Canadian barley, a survey was c a r r i e d out to t e s t for possible s i g n i f i c a n t l e v e l s of contamination. A l l samples were found to be negative at a detection l e v e l of 500 ppb; therefore, feeds formulated from the barley samples sources would not l i k e l y cause zearalenone t o x i c o s i s problems i n livestock. Also, an ELISA method, Agri-Screen™, developed by Neogen Corporation (Lansing, Michigan) was tested and found to be a simple and economical method for pre-screening of feed samples i n the f i e l d . To study d i r e c t t o x i c o l o g i c a l e f f e c t s of zearalenone on i n v i t r o murine blastocyst development, murine embryos were cultured i n medium (Ham's F-10 + estrous cow serum) containing various l e v e l s of the mycotoxin. The c r i t i c a l concentration range for zearalenone to cause detrimental e f f e c t s on blastocyst development was determined to be between 70-160 /ig/ml medium. Add i t i o n a l l y , a concentration e f f e c t on the length of time required to exert deleterious actions was demonstrated. At mycotoxin concentrations of 500 /ug/ml medium and above, blastocysts degenerated a f t e r 6 h of culture. At a lower i i i concentration l e v e l of 160 jug/ml, blastocysts were not affected u n t i l 28 h of culture. In order to investigate the d i r e c t t o x i c o l o g i c a l e f f e c t s of zearalenone on i n v i t r o porcine pre-implantation embryo development, attempts were made to develop a successful culture system. Since a suitable system was not developed, t o x i c o l o g i c a l studies were not possible. Possibly, steps i n the recovery process could have resulted i n detrimental e f f e c t s before the embryos were placed i n culture. A l t e r n a t i v e l y , the media chosen (Ham's F-10 + estrous cow serum; Minimum es s e n t i a l medium + f e t a l c a l f serum) may not be suitable for i n v i t r o culture of porcine pre-implantation embryos. F i n a l l y , at a zearalenone concentration l e v e l (250 /xg/ml medium) found to cause degeneration of murine blastocysts, the in v i t r o maturation of bovine oocytes i n Tissue Culture Medium 199 was not affected. I t was suggested that the surrounding cumulus layer acts as a b a r r i e r to prevent the mycotoxin from d i r e c t l y acting on the oocyte. i v TABLE OF CONTENTS page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF APPENDICES i x LIST OF ABBREVIATIONS AND SYMBOLS X ACKNOWLEDGEMENT x i i 1. 0 LITERATURE REVIEW 1-20 1.1. INTRODUCTION 1 1.2. DISCOVERY 1 1.3. CHEMICAL AND PHYSICAL PROPERTIES 3 1.4. BIOSYNTHESIS 5 1.5. DERIVATIVES 7 1.6. FUNGAL ESTABLISHMENT AND ZEARALENONE PRODUCTION 7 1.7. METABOLISM OF ZEARALENONE BY ANIMALS 9 1.8. MECHANISM OF ACTION OF ZEARALENONE IN ANIMALS 14 1.9. TOXICOLOGICAL EFFECTS OF ZEARALENONE IN ANIMALS 17 2.0 SURVEY OF BARLEY FROM ALBERTA AND BRITISH COLUMBIA GRAIN ELEVATORS FOR ZEARALENONE CONTAMINATION 21-34 2.1. INTRODUCTION 21 2.1.1. RESULTS OF PREVIOUS SURVEYS FOR ZEARALENONE CONTAMINATION. . 21 2.1.2. CONVENTIONAL METHODS OF ZEARALENONE DETERMINATION IN FEED 24 V 2.1.3. IMMUNOCHEMICAL TECHNIQUES FOR ZEARALENONE DETERMINATION 26 2.1.4. AGRI-SCREEN™ FIELD TEST KIT FOR ZEARALENONE DETERMINATION 27 2.2. OBJECTIVES FOR BARLEY ZEARALENONE CONTAMINATION SURVEY 28 2.3. MATERIALS AND METHODS 29 2.3.1. Sample C o l l e c t i o n , Storage and P r e p a r a t i o n 29 2.3.2. E x t r a c t i o n Procedure 29 2.3.3. Q u a l i t a t i v e A n a l y s i s 29 2.4. RESULTS 31 2.5. DISCUSSION 31 3.0. STUDIES ON THE DIRECT EFFECTS OF ZEARALENONE ON EMBR YON ICMORTALIT Y 35-68 3.1. INTRODUCTION ...35 3.2. PRIMARY OBJECTIVE OF STUDIES 37 3.3. PRELIMINARY STUDIES ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO MURINE PRE-IMPLANTATION EMBRYO DEVELOPMENT 37 3.3.1. OBJECTIVE OF PRELIMINARY STUDIES 37 3.3.2. MATERIALS AND METHODS 37 3.3.3. RESULTS 42 3.3.4. DISCUSSION 43 3.4. STUDIES ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO MURINE PRE-IMPLANTATION EMBRYO DEVELOPMENT 4 3 3.4.1. INTRODUCTION 4 3 3.4.2. MATERIALS AND METHODS 44 3.4.3. RESULTS 49 3.4.4. DISCUSSION 66 v i 3.5. DEVELOPMENT OF A PORCINE PRE-IMPLANTATION EMBRYO CULTURE SYSTEM 69 3.5.1. INTRODUCTION 69 3.5.2. MATERIALS AND METHODS 69 3.5.3. RESULTS 72 3.5.4. DISCUSSION 77 4.0 STUDY ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO BOVINE OOCYTE MATURATION 79-87 4.1. INTRODUCTION 79 4.2. OBJECTIVE OF STUDY 80 4.3. MATERIALS AND METHODS 80 4.3.1. C u l t u r e Medium P r e p a r a t i o n 80 4.3.2. Ovary R e t r i e v a l from the slaughterhouse 81 4.3.3. Oocyte Recovery 81 4.3.4. Washing of Oocytes 81 4.3.5. In c u b a t i o n and Oocyte M a t u r a t i o n 81 4.3.6. S t a i n i n g Procedure 82 4.3.7. M a t u r a t i o n E v a l u a t i o n . . 8 3 4.4. RESULTS 85 4.5. DISCUSSION 87 5.0 GENERAL DISCUSSION 88-90 BIBLIOGRAPHY 91 APPENDICES 108 LIST OF TABLES Table page 1. B a r l e y sample sources 3 0 2. P r e l i m i n a r y Study 1: e f f e c t o f zearalenone on i n v i t r o murine b l a s t o c y s t development 42 3. P r e l i m i n a r y Study 2: e f f e c t of zearalenone on i n v i t r o murine b l a s t o c y s t development 42 4. Summary of zearalenone c o n c e n t r a t i o n f o r a l l t h r e e s t u d i e s 47 5. Development stage a l p h a b e t i c a l s c o r i n g . . . 48 6. Study 1: summary of murine b l a s t o c y s t development 54 7. Study 2: summary of murine b l a s t o c y s t development 58 8. Study 3: summary of murine b l a s t o c y s t development 62 9. Reproductive t r a c t e v a l u a t i o n o f g i l t s 73 10. Oocyte and embryo recover y from g i l t r e p r o d u c t i v e t r a c t s 74 11. P o r c i n e morulae c u l t u r e r e s u l t s 75 v i i i LIST OF FIGURES F i g u r e page I. Zearalenone and i t s i s o m e r i c m e t a b o l i t e s 10 2 (A-F). Study 1: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 51 3 (A-F). Study 2: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 55 4 (A-F). Study 3: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 59 5. E a r l y murine b l a s t o c y s t 63 6. Mature murine b l a s t o c y s t 63 7. Expanded murine b l a s t o c y s t 64 8. Hatching murine b l a s t o c y s t 64 9. Hatching murine b l a s t o c y s t 65 10. Degenerating murine b l a s t o c y s t 65 I I . Degenerating p o r c i n e morulae 76 12. Cumulus c e l l expansion of bovine oocyte 84 13. Naked bovine oocyte 84 14. Metaphase I I chromosome c o n f i g u r a t i o n of a mature bovine oocyte 8 6 i x LIST OF APPENDICES Appendix page 1. B a r l e y zearalenone contamination survey r e s u l t s 108 2. Dulbecco's phosphate-buffered s a l i n e (PBS) composition... 110 3. Ham's F-10 n u t r i e n t mixture - Powder form composition.... I l l 4. UBC human i n v i t r o f e r t i l i z a t i o n program Ham's F-10 p r e p a r a t i o n p r o t o c o l 113 5 (A-F). Study 1: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 114 6 (A-F). Study 2: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 116 7 (A-F). Study 3: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development 118 8. Minimum e s s e n t i a l medium (MEM) composition 120 9. T i s s u e c u l t u r e medium (TCM) 199 composition 122 X LIST OF ABBREVIATIONS AND SYMBOLS a - alpha 3a-HSD = 3alpha h y d r o x y s t e r o i d dehydrogenase AOAC = A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists AWP = A l b e r t a Wheat Pool B = beta B.C. = B r i t i s h Columbia BSA = bovine serum albumin °C = degrees C e l s i u s cm = ce n t i m e t r e DNA = d e o x y r i b o n u c l e i c a c i d ELISA = enzyme-linked immunosorbent assay FSH = f o l l i c l e s t i m u l a t i n g hormone G = a c c e l e r a t i o n f o r c e of g r a v i t y g = gram GCMS = gas chromatography-mass spectrometry GLCMS = g a s - l i q u i d chromatography-mass spectrometry h = hour hCG = human c h o r i o n i c gonadotropin HIFCS = h e a t - i n a c t i v a t e d f e t a l c a l f serum HPLC = h i g h - p r e s s u r e l i q u i d chromatography IU = i n t e r n a t i o n a l u n i t s IUPAC = I n t e r n a t i o n a l Union of Pure and A p p l i e d Chemistry LH = l u t e i n i z i n g hormone M = molar M+ = m o l e c u l a r i o n x i m/e — mass-to-charge r a t i o MEM minimum e s s e n t i a l medium mg = m i l l i g r a m min = minute ml = m i l l i l i t r e mm = m i l l i m e t r e NAD = n i c o t i n a m i d e adenine d i n u c l e o t i d e NADP ni c o t i n a m i d e adenine d i n u c l e o t i d e phosphate nm nanometre PBS - phosphate-buffered s a l i n e PMSG = pregnant mares' serum gonadotropin ppb = p a r t s per b i l l i o n ; (1 /ig/kg = 1 ppb) ppm = p a r t s per m i l l i o n ; (1 mg/kg = 1 ppm) RIA = radioimmuno assay RNA r i b o n u c l e i c a c i d TCM-199 - t i s s u e c u l t u r e medium 199 TLC = t h i n - l a y e r chromatography TM = trademark UBC = U n i v e r s i t y of B r i t i s h Columbia UDP-G = u r i d i n e diphosphate g l u c u r o n y l t r a n s f e r a s e Mg = microgram /xl = m i c r o l i t r e UGG = U n i t e d G r a i n Growers U.S. = U n i t e d S t a t e s US DA = U n i t e d S t a t e s Department of A g r i c u l t u r e X — times ACKNOWLEDGEMENT x i i S i n c e nobody can p o s s i b l y know and do e v e r y t h i n g , we a l l have t o r e l y on oth e r s , whether we l i k e i t o r not. T h e r e f o r e , I d e d i c a t e t h i s t h e s i s t o my f a m i l y and t o the UBC Animal S c i e n c e Department ( f a c u l t y , s t a f f , and s t u d e n t s ) . A l s o , I wish t o thank E a s t C h i l l i w a c k A g r i c u l t u r a l Co-op f o r p r o v i d i n g the b a r l e y samples used i n the zearalenone contamination survey. 1 1.0 LITERATURE REVIEW 1.1. INTRODUCTION Mycotoxin r e s e a r c h has been r e s p o n s i b l e f o r e x p l a i n i n g d i s e a s e s p r e v i o u s l y r e f e r r e d t o as i d i o p a t h i c by human and v e t e r i n a r y medicine. The word mycotoxin o r i g i n a t e s from the Greek words "mykes" (fungus) and " t o x i c o n " (a noxious or poisonous substance) ( F r i e n d and Trenholm, 1988). Fungi which produce these t o x i c chemicals can become e s t a b l i s h e d i n f i e l d crops or h a r v e s t e d feed sources under a p p r o p r i a t e moisture and temperature c o n d i t i o n s . Mycotoxin contamination of feeds causes s e r i o u s economic l o s s e s r e s u l t i n g from decreased animal p r o d u c t i v i t y through decreases i n n u t r i t i o n a l v a l u e and t o x i c o l o g i c a l e f f e c t s . The s u b j e c t of t h i s t h e s i s i s the phytoestrogen zearalenone which d e r i v e s i t s name from the p e r f e c t stage of the fungus t h a t s y n t h e s i z e s i t , G i b b e r e l l a zeae. and the host crop (corn= Zea  mays), which i s commonly c o l o n i z e d ( C o u n c i l f o r A g r i c u l t u r a l S c i e nce and Technology, 1979). A l s o , the r e s o r c y c l i c a c i d l a c t o n e ' s chemical s t r u c t u r e (en = double bond a t C - l ' - 2 ' and one = ketone) c o n t r i b u t e s t o the g e n e r i c name (Mirocha e t a l . , 1980). 1.2. DISCOVERY Symptoms of zearalenone t o x i c o s i s i n swine were f i r s t r e p o r t e d by Buxton (1927) , Legenhausen (1928) and McNutt e t aJL. 2 (1928) i n herds l o c a t e d i n the S t a t e of Iowa. In g i l t s and sows a l l t h r e e authors d e s c r i b e d s w e l l i n g of the v a g i n a , s w e l l i n g of the mammae, as w e l l as p r o l a p s e s of the vagina and rectum. McNutt e t a l . (1928) i n t r o d u c e d the term v u l v o v a g i n i t i s t o d e s c r i b e the symptoms he observed. A d d i t i o n a l l y , s w e l l i n g of the prepuce i n males was r e p o r t e d (Legenhausen, 1928; McNutt e t a l . , 1928). Although the cause of these problems was not i d e n t i f i e d , McNutt e t a l . (1928) proposed a c a u s e - e f f e c t r e l a t i o n s h i p between f e e d i n g s p o i l e d corn and the d i s e a s e symptoms observed. As a r e s u l t of e x c e s s i v e r a i n f a l l i n September, corn had been h a r v e s t e d wet and s t o r e d over the w i n t e r w i t h a moisture content f a v o u r a b l e t o the growth of mold s p e c i e s . N e a r l y t w e n t y - f i v e years l a t e r , McErlean (1952) a s s o c i a t e d the f e e d i n g of fungus (Fusarium qraminearum) i n f e c t e d b a r l e y with v u l v o v a g i n i t i s i n swine. The author suggested t h a t t h i s fungus produced a t o x i c m e t a b o l i t e r e s p o n s i b l e f o r the adverse e f f e c t s . A f t e r s e v e r a l r e p o r t s i n 1957 and 1958 of e s t r o g e n i c e f f e c t s i n swine consuming moldy corn, Stob e t a l . (1962) a t Purdue U n i v e r s i t y i n v e s t i g a t e d the r e l a t i o n s h i p between microorganisms i n the feed and symptoms r e p o r t e d . From s p o i l e d g r a i n samples ob t a i n e d from a f f e c t e d farms, f u n g a l s p e c i e s were i s o l a t e d and c u l t u r e d on s t e r i l e c orn. Feeding of G i b b e r e l l a zeae i n f e c t e d c o r n t o p r e p u b e r a l g i l t s r e s u l t e d i n producing the e s t r o g e n i c e f f e c t s observed i n n a t u r a l contamination of feed. 3 A d d i t i o n a l l y , the group a t Purdue U n i v e r s i t y i s o l a t e d the t o x i c m e t a b o l i t e o f G i b b e r e l l a zeae and found t h a t i t induced s i g n i f i c a n t u t e r o t r o p h i c responses i n l a b o r a t o r y animals. These s t u d i e s were confirmed by the work of ot h e r s (Urry e t a l . , 1966; Mirocha e t a l . , 1967). In 1966 the s t r u c t u r e of the mycotoxin was e s t a b l i s h e d u s i n g mass s p e c t r o m e t r i c and n u c l e a r magnetic resonance methods. A n a l y s i s showed the substance t o be one of the enantiomorphs of 6 - ( 1 0 - h y d r o x y - 6 - o x o - t r a n s - l - u n d e c e n y l ) - b e t a - r e s o r c y c l i c a c i d l a c t o n e (Urry e t a l . , 1966). I t was g i v e n the t r i v i a l name zearalenone based on i t s being a r e s o r c y l i c a c i d l a c t o n e d e r i v a t i v e produced as a m e t a b o l i t e of G i b b e r e l l a zeae. S i n c e the i s o l a t i o n and i d e n t i f i c a t i o n o f zearalenone, much has been d i s c o v e r e d about t h i s mycotoxin's p h y s i c a l and chemical p r o p e r t i e s , b i o s y n t h e s i s , and d e r i v a t i v e s . 1.3. CHEMICAL AND PHYSICAL PROPERTIES Zearalenone i s desi g n a t e d i n Chemical A b s t r a c t s as [S-(E)]-3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-lH-2-benzoxacy-clotetradecin-1,7(8H)-dione] (Schoental, 1985). I t i s a white c r y s t a l l i n e compound with a mol e c u l a r weight of 318, an e m p i r i c a l formula o f C 1 8H 2 20 5, a m e l t i n g p o i n t o f 164-165°C, and an o p t i c a l a c t i v i t y of [alpha] 2 5= -170.5°, c= 1.0 M i n methyl 546 a l c o h o l (Urry e t a l . , 1966). At 25°C, the compound i s s l i g h t l y s o l u b l e i n water (2.0 4 mg/100 g ) , s o l u b l e i n ethanol (24 g/100 g ) , methanol (18 g/lOOg), a c e t o n i t r i l e (8.6 g/lOOg), acetone (58 g/lOOg), methylene c h l o r i d e (17.5 g/lOOg), benzene (1.13 g/lOOg) (Hidy e t a l . , 1977). The u l t r a v i o l e t maxima measured i n e t h y l a l c o h o l are a t 236, 274, and 316 nm w i t h the absorbance a t 274 nm b e i n g most u s e f u l f o r q u a n t i t a t i v e a n a l y s i s (Mirocha e t a l . , 1971) Z e a r a l e n o n e 1 s c h a r a c t e r i s t i c a b s o r p t i o n i s dependent upon the e l e c t r o n i c c o n f i g u r a t i o n around the aromatic r i n g , l a c t o n e , and extended c o n j u g a t i o n i n the undecenyl s i d e r i n g (Mirocha e t a l . , 1971). At a l l t h r e e wavelengths, t h e r e e x i s t s a l i n e a r r e l a t i o n s h i p between absorbance and c o n c e n t r a t i o n . Another p h y s i c a l p r o p e r t y used f o r a n a l y t i c a l work i s i t s f l u o r e s c e n c e when i r r a d i a t e d by u l t r a v i o l e t l i g h t . I t i s the h y d r o x y l group at C2 which i s r e s p o n s i b l e f o r t h i s p r o p e r t y (Mirocha e t a l . , 1977). When i r r a d i a t e d with long wavelength (3 60 nm) u l t r a v i o l e t l i g h t , zearalenone f l u o r e s c e s a blue-green which becomes more i n t e n s e when exposed t o s h o r t wavelength (2 60 nm) u l t r a v i o l e t l i g h t ( C h r i s t e n s e n , 1979). F l u o r e s c e n c e can be used t o d e t e c t the t o x i n i n a sample separated by t h i n - l a y e r chromatography. The i n f r a r e d spectrum of zearalenone shows prominent a b s o r p t i o n bands at 3300 cm"1 (C-2-OH, i n t r a m o l e c u l a r hydrogen bonding wi t h the l a c t o n e c a r b o n y l ) ; 1645 cm"1 ( l a c t o n e c a r b o n y l ) ; 1612 cm"1, 1578 cm"1 (aromatic r i n g ) and 968 cm"1 ( t r a n s o l e f i n i c 5 double bond ( C h r i s t e n s e n , 1979). U n d e r i v a t i z e d zearalenone•s mass spectrum i s c h a r a c t e r i z e d by i n t e n s e fragments m/e+ 188, 161, 151, 125, 112, and 97 w h i l e i t s m o l e c u l a r i o n (M*) i s 318 ( C h r i s t e n s e n , 1979) . For r e s o l u t i o n by g a s - l i q u i d chromatography, the d i - t r i m e t h y l s i l y l e t h e r -zearalenone d e r i v a t i v e i s used which has a M* a t 462 and a base peak a t m/e+ 151 ( C h r i s t e n s e n , 1979). Other s i g n i f i c a n t fragment ions are 260, 305, 333, 429, and 447 (M+-15) w i t h a metastable i o n a t 412. Knowledge of the p h y s i c a l and chemical p r o p e r t i e s of zearalenone can be a p p l i e d i n experimental methods i n v o l v i n g the mycotoxin. A l s o , these p r o p e r t i e s can be used as t o o l s i n the q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s of samples f o r zearalenone. 1.4. BIOSYNTHESIS O r i g i n a l l y , s y n t h e s i s of zearalenone was o n l y a s s o c i a t e d with the ascospore-producing stage ( G i b b e r e l l a zeae) of the fungus Fusarium roseum. However, the mycotoxin has been demonstrated t o be a m e t a b o l i t e of other Fusarium s p e c i e s i n c l u d i n g F. avenaceum, F. crookwellense, F. culmorum, F. e q u i s e t i , F. qibbosum. F. moniliforme, F. n i v a l e , F. oxysporum, F. sambucinum, and F. s p o r o t r i c h i o i d e s ( C a l d w e l l and T u i t e , 1970; Hacking, 1976; Food and A g r i c u l t u r e O r g a n i z a t i o n , 1977; G o l i n s k i e t a l . , 1988). The s p e c i e s most o f t e n a s s o c i a t e d w i t h zearalenone contamination of crops i n North America i s F. roseum which i s a l s o r e f e r r e d t o as 6 F. qraminearum. Lieberman (1971) and S t e e l e e t a l . (1974) r e p o r t e d zearalenone t o be formed by the h e a d - t o - t a i l condensation of 9 a c e t a t e u n i t s v i a the acetate-malonyl-CoA ( p o l y k e t i d e ) pathway. Once the a c e t a t e u n i t s have formed a p o l y k e t i d e c h a i n , i t subsequently c y c l i z e s a t the C - l , C-6, and the C-10, C-12 p o s i t i o n s ( B l a c k w e l l e t a l . , 1985). The s t r u c t u r e of the m e t a b o l i t e suggests t h i s s i n c e a l l even-numbered carbons are so arranged as t o have a h y d r o x y l or ketone group c o r r e s p o n d i n g t o the CI carbon of a c e t a t e (Mirocha and Pathre, 1979). In the f u n g i which produce the m e t a b o l i t e , zearalenone p l a y s a v i t a l r o l e i n the l i f e c y c l e as f i r s t suggested by Eugenio (1968) and shown t o be t r u e by Wolf e t a l . (1972) . The m e t a b o l i t e a c t s as a hormone of sexual r e p r o d u c t i o n by r e g u l a t i n g the s e r i e s of events r e q u i r e d f o r i n i t i a t i o n of p e r i t h e c i a . R e g u l a t i o n of p e r i t h e c i a f ormation may i n v o l v e the b i n d i n g of zearalenone t o a r e c e p t o r p r o t e i n and then t o DNA, r e s u l t i n g i n the c o n t r o l of a p r o c e s s of t r a n s c r i p t i o n necessary f o r p e r i t h e c i a p r o d u c t i o n (Wolf and Mirocha, 1977). Zearalenone s y n t h e s i s i s r e g u l a t e d by c y c l i c 3 *-5 1-adenosine monophophate (cAMP) found i n f u n g a l mycelium and t o g e t h e r the two are an important p a r t of the r e p r o d u c t i v e c o n t r o l mechanism (Wolf and Mirocha, 1977). To date, zearalenone i s the o n l y mycotoxin whose f u n c t i o n i n the producing s p e c i e s has been determined. 7 1.5. DERIVATIVES Examination o f g r a i n samples contaminated w i t h the mycotoxin a l s o demonstrates the presence o f zearalenone d e r i v a t i v e s . These d e r i v a t i v e s are i n v o l v e d i n the formation o f the m e t a b o l i t e o r are formed when i t i s metabolized by the producing f u n g i . For example, c o n v e r s i o n o f r a d i o a c t i v e l a b e l l e d a l p h a - and b e t a - z e a r a l e n o l s t o zearalenone by Fusarium qraminearum suggests t h a t they are p r e c u r s o r s of the mycotoxin (Richardson e t a l . , 1984). In zearalenone metabolism s t u d i e s , s e v e r a l d e r i v a t i v e s i n c l u d i n g the f o l l o w i n g have been i d e n t i f i e d : two isomers o f 8'-hydroxyzearalenone, 6',8'-dihydroxyzearalene, 7 1 - z e a r a l d i e n o n e , 6 - ( c a r b o x y p e n t y l ) - b e t a - r e s o r c y l i c a c i d , 3 1-hydroxyzearalenone ( S t e e l e e t a l . , 1974; S t e e l e e t a l . , 1976; Pathre e t a l . , 1980). 1.6. FUNGAL ESTABLISHMENT AND ZEARALENONE PRODUCTION Fusarium s p e c i e s invade and cause d i s e a s e s i n v a r i o u s North American f i e l d crops i n c l u d i n g corn, b a r l e y , wheat, oats, sorghum, and soybeans (Bennett and Shot w e l l , 1979; Wicklow e t a l . , 1987). I n f e c t i o n i n corn l e a d s t o ear r o t wh i l e i n wheat, b a r l e y and sorghum the r e s u l t i s head and k e r n e l b l i g h t which i s commonly r e f e r r e d t o as "scab" (Schroeder and C h r i s t e n s e n , 1963; Schroeder and Hein, 1975; C o u n c i l f o r A g r i c u l t u r a l S c i e n c e and Technology 1979; Sutton, 1982). Corn crops are p a r t i c u l a r l y 8 s u s c e p t i b l e t o f u n g a l i n v a s i o n . As a consequence, r e g i o n s which r e l y on corn as the primary animal feed are more l i k e l y t o e x p e r i e n c e mycotoxicoses problems. Favourable f i e l d c o n d i t i o n s f o r f u n g a l c o l o n i z a t i o n and e s t a b l i s h m e n t i n c l u d e p e r s i s t e n t wetness and warm temperatures (15-35°C) ( C h r i s t e n s e n and Kaufmann, 1969; Sutton, 1982). C o o l e r temperatures (8-10°C) are r e q u i r e d f o r optimal zearalenone p r o d u c t i o n ( C h r i s t e n s e n and Kaufmann, 1969). S t u d i e s by Mirocha and C h r i s t e n s e n (1974) suggest t h a t the enzymes r e s p o n s i b l e f o r zearalenone s y n t h e s i s are induced or a c t i v a t e d a t these lower temperatures. In Canada, the r e g i o n which experiences weather c o n d i t i o n s most s u i t a b l e f o r zearalenone p r o d u c t i o n i n the f i e l d i s Southern O n t a r i o (Sutton e t a l . , 1976; Sutton e t a l . , 1980). P e r s i s t e n t r a i n f a l l i n August or September r e s u l t s i n h i g h p l a n t moisture content which supports fungal e s t a b l i s h m e n t . S i g n i f i c a n t zearalenone p r o d u c t i o n occurs w i t h c o o l e r temperatures experienced d u r i n g l a t e September or October. In 1971, zearalenone m y c o t o x i c o s i s was f i r s t r e p o r t e d i n Canada when cases of estrogenism i n Southern O n t a r i o swine herds were d i s c o v e r e d ( B r i s t o l and D j u r i c k o v i c , 1971). Other areas of Canada experience environmental c o n d i t i o n s conducive t o f u n g a l e s t a b l i s h m e n t and mycotoxin p r o d u c t i o n . These areas i n c l u d e the Peace R i v e r V a l l e y of A l b e r t a and B r i t i s h Columbia and r e g i o n s of Southern Manitoba, e s p e c i a l l y i n the Red 9 R i v e r V a l l e y (Trenholm e t a l . , 1985). Mold growth and zearalenone p r o d u c t i o n a l s o occur d u r i n g s t o r a g e i f a p p r o p r i a t e c o n d i t i o n s e x i s t . In f a c t , zearalenone b i o s y n t h e s i s under storage c o n d i t i o n s i s more s i g n i f i c a n t than i n 2 the f i e l d ( C a l d w e l l and T u i t e , 1974; Hacking e t a l . , 1976). Crops h a r v e s t e d under wet c o n d i t i o n s and s t o r e d without d r y i n g are s u s c e p t i b l e t o fu n g a l growth. A l s o , s t o r e d feed exposed t o r a i n , snow or condensation i s a l s o a t r i s k (Shotwell e t a l . , 1975; Trenholm e t a l . , 1989). A moisture content o f 14% or h i g h e r a l l o w s f u n g a l growth a t temperatures g r e a t e r than 5-10°C (Sherwood and Peberdy, 1974; Trenholm e t a l . , 1988). Once the fungus has been e s t a b l i s h e d , s i g n i f i c a n t zearalenone p r o d u c t i o n can occur a t temperatures above 0°C (Sherwood and Peberdy, 1974). 1.7. METABOLISM OF ZEARALENONE BY ANIMALS In order t o understand how zearalenone exposure causes adverse e f f e c t s , i n f o r m a t i o n p e r t a i n i n g t o i t s metabolism and mechanism of a c t i o n i n animals i s be i n g o b t a i n e d and s t u d i e d . Zearalenone metabolism i n s p e c i e s s t u d i e d so f a r i n v o l v e s e i t h e r a r e d u c t i o n r e a c t i o n t o an a l c o h o l o r c o n j u g a t i o n with g l u c u r o n i c a c i d or sul p h a t e . The r e d u c t i o n of zearalenone occurs a t the keto group i n the 6' p o s i t i o n and r e s u l t s i n the formation o f i s o m e r i c m e t a b o l i t e s known as a l p h a ( a ) - and b e t a ( 6 ) - z e a r a l e n o l ( F i g u r e 1). C a t a l y s a t i o n of the r e a c t i o n i s c a r r i e d out by the enzyme 3alpha-10 11 hydroxy s t e r o i d dehydrogenase (3a-HSD) and predominately occurs i n the l i v e r ( K i e s s l i n g e t a l . , 1978; Thouvenot and M o r f i n , 1980; Olsen e t a l . , 1981). The enzyme e x i s t s i n m u l t i p l e forms wi t h v a r i o u s pH optima and i s l o c a l i z e d i n one of two s u b c e l l u l a r l o c a t i o n s , e i t h e r microsome or c y t o s o l (Olsen e t a l . , 1981). S t u d i e s on zearalenone r e d u c i n g a c t i v i t y have r e v e a l e d a s p e c i e s e f f e c t on h e p a t i c metabolism. In v i t r o s t u d i e s on r a t , mouse, p i g , cow and r a b b i t l i v e r t i s s u e show t h a t a - z e a r a l e n o l i s the major m e t a b o l i t e produced a t pH 4.5 wit h e i t h e r NADH or NADPH as c o f a c t o r i n the r e a c t i o n (Ueno e t a l . , 1983). For these s p e c i e s the same isomer i s predominant a t pH 7.4 w i t h NADH. However, 6 - z e a r a l e n o l becomes the major product a t pH 7.4 wit h NADPH. In c o n t r a s t , the 6 - z e a r a l e n o l form i s always the major m e t a b o l i t e i n hamsters (Ueno e t a l . , 1983). While i n guinea p i g s , s i m i l a r amounts of a- and 6 - z e a r a l e n o l are produced r e g a r d l e s s o f pH and c o f a c t o r p r e s e n t (Ueno e t a l . , 1983). As f o r the s u b c e l l u l a r s i t e f o r r e d u c t i o n , t h i s too i s i n f l u e n c e d by s p e c i e s . In p i g s and goats both isomers of z e a r a l e n o l are produced i n the microsomes (endoplasmic reticulum) independent of the coenzyme prese n t (Olsen and K i e s s l i n g , 1983). For sheep the c y t o s o l i s the l o c a t i o n o f g r e a t e s t a - z e a r a l e n o l producing a c t i v i t y w i t h NADPH as the coenzyme (Olsen and K i e s s l i n g , 1983) . However, cows and hens form the m a j o r i t y o f the a form microsomally and produce the & isomer i n the c y t o s o l only when NADPH i s presen t (Olsen and K i e s s l i n g , 1983) . 12 Although the l i v e r i s the predominant l o c a t i o n of zearalenone r e d u c t i o n f o r non-ruminant s p e c i e s , p r o s t a t e glands (Thouvenot and M o r f i n , 1980) as w e l l as e r y t h r o c y t e s (Chang and L i n , 1984) are a l s o capable of t h i s r e a c t i o n . In ruminants, the m a j o r i t y of zearalenone r e d u c t i o n occurs i n the rumen v i a p r o t o z o a l a c t i o n and the main m e t a b o l i t e formed i s a - z e a r a l e n o l w i t h l e s s e r amounts of the & form ( K i e s s l i n g e t a l . , 1984). By i n c r e a s i n g the p o l a r i t y of the mycotoxin v i a r e d u c t i o n , a b s o r p t i o n from the s m a l l i n t e s t i n e i n t o the bloodstream i s f a c i l i t a t e d . S i n c e p r o t o z o a l a c t i v i t y r e s u l t s i n the p r o d u c t i o n of a m e t a b o l i t e even more e s t r o g e n i c than the parent compound, rumination does not o f f e r p r o t e c t i o n from the harmful e f f e c t s of zearalenone. The a l t e r n a t i v e m e t a b o l i c pathway i n v o l v e s c o n j u g a t i o n of zearalenone i n t o b i o l o g i c a l l y i n a c t i v e g l u c u r o n i c or s u l p h a t e d e r i v a t i v e s i n the l i v e r . As w i t h the r e d u c t i o n r e a c t i o n , s p e c i e s d i f f e r e n c e s e x i s t . To i l l u s t r a t e , p i g s and goats have a g r e a t e r c a p a c i t y f o r c o n j u g a t i o n than c a t t l e ( K i e s s l i n g e t a l . , 1979). P o s s i b l y , t h i s c o u l d be the r e s u l t of a v a i l a b i l i t y and e x i s t e n c e of f a v o u r a b l e c o n d i t i o n s f o r c o n j u g a t i o n enzymes such as u r i d i n e diphosphate g l u c u r o n y l t r a n s f e r a s e (UDP-G) ( K i e s s l i n g and P e t t e r s s o n , 1978). I n c i d e n t a l l y , o n l y i n c a t t l e i s t h e r e s i g n i f i c a n t p r o d u c t i o n of the s u l p h a t e d e r i v a t i v e (Mirocha e t a l . , 1981). Glu c u r o n i d e conjugates of zearalenone and i t s m e t a b o l i t e s 13 are found i n the blood. In p i g s , Olsen e t a l . (1985) found conjugated a - z e a r a l e n o l l e v e l s t o be 3-4 times h i g h e r than conjugated zearalenone. Regarding the e l i m i n a t i o n of the mycotoxin and i t s d e r i v a t i v e s , both u r i n e and f e c e s are s i g n i f i c a n t r o u t e s of e x c r e t i o n . Both r e d u c t i o n and c o n j u g a t i o n i n c r e a s e the mycotoxin's p o l a r i t y and, t h e r e f o r e , i n c r e a s e i t s s o l u b i l i t y r e s u l t i n g i n f a c i l i t a t e d u r i n e e x c r e t i o n ( F i t z p a t r i c k e t a l . , 1988). In u r i n e , zearalenone and i t s m e t a b o l i t e s are e l i m i n a t e d as f r e e and conjugated forms (Mirocha e t a l . , 1981). S t u d i e s by Mirocha e t a l . (1981) i n swine demonstrated t h a t 63% of the mycotoxin was e x c r e t e d as the parent compound, 32% as a - z e a r a l e n o l , and 5% as 6 - z e a r a l e n o l . In c o n t r a s t , the predominant e x c r e t o r y product i n c a t t l e was B - z e a r a l e n o l (51%) f o l l o w e d by zearalenone (29%), and a - z e a r a l e n o l (20%) (Mirocha e t a l . , 1981). B i l i a r y e x c r e t i o n i s promoted v i a c o n j u g a t i o n s i n c e m o l e c u l a r weight i s i n c r e a s e d (Mulder e t a l . , 1981). Because of the e x t e n s i v e b i l i a r y e x c r e t i o n , s i g n i f i c a n t e l i m i n a t i o n occurs v i a f e c e s . In c a t t l e f e c e s , m e t a b o l i t e d i s t r i b u t i o n i s s i m i l a r t o t h a t found i n u r i n e (Mirocha e t a l . , 1981). However, i n swine f e c e s , 91% i s e x c r e t e d as zearalenone and 9% as a - z e a r a l e n o l which d i f f e r s from the d i s t r i b u t i o n found i n u r i n e (Mirocha e t a l . , 1981). 14 A d d i t i o n a l l y , s i g n i f i c a n t e x c r e t i o n can occur v i a m i l k i n a l a c t a t i n g animal. When hig h c o n c e n t r a t i o n s o f zearalenone are fed, t h e r e i s t r a n s m i s s i o n of zearalenone, a- and 6 - z e a r a l e n o l s as w e l l as conjugated forms i n t o the m i l k of sheep, cows, and p i g s (Hagler e t a l . , 1980; P a l y u s i k e t a l . , 1980; Mirocha e t a l . , 1981; Vanyi e t a l . , 1983). 1.8. MECHANISM OF ACTION OF ZEARALENONE IN ANIMALS E s t r o g e n i c a c t i v i t y of zearalenone and i t s analogues i s caused by mechanisms s i m i l a r t o t h a t of s t e r o i d hormones. The mycotoxin f i r s t binds t o estrogen r e c e p t o r p r o t e i n i n the c y t o s o l of t a r g e t t i s s u e c e l l s . Then, the mycotoxin-receptor complex i s t r a n s l o c a t e d t o the nucleus where i t binds t o the DNA template and promotes RNA and p r o t e i n s y n t h e s i s as w e l l as i n c r e a s e s i n organ weight (Ueno and Yagasaki, 1975; W i t t l i f f e t a l . , 1976; Ueno e t a l . , 1977; Boyd and W i t t l i f f , 1978; Kiang e t a l . , 1978; Greenman e t a l . , 1979; Katzenbellenbogen e t a l . , 1979). In e s t r o g e n r e s p o n s i v e t i s s u e s such as the l i v e r , mammary glands and u t e r u s , zearalenone and i t s d e r i v a t i v e s have been demonstrated t o compete wi t h 1 7 6 - e s t r a d i o l f o r c y t o s o l r e c e p t o r b i n d i n g (Boyd and W i t t l i f f , 1978; Kiang e t a l . , 1978; Greenman e t a l . , 1979; Katzenbellenbogen e t a l . , 1979; Powell-Jones e t a l . , 1981). Based on b i n d i n g a f f i n i t y , the d e r i v a t i v e s a - z e a r a l a n o l , a - z e a r a l e n o l , and B - z e a r a l a n o l are even b e t t e r i n h i b i t o r s of endogenous estrogen b i n d i n g than the parent compound, w h i l e R-z e a r a l e n o l i s l e s s e f f e c t i v e ( T a s h i r o e t a l . , 1980). As a 15 consequence of t h e i r r e l a t i v e l y h i g h b i n d i n g a f f i n i t y , the e s t r o g e n i c potency of the s t r o n g e r i n h i b i t o r s i s g r e a t e r than t h a t o f zearalenone. Because of b i n d i n g a f f i n i t y r e l a t i v e t o zearalenone, r e d u c t i o n o f the mycotoxin t o a - z e a r a l e n o l r e p r e s e n t s an a c t i v a t i o n p r o c e s s , w h i l e p r o d u c t i o n of the R form i s d e a c t i v a t i o n . A d d i t i o n a l l y , e s t r o g e n i c potency i s a f f e c t e d by the r e l a t i v e s t a b i l i t y of the mycotoxin-receptor complex (Powell-Jones e t a l . , 1981). Ligands which s l o w l y d i s s o c i a t e w i t h t h e i r r e c e p t o r have a g r e a t e r a b i l i t y t o t r a n s l o c a t e c y t o s o l r e c e p t o r s and m a i n t a i n nucleus r e c e p t o r s . The r e s u l t i s i n c r e a s e d potency through the i n i t i a t i o n and maintenance of e s t r o g e n i c e f f e c t s . Although zearalenone's b i n d i n g a f f i n i t y t o estrogen r e c e p t o r s i s 5% or l e s s than t h a t of 1 7 B - e s t r a d i o l (Kuiper-Goodman e t a l . , 1987), t h i s apparent l a c k o f potency i s co u n t e r a c t e d by oth e r f a c t o r s . F i r s t , the d u r a t i o n o f n u c l e a r r e t e n t i o n f o r the r e c e p t o r complex i s l o n g e r w i t h zearalenone than w i t h 1 7 B - e s t r a d i o l (Kiang e t a l . , 1978). Secondly, zearalenone has low a f f i n i t y f o r sex s t e r o i d b i n d i n g g l o b u l i n s (Martin e t a l . , 1978), which enhances b i o l o g i c a l e f f e c t i v e n e s s by i n c r e a s i n g the c o n c e n t r a t i o n of f r e e zearalenone a t t a r g e t c e l l s . F i n a l l y , as d i s c u s s e d e a r l i e r , zearalenone i s metabolized i n the l i v e r t o a - z e a r a l e n o l which i s a more potent d e r i v a t i v e based on r e c e p t o r b i n d i n g a f f i n i t y s t u d i e s . 16 L i k e estrogens, zearalenone causes an i n c r e a s e i n c e l l u l a r p e r m e a b i l i t y t o the p r e c u r s o r s of macromolecule syntheses such as amino a c i d s , sugars, and n u c l e o s i d e s (Ueno and Yagasaki, 1975; Ueno e t a l . , 1977). Furthermore, harmful e f f e c t s of zearalenone may be p a r t i a l l y a t t r i b u t e d t o an e f f e c t on the metabolism of endogenous s t e r o i d s . For example, r e d u c t i o n of zearalenone i n t o a- and 6 - z e a r a l e n o l i n t e r f e r e s with the metabolism of s t e r o i d s through s u b s t r a t e c o m p e t i t i o n . The enzyme, (3a-HSD), r e q u i r e d f o r r e d u c t i o n i s a l s o i n v o l v e d i n the c o n v e r s i o n of androsterone t o 5a-androsterone-3, 17-dione and the t r a n s f o r m a t i o n of t e s t o s t e r o n e i n t o 5a-a n d r o s t a n e d i o l s (Thouvenot and M o r f i n , 1980; Olsen e t a l . , 1981; Olsen, 1985). S i n c e zearalenone and i t s analogues are n o n - s t e r o i d a l , the q u e s t i o n remains as t o how they cause responses i n t a r g e t t i s s u e through mechanisms s i m i l a r t o t h a t of estrogens. B i o l o g i c a l a c t i o n of these e s t r o g e n i c mycotoxins i s l i k e l y caused by c y t o s o l e s trogen r e c e p t o r r e c o g n i t i o n and b i n d i n g of t h e i r p h e n o l i c and a l c o h o l i c f u n c t i o n s . Based on the r e s u l t s of e s t r o g e n r e c e p t o r a f f i n i t y s t u d i e s , an a l p h a - h y d r o x y l group a t C-6' and a double bond between C - l 1 and C-2 1 i n f l u e n c e e s t r o g e n i c a c t i o n (Kawabata e t a l . , 1982). I n t e r a c t i o n of zearalenone w i t h r e c e p t o r s has been suggested t o be the r e s u l t of f o l d i n g of the molecule i n such a way t h a t the h y d r o x y l group or p o t e n t i a l h y d r o x y l groups are a p p r o p r i a t e l y o r i e n t a t e d f o r b i n d i n g (Powell-Jones e t a l . , 1981). 17 1.9. TOXICOLOGICAL EFFECTS OF ZEARALENONE IN ANIMALS As a consequence of z e a r a l e n o n e 1 s e s t r o g e n i c a c t i o n , the m a j o r i t y o f i t s e f f e c t s occur i n the r e p r o d u c t i v e system, w i t h females b e i n g more s e r i o u s l y a f f e c t e d . Both on-farm and experimental exposure have shown swine t o be the domestic s p e c i e s most s u s c e p t i b l e t o zearalenone m y c o t o x i c o s i s . T h e i r s e n s i t i v i t y may be of systemic o r i g i n s i n c e t h e r e e x i s t s a hig h r a t e of t o x i n a b s o r p t i o n from the g a s t r o i n t e s t i n a l t r a c t ( F r i e n d e t a l . , 1986). Estrogenism i n prep u b e r a l g i l t s i s the be s t known c o n d i t i o n caused by the mycotoxin. E x t e r n a l l y , estrogenism i s c h a r a c t e r i z e d by an edematous and hyperemic v u l v a , v a g i n a l d i s c h a r g e , and mammary enlargement (McNutt et a l . , 1928; B r i s t o l and D j u r i c k o v i c , 1971). A d d i t i o n a l l y , v a g i n a l and or r e c t a l p r o l a p s e o f t e n o c c u r s . Secondary c o m p l i c a t i o n s such as hemorrhage or i n f e c t i o n caused by t r a u m a t i s a t i o n of a p r o l a p s e can r e s u l t i n death (McNutt e t a l . , 1928). H i s t o l o g i c a l examination o f a f f e c t e d g i l t s r e v e a l s e s t r o g e n i c - l i k e e f f e c t s on t i s s u e s i n c l u d i n g : edema and c e l l u l a r p r o l i f e r a t i o n i n a l l l a y e r s of the u t e r u s , but e s p e c i a l l y the myometrium v i a hypertrophy and h y p e r p l a s i a ; m e t a p l a s i a of the c e r v i x mucosal e p i t h e l i u m from a normal l a y e r of columnar type c e l l s t o a s t r a t i f i e d squamous c e l l u l a r l a y e r ; squamous m e t a p l a s i a of v a g i n a l e p i t h e l i u m ; and d u c t a l h y p e r p l a s i a and i n c r e a s e d m i t o t i c index i n the mammary gland (Kurtz e t a l . , 1969). 18 Once zearalenone exposure has ceased, t h e r e i s no d e l a y of puberty attainment or e f f e c t on subsequent r e p r o d u c t i v e parameters such as c o n c e p t i o n r a t e , o v u l a t i o n r a t e , embryonic m o r t a l i t y , and l i t t e r s i z e (Green e t a l . , 1990; Rainey e t a l . , 1990). T h e r e f o r e , zearalenone m y c o t o x i c o s i s d u r i n g the p r e p u b e r a l p e r i o d does not permanently a f f e c t female r e p r o d u c t i v e c a p a b i l i t i e s . In both mature g i l t s and sows, zearalenone can d i s r u p t c y c l i c i t y by c a u s i n g a n e s t r u s . The anestrus s t a t e i s the r e s u l t of a prolonged e s t r o u s c y c l e through maintenance of f u n c t i o n a l c o r p o r a l u t e a (Cantley e t a l . , 1982; E t l e n n e and Jemmali, 1982; Young and King, 1983; Edwards e t a l . , 1984). L u t e a l maintenance promotes u t e r i n e hypertrophy l e a d i n g t o a pseudopregnant c o n d i t i o n . S i m i l a r l u t e o t r o p h i c e f f e c t s can be induced through a d m i n i s t r a t i o n of n a t u r a l or s y n t h e t i c estrogens (Kidder e t a l . , 1955; Gardner e t a l . , 1963). Whether the l u t e o t r o p h i c a c t i o n i s caused hormonally or by a d i r e c t e f f e c t on the ovary i s y e t t o be determined (Flowers e t a l . , 1987). I n g e s t i o n of zearalenone by pregnant sows has been a s s o c i a t e d w i t h embryonic m o r t a l i t y , s t i l l b i r t h s , mummification, sma l l l i t t e r s , and s p l a y l e g i n p i g l e t s (Wilson e t a l . , 1967; M i l l e r e t a l . , 1973). Exposure of l a c t a t i n g sows t o the mycotoxin i n c r e a s e s the 19 weaning-to-estrus i n t e r v a l (Edwards e t a l . , 1987; Young e t a l . , 1990). P o s s i b l y , the d e l a y i n r e t u r n t o e s t r u s i n v o l v e s a blockade of gonadotropin r e l e a s e a t weaning, thus p r e v e n t i n g f o l l i c u l a r development. A l t e r n a t i v e l y , t h e r e i s a d i r e c t e f f e c t on the ovary i t s e l f . In c a t t l e and sheep, r e p r o d u c t i v e problems such as i n f e r t i l i t y , a b o r t i o n , and p r e c o c i o u s mammary development have been l i n k e d t o zearalenone contamination of feed (Mirocha e t a l . , 1968; M i t t o n e t a l . , 1975; Bloomquist e t a l . , 1982; K a l l e l a and E t t a l a , 1984; Smith e t a l . , 1986; Weaver e t a l . , 1986). Although known f o r i t s adverse e f f e c t s on l i v e s t o c k , zearalenone m y c o t o x i c o s i s a l s o occurs i n domestic fowl. In chicken s , o v i d u c t hypertrophy and reduced egg p r o d u c t i o n have been r e p o r t e d (Adams and T u i t e , 1976; Chi e t a l . , 1980a; C h i e t a l . , 1980b). While i n tu r k e y s , zearalenone exposure can cause vent edema and p r o l a p s e o f the c l o a c a (Meronuck e t a l . , 1970). G e n e r a l l y , males of a l l s p e c i e s are l e s s s e n s i t i v e t o the a c t i o n o f zearalenone. However, they too can s u f f e r from the e f f e c t s of the mycotoxin's d e l e t e r i o u s a c t i o n s . For example, tur k e y cocks consuming zearalenone contaminated feed e x p e r i e n c e reduced f e r t i l i t y i n terms o f sperm q u a n t i t y and v i a b i l i t y of spermatozoa ( P a l y u s i k e t a l . , 1971). In pr e p u b e r a l boars, zearalenone can cause prepuce and mammary gland enlargement as w e l l as a decrease i n t e s t i c u l a r weights (Koen and Smith, 1945; C h r i s t e n s e n et a l . , 1972). In both t u r k e y s and swine, i n v i v o and i n v i t r o s t u d i e s have demonstrated t h a t the mycotoxin causes degeneration of t e s t i c u l a r c e l l s ( P a l y u s i k e t a l . , 1971; Vanyi and S z a i l e r , 1974). A d d i t i o n a l l y , decreased l i b i d o as w e l l as depressed plasma t e s t o s t e r o n e c o n c e n t r a t i o n s have been r e p o r t e d i n both mature and immature boars ( B r i s t o l and D j u r i c k o v i c , 1971; Berger e t a l . , 1981). In v i t r o s t u d i e s w i t h g e r b i l t e s t i c u l a r t i s s u e have demonstrated t h a t exposure of i n t e r s t i t i a l c e l l s t o zearalenone i n h i b i t s t e s t o s t e r o n e p r o d u c t i o n (Fenske and Fink-Gremmels, 1990). The major non-reproductive e f f e c t s of zearalenone on l i v e s t o c k and domestic fowl are decreases i n feed consumption, feed e f f i c i e n c y , and growth r a t e (Wilson e t a l . , 1967; Adams and T u i t e , 1976; Young e t a l . , 1979). The reduced p a l a t a b i l i t y and n u t r i t i o n a l v a l u e of a moldy feed would account f o r these e f f e c t s . 21 2.0 SURVEY OF BARLEY FROM ALBERTA AND BRITISH COLUMBIA GRAIN ELEVATORS FOR ZEARALENONE CONTAMINATION 2.1. INTRODUCTION 2.1.1. RESULTS OF PREVIOUS SURVEYS FOR ZEARALENONE CONTAMINATION The i n i t i a l surveys on zearalenone contamination of feed sources were conducted i n the 1960's by the U n i t e d S t a t e s Department of A g r i c u l t u r e (USDA). In 1967, the corn crops of Iowa, I l l i n o i s , Alabama, and Tennessee were surveyed. Of the 283 samples t e s t e d a t a d e t e c t i o n l i m i t o f 2 00 ppb, p o s i t i v e r e s u l t s were found i n 2 low grade q u a l i t y samples (Odette e t a_l. , 1970) . The USDA l a t e r surveyed export corn c o l l e c t e d d u r i n g 1968 and 1969 from 8 U.S. and 2 Canadian p o r t s (Shotwell e t a l . , 1971). Of the 293 samples t e s t e d , 5 were p o s i t i v e f o r zearalenone at the 200 ppb l e v e l . P o s i t i v e r e s u l t s were ob t a i n e d i n v a r i o u s grades of corn and the l e v e l of contamination ranged from 450-750 ppb. By the 1970's, improved methods f o r d e t e c t i n g zearalenone i n corn and o t h e r feed sources had been developed which l e d t o more e x t e n s i v e t e s t i n g . During the s p r i n g of 1973, samples of the 1972 corn crop were c o l l e c t e d by the U.S. Food and Drug A d m i n i s t r a t i o n from g r a i n e l e v a t o r s i n the Corn B e l t (Eppley e t a l . , 1974). At a d e t e c t i o n l i m i t o f 50-100 ppb, 17% of samples c o n t a i n e d 22 zearalenone w i t h a c o n c e n t r a t i o n range of 0.1-5.0 ppm. A survey of zearalenone contamination of 1973 marketable corn s t o r e d on farms and i n country e l e v a t o r s r e v e a l e d t h a t 10% of samples from the Corn B e l t were contaminated ( S t o l o f f e t a l . , 1976). The d e t e c t i o n l i m i t was 50-100 ppb and the average l e v e l of contamination was 117 ppb wi t h a range of 38-204 ppb. The same survey showed t h a t 7% of damaged or moldy corn c o n t a i n e d the mycotoxin a t l e v e l s r a n g i n g from 97-10,400 ppb wi t h the average being 2100 ppb. A survey of U.S. sorghum crops from 1975 and 1976 showed 28% of samples t o be zearalenone contaminated (Shotwell e t a l . , 1980). The d e t e c t i o n l i m i t was 100-200 ppb and p o s i t i v e r e s u l t s ranged from 200-6900 ppb. In V i r g i n i a , 45% of samples from the 1975 wheat crop c o n t a i n e d the mycotoxin a t a d e t e c t i o n l i m i t of 0.25 ppm (Shotwell e t a l . , 1977). Contamination l e v e l s ranged from 0.36 t o 11.05 ppm. More r e c e n t l y , feeds formulated from the 1981 corn h a r v e s t i n I l l i n o i s and surrounding s t a t e s were submitted f o r l a b o r a t o r y a n a l y s i s a f t e r complaints of poor animal h e a l t h and performance (Cote e t a l . , 1984). Of the 274 samples analyzed, 40 t e s t e d p o s i t i v e f o r zearalenone a t a d e t e c t i o n l e v e l o f 0.02 ppm. Mycotoxin c o n c e n t r a t i o n ranged from 0.1 t o 8.0 ppm wi t h the average being 0.66 ppm. 23 In Canada, a l i m i t e d number of mycotoxin surveys have been conducted. In one study, r e c o r d s kept on corn samples submitted t o the O n t a r i o M i n i s t r y of A g r i c u l t u r e and Food's T o x i c o l o g y Laboratory were e v a l u a t e d f o r the years 1972 t o 1977 ( F u n n e l l , 1979). For the years 1972, 1973, 1974, 1975, 1976, and 1977, the presence of zearalenone was d e t e c t e d i n 16.3%, 4.1%, 5.5%, 22.4%, 9.5%, and 13.0% of samples r e s p e c t i v e l y . The average l e v e l of contamination was 3.85 ppm with a range of 0.01 ppm t o 141 ppm. A study by A g r i c u l t u r e Canada i n v e s t i g a t e d the i n c i d e n c e of zearalenone i n Western Canada ( P r i o r , 1981). Information on 499 p l a n t and animal specimens submitted t o the Animal Pathology Laboratory i n Saskatoon f o r mycotoxin a n a l y s i s d u r i n g the years 1975 t o 1979 was e v a l u a t e d . Only one sample of b a r l e y and two samples of corn were found t o be p o s i t i v e a t a d e t e c t i o n l e v e l of 500 ppb. S i n c e zearalenone contamination as low as 0.01 ppm has been demonstrated t o cause adverse e f f e c t s (Mirocha e t a l . , 1974) , c o n c e n t r a t i o n l e v e l s r e p o r t e d i n these surveys would pose a h e a l t h t h r e a t . As d i s c u s s e d e a r l i e r , zearalenone contamination i s not a random occurrence, but i s i n f a c t c o r r e l a t e d t o weather and storage c o n d i t i o n s . Under the a p p r o p r i a t e c o n d i t i o n s , corn, i n p a r t i c u l a r , i s v e r y s u s c e p t i b l e t o Fusarium i n v a s i o n and both the 24 i n c i d e n c e and l e v e l s of contamination can be h i g h . T h i s i s of p a r t i c u l a r concern t o E a s t e r n Canada where corn i s the main component i n r a t i o n f o r m u l a t i o n . S i n c e Western Canada r e l i e s on b a r l e y as a feed source, t h e r e i s l e s s p o t e n t i a l f o r zearalenone m y c o t o x i c o s i s i n l i v e s t o c k . However, s i g n i f i c a n t contamination o f b a r l e y by zearalenone producing s p e c i e s o f Fusarium has been r e p o r t e d i n B r i t i s h Columbia (Greenway and Pu i s , 1976). In 1973, b a r l e y o b t a i n e d from a boxcar from the. Peace R i v e r D i s t r i c t of A l b e r t a and B r i t i s h Columbia caused f u s a r i o t o x i c o s i s i n s e v e r a l farm s p e c i e s . Although zearalenone was not determined t o be one of the c o n t r i b u t i n g mycotoxins, the Fusarium s p e c i e s ( G i b b e r e l l a zeae) i s o l a t e d from s e l e c t e d samples i s capable of producing the mycotoxin. 2.1.2. CONVENTIONAL METHODS OF ZEARALENONE DETERMINATION IN FEED A l l of the surveys d i s c u s s e d were conducted u s i n g c o n v e n t i o n a l methods of zearalenone a n a l y s i s i n feed which i n c l u d e s : t h i n - l a y e r chromatography (TLC), h i g h - p r e s s u r e l i q u i d chromatography (HPLC), g a s - l i q u i d chromatography (GLC), and combination g a s - l i q u i d chromatography-mass spectrometry (GLC-MS) . In the past, TLC was the method of c h o i c e i n mycotoxin a n a l y s i s . In f a c t , the method of Eppley (1968) was the f i r s t t e chnique accepted by the A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists f o r zearalenone d e t e r m i n a t i o n . A f t e r s o l v e n t s e p a r a t i o n 25 of zearalenone from o t h e r components i n an e x t r a c t a p p l i e d t o s i l i c a g e l , the mycotoxin i s d e t e c t e d by i t s c h a r a c t e r i s t i c f l u o r e s c e n c e when i r r a d i a t e d with u l t r a v i o l e t l i g h t ( S t o l o f f e t a l . , 1971; Mirocha e t a l . , 1974; Thomas e t a l . , 1975). A l t e r n a t i v e l y , zearalenone can be d e t e c t e d a f t e r s e p a r a t i o n by s p r a y i n g w i t h a chromogenic reagent ( M a l a i y a n d i e t a l . , 1976; S c o t t e t a l . , 1978). In the HPLC method, the sample e x t r a c t i s separated by column chromatography and mycotoxin d e t e r m i n a t i o n i s done by the system's u l t r a v i o l e t and f l u o r e s c e n c e d e t e c t o r s (Holder e t a l . . 1977; M a l a i y a n d i and B a r r e t t e , 1978; Ware and Thorpe, 1978). In c o n t r a s t t o TLC, t h i s method i s capable of v e r y f i n e r e s o l u t i o n and e x c e l l e n t q u a n t i t a t i o n . L i k e HPLC procedures, GLC i s used f o r i t s r e s o l u t i o n and q u a n t i t a t i o n c a p a b i l i t i e s . The f i r s t s tep of GLC i n v o l v e s c o n v e r t i n g zearalenone's two hydroxy groups t o t h e i r c o r r e s p o n d i n g t r i - o r d i m e t h y l s i l y l e t h e r i n o r d e r t o i n c r e a s e v o l a t i l i t y . Then, zearalenone d e r i v a t i v e s are measured i n s o l u t i o n u s i n g flame i o n i z a t i o n d e t e c t i o n (Mirocha e t a l . , 1974; Holder e t a l . , 1977; Thouvenot and M o r f i n , 1979). The GLC-MS method i s used i n the c o n f i r m a t i o n o f suspect t o x i n s d e t e c t e d by other methods s i n c e i t p r o v i d e s d e f i n i t i v e i d e n t i f i c a t i o n of molecules. As i n GLC, zearalenone i s f i r s t c o n verted i n t o t r i - or d i m e t h y l s i l y l e t h e r d e r i v a t i v e s . A n a l y s i s i s then done by the mass spectrometer which focuses on the 26 c h a r a c t e r i s t i c fragment i o n s o f the mycotoxin ( S t e e l e e t a l . , 1976; S c o t t e t a l . , 1978). The development of these c o n v e n t i o n a l a n a l y t i c a l techniques f o r mycotoxin d e t e r m i n a t i o n i n samples has r e s u l t e d i n a c c u r a t e and s e n s i t i v e t e s t i n g procedures. However, these methods are time-consuming and r e q u i r e expensive equipment used by t r a i n e d t e c h n i c i a n s . As a r e s u l t , c o n v e n t i o n a l techniques are not s u i t a b l e f o r mycotoxin t e s t i n g o u t s i d e o f the l a b o r a t o r y where economical procedures simple enough f o r use by i n e x p e r i e n c e d personnel are r e q u i r e d . 2.1.3. IMMUNOCHEMICAL TECHNIQUES FOR ZEARALENONE DETERMINATION Recently, a l t e r n a t i v e techniques employing radioimmuno assay (RIA) o r enzyme-linked immunosorbent assay (ELISA) have been developed f o r zearalenone d e t e r m i n a t i o n . S p e c i f i c p o l y c l o n a l and monoclonal a n t i b o d i e s have been r a i s e d a g a i n s t z e a r a l e n o n e - p r o t e i n conjugates and used f o r immunochemical d e t e c t i o n of the mycotoxin i n samples (Thouvenot and M o r f i n , 1983; L i u e t a l . , 1985; Warner e t a l . , 1986; Dixon e t a l . , 1987). Both assays are based on c o m p e t i t i o n o f b i n d i n g between u n l a b e l l e d zearalenone i n the sample and l a b e l l e d zearalenone i n the assay system f o r s p e c i f i c b i n d i n g s i t e s o f antibody molecules (Chu, 1984). In the RIA system, the l a b e l f o r zearalenone i n the assay system i s r a d i o a c t i v i t y . A f t e r c o m p e t i t i o n f o r antibody b i n d i n g s i t e s , f r e e zearalenone and bound zearalenone are separated and r a d i o a c t i v i t y i n the two f r a c t i o n s i s determined. Zearalenone c o n c e n t r a t i o n i n the unknown sample i s determined by comparing r e s u l t s t o a standard curve. The standard curve i s c o n s t r u c t e d by p l o t t i n g the r a t i o of r a d i o a c t i v i t i e s i n the bound and f r e e f r a c t i o n a g a i n s t the l o g 1 0 c o n c e n t r a t i o n of u n l a b e l l e d standard t o x i n . The ELISA method employs zearalenone conjugated t o an enzyme (e.g. h o r s e r a d i s h peroxidase) as a l a b e l i n s t e a d of r a d i o a c t i v i t y . Instead of c o u n t i n g r a d i o a c t i v i t y , c o l o r i s measured when the enzyme r e a c t s with s u b s t r a t e . To a s o l i d phase coated w i t h a n t i b o d i e s , both sample s o l u t i o n c o n t a i n i n g unknown mycotoxin as w e l l as c o n t r o l s o l u t i o n c o n t a i n i n g enzyme conjugate are added and incubated. A f t e r the i n c u b a t i o n p e r i o d where c o m p e t i t i v e b i n d i n g with a n t i b o d i e s occurs, the s o l i d phase i s washed and the bound enzyme conjugate of mycotoxin i s determined. T h i s d e t e r m i n a t i o n i s accomplished by i n c u b a t i n g the enzyme conjugate w i t h a s u b s t r a t e s o l u t i o n c o n t a i n i n g hydrogen peroxide and a chromogen. The c o l o r r e a c t i o n i s measured s s p e c t r o p h o t o m e t r i c a l l y or by v i s u a l comparison w i t h standards. 2.1.4. AGRI-SCREEN™ FIELD TEST KIT FOR ZEARALENONE DETERMINATION Advances i n immunochemical techniques f o r mycotoxin d e t e c t i o n have l e d t o the development of Neogen C o r p o r a t i o n ' s Agri-screen™, a p r a c t i c a l f i e l d t e s t k i t . B a s i c a l l y , A g r i -screen™ i s an ELISA procedure i n v o l v i n g e x t r a c t i o n , f i l t r a t i o n , 28 a b s o r p t i o n w i t h antibody, a washing procedure, and q u a n t i f y i n g w i t h a c o l o r change. A l l the m a t e r i a l s r e q u i r e d f o r a n a l y s i s and i n s t r u c t i o n s f o r t h e i r use are s u p p l i e d by Neogen. The a b i l i t y o f the t e s t k i t t o a c c u r a t e l y d e t e c t zearalenone has been t e s t e d by the USDA and i s the o n l y r a p i d s c r e e n i n g procedure w i t h both AOAC and IUPAC appro v a l (Herbert, 1989). While the standard k i t i s s u p p l i e d w i t h a 500 ppb c o n t r o l , o p t i o n a l l e v e l s of 250, 1000, and 3000 ppb are a v a i l a b l e . Although the b a s i c procedure g i v e s q u a l i t a t i v e r e s u l t s , q u a n t i t a t i v e measurements can be obt a i n e d by c o n s t r u c t i n g a standard curve and u s i n g a m i c r b p l a t e reader. 2.2. OBJECTIVES FOR BARLEY ZEARALENONE CONTAMINATION SURVEY Ba r l e y serves as the main feed component of animal r a t i o n s formulated i n Western Canada. Although feed manufacturers r e c o g n i z e the p o t e n t i a l f o r zearalenone contamination, r e g u l a r t e s t i n g procedures f o r the mycotoxin are not c a r r i e d out on b a r l e y r e c e i v e d from g r a i n e l e v a t o r s . T h e r e f o r e , the f i r s t o b j e c t i v e of the survey was t o t e s t b a r l e y c o l l e c t e d from A l b e r t a and B r i t i s h Columbia e l e v a t o r s f o r p o s s i b l e zearalenone contamination. The second o b j e c t i v e was t o ev a l u a t e Neogen C o r p o r a t i o n s 1 s c l a i m t h a t the Agri-screen™ t e s t k i t i s a p r a c t i c a l method of zearalenone d e t e r m i n a t i o n i n the f i e l d . 29 2.3. MATERIALS AND METHODS 2.3.1. Sample C o l l e c t i o n , Storage and P r e p a r a t i o n Samples of b a r l e y (approximately 500 g) intended f o r animal r a t i o n f o r m u l a t i o n were c o l l e c t e d by E a s t C h i l l i w a c k A g r i c u l t u r a l Co-op from g r a i n e l e v a t o r l o c a t i o n s i n A l b e r t a and B r i t i s h Columbia (Table 1) d u r i n g October of 1988. An automated c r o s s - c u t sampler was used t o take samples a t predetermined i n t e r v a l s from a moving stream e n t e r i n g e l e v a t o r s . A f t e r c o l l e c t i o n , samples were s t o r e d a t 4°C. Immediately p r i o r t o e v a l u a t i o n , samples were ground t o a 1.0 mm screen s i z e u s i n g a Brinkmann g r i n d e r . 2.3.2. E x t r a c t i o n Procedure Two 5 g subsamples of each b a r l e y sample were mixed w i t h 50 ml of 70% methanol and the mixture was shaken v i g o r o u s l y f o r 2 min. Then, the mixture was passed through Whatman #1 f i l t e r paper and c o l l e c t e d . 2.3.3. Q u a l i t a t i v e A n a l y s i s B a r l e y subsample e x t r a c t s were analyzed f o r zearalenone contamination a t the 500 ppb l e v e l u s i n g m a t e r i a l s and i n s t r u c t i o n s p r o v i d e d by the Agri-Screen™ (Neogen C o r p o r a t i o n , Lansing, Michigan) t e s t k i t , an ELISA based method. Step 1: Zearalenone-enzyme conjugate was added t o a s t r i p of mixing w e l l s . To the c o n t r o l w e l l , 500 ppb zearalenone was added and mixed. To the remaining w e l l s , subsample e x t r a c t s were added and mixed. 30 Tabl e 1. B a r l e y sample sources ELEVATOR ALBERTA LOCATION ELEVATOR B.C. LOCATION AWP1 Bashaw AWP Dawson Creek AWP Chipman AWP F o r t S t . John AWP E l n o r a CARGILL F o r t S t . John AWP I r r i c a n a UGG Dawson Creek AWP Kathyrn UGG F o r t S t . John AWP Spruce Grove AWP Stony P l a i n AWP Three H i l l s AWP Trochu CARGILL Be i s e k e r CARGILL T a y l o r UGG2 B e i s e k e r UGG Camrose UGG Gaudin UGG Gibbons UGG Huxley UGG Lamont UGG M o r i n v i l l e UGG Olds 1 A l b e r t a Wheat Pool 2 U n i t e d G r a i n Growers 31 Step 2: From the mixing w e l l s , c o n t r o l and sample e x t r a c t s were t r a n s f e r r e d t o i n d i v i d u a l a n t i b o d y - c o a t e d w e l l s and 15 min of i n c u b a t i o n was allowed. Step 3: A f t e r i n c u b a t i o n , antibody-coated w e l l s were emptied and g e n t l y r i n s e d w i t h water 10 times. A l l remaining water d r o p l e t s were removed by t u r n i n g the w e l l s u p s i d e down and t a p p i n g them. Step 4: F o l l o w i n g the r i n s i n g procedure, s u b s t r a t e s o l u t i o n c o n s i s t i n g of hydrogen peroxide and the chromogen t r i m e t h y l b l u e were added t o each a n t i b o d y - c o a t e d w e l l . Step 5: A f t e r a l l o w i n g 15 min of r e a c t i o n time, s t o p p i n g reagent was added t o the antibody-coated w e l l s . Step 6: To e v a l u a t e the r e s u l t s , subsample w e l l c o l o r was compared with the 500 ppb c o n t r o l w e l l . A sample t h a t was b l u e r than the c o n t r o l c o n t a i n e d l e s s zearalenone than the c o n t r o l , w h ile a sample t h a t was l i g h t e r b l u e (or pinker) c o n t a i n e d more zearalenone than the c o n t r o l . 2.4. RESULTS A l l the subsample t e s t w e l l s were b l u e r than the c o n t r o l w e l l (Appendix 1). T h e r e f o r e , none of the b a r l e y samples t e s t e d p o s i t i v e f o r zearalenone contamination a t the 500 ppb l e v e l . 2.5. DISCUSSION B a r l e y o b t a i n e d from the g r a i n e l e v a t o r s l i k e l y would not have caused zearalenone contamination r e l a t e d problems. However, 32 s i n c e the zearalenone d e t e c t i o n l e v e l i n the survey was o n l y 500 ppb, samples c o u l d have c o n t a i n e d lower, u n d e t e c t a b l e l e v e l s of the mycotoxin. A d d i t i o n a l l y , the absence of s i g n i f i c a n t zearalenone contamination does not guarantee the b a r l e y samples t o be f r e e of o t h e r mycotoxins. I d e a l l y , commodity s c r e e n i n g procedures should be capable of r a p i d l y t e s t i n g f o r s e v e r a l mycotoxins. Feed sources are o f t e n s i m u l t a n e o u s l y contaminated w i t h v a r i o u s mycotoxin producing f u n g a l s p e c i e s , or f u n g a l s p e c i e s which produce more than one mycotoxin. For example, the mycotoxins d e o x y n i v a l e n o l and n i v a l e n o l are found n a t u r a l l y o c c u r r i n g t o g e t h e r with zearalenone (Mirocha e t a l . , 1976; Tanaka e t a l . , 1988). Together these two mycotoxins may e x e r t a d d i t i v e or s y n e r g i s t i c d e l e t e r i o u s e f f e c t s on animals. C o n v e n t i o n a l a n a l y t i c a l techniques have been designed t o a l l o w f o r simultaneous multi-mycotoxin t e s t i n g (Eppley, 1968; S t o l o f f e t a_l. , 1971; Wilson e t a l . , 1976; J o s e f s s o n and M o l l e r , 1977; Takeda e t a l . , 1979; Howell and T a y l o r , 1981). U n f o r t u n a t e l y , the t e c h n o l o g i c a l a b i l i t y t o d e t e c t s e v e r a l mycotoxins s i m u l t a n e o u s l y i s not simple enough t o apply t o the f i e l d s i t u a t i o n . E v a l u a t i o n of the Agri-Screen™ t e s t k i t showed i t t o be a simple procedure a l l o w i n g the s c r e e n i n g of s e v e r a l samples s i m u l t a n e o u s l y . Assuming samples a l r e a d y have been prepared f o r 33 a n a l y s i s , the e n t i r e procedure can be completed i n l e s s than an hour a t a c o s t of approximately $12 per sample. The o n l y n e g a t i v e comment concerns the s i z e of the mixing and antibody-coated w e l l s . S i n c e the w e l l s are even s m a l l e r than a thimble, h a n d l i n g them was not easy, and they o f t e n t i p p e d over by a c c i d e n t . In a d d i t i o n t o the zearalenone t e s t , Neogen C o r p o r a t i o n a l s o s e l l s Agri-Screen™ k i t s f o r a f l a t o x i n , T2 t o x i n and d e o x y n i v a l e n o l . The development of these ELISA based procedures has made s c r e e n i n g of feed components f e a s i b l e a t both g r a i n e l e v a t o r s and farms. For feed companies, i n c l u d i n g new mycotoxin t e s t i n g procedures as p a r t of r e g u l a r g r a i n a n a l y s i s would be prudent from a l e g a l aspect. A farmer whose animals s u f f e r from the symptoms of m y c o t o x i c o s i s i n c u r r e d by the consumption of s p o i l e d feed may seek l e g a l a c t i o n ( S c h i e f e r and O ' F e r r a l l , 1981). S i n c e the Agri-Screen™ t e s t k i t s are more q u a l i t a t i v e than q u a n t i t a t i v e i n nature, a sample found p o s i t i v e f o r mycotoxin contamination should be r e f e r r e d t o a l a b o r a t o r y f o r more thorough a n a l y s i s and c o n f i r m a t i o n . Although improvements i n technology w i l l r e s u l t i n e f f e c t i v e g r a i n m o n i t o r i n g programs i n the f u t u r e , proper sampling procedures must be employed t o ensure the v a l i d i t y of r e s u l t s . Problems i n h e r e n t t o mycotoxin a n a l y s i s make i t d i f f i c u l t t o o b t a i n a r e p r e s e n t a t i v e sample. A p p r o p r i a t e sampling methods take i n t o c o n s i d e r a t i o n problems such as low mycotoxin c o n c e n t r a t i o n and uneven d i s t r i b u t i o n o f mycotoxin i n g r a i n s . For i n s t a n c e , when sampling g r a i n e n t e r i n g an e l e v a t o r , stream sampling i s adv i s e d . An automated c r o s s - c u t sampler can be used which takes sm a l l p o r t i o n s from a moving stream a t predetermined i n t e r v a l s (Kramer, 1968). Then, a l l the i n d i v i d u a l p o r t i o n s a re combined t o g i v e a r e p r e s e n t a t i v e sample. T h i s removes the b i a s e s i n v o l v e d with sampling a s t a t i c source. 35 3.0 STUDIES ON THE DIRECT EFFECTS OF ZEARALENONE ON EMBRYONIC MORTALITY 3.1. INTRODUCTION Exposure of pregnant sows t o zearalenone as a r e s u l t of n a t u r a l contamination of feeds or through experimental a d m i n i s t r a t i o n can r e s u l t i n embryonic m o r t a l i t y . Experimental a d m i n i s t r a t i o n of zearalenone a t 60 ppm on days 2-15 postmating r e s u l t e d i n the absence of v i a b l e f e t u s e s when r e p r o d u c t i v e t r a c t s were examined on days 40-44 (Long and Diekman, 1983). In a subsequent i n v e s t i g a t i o n by Diekman and Long (1984) , 60 ppm of the mycotoxin was f e d d a i l y on days 2-6, 7-10, or 11-15 postmating. At the time of embryo development assessment on days 30-32 a f t e r breeding, a h i g h p r o p o r t i o n of animals g i v e n zearalenone on days 7-10 postmating were not pregnant. The 7-10 day postmating stage r e p r e s e n t s the time of embryo m i g r a t i o n w i t h i n the u t e r u s . From the r e s u l t s of hormonal s t u d i e s , i t was concluded t h a t m o r t a l i t y was not mediated by changes i n the s e c r e t o r y p a t t e r n of e i t h e r l u t e i n i z i n g hormone (LH) or f o l l i c l e s t i m u l a t i n g hormone (FSH) (Diekman and Long, 1984). In a more re c e n t study by Diekman and Long (1989), b l a s t o c y s t s were recovered and examined on days 10 and 14 of pregnancy from t r e a t e d animals g i v e n zearalenone on days 7-10 postmating and from a c o n t r o l group. On day 10, no s i g n i f i c a n t d i f f e r e n c e s were observed between c o n t r o l and treatment embryos. 36 However, day 14 b l a s t o c y s t s from the c o n t r o l group were fi l a m e n t o u s w h i l e those from t r e a t e d sows were fragmented and had f o c i of n e c r o s i s . Although the 7-10 day p e r i o d of pregnancy has been demonstrated t o be the c r i t i c a l time f o r zearalenone a d m i n i s t r a t i o n t o be harmful, adverse e f f e c t s on embryos are not m a n i f e s t a t e d u n t i l a few days l a t e r . T h e r e f o r e , d e l e t e r i o u s e f f e c t s on development occur d u r i n g or a f t e r b l a s t o c y s t e l o n g a t i o n which occurs on days 11-13 ( G e i s e r t e t a l . , 1982). Diekman and Long's (1989) study a l s o showed a change i n LH s e c r e t o r y p a t t e r n s i n the t r e a t e d group i n d i c a t i n g t h a t hormonal changes are a f a c t o r c o n t r i b u t i n g t o embryonic death. A d m i n i s t r a t i o n of e s t r a d i o l on days 7-10 a f t e r b r e e d i n g causes embryonic m o r t a l i t y i n sows (Gardner e t a l . , 1963; Morgan et a l . , 1987). I t has been proposed t h a t g i v i n g exogenous hormone may i n t e r f e r e w i t h s y n t h e s i s and s e c r e t i o n of endogenous e s t r a d i o l on days 11 and 12 of pregnancy (Heap e t a l . , 1975). Perhaps the e s t r o g e n i c a c t i o n of zearalenone i s r e s p o n s i b l e f o r the d e t r i m e n t a l e f f e c t s on embryos. Unpublished s t u d i e s by M a r t i n (1990) suggest t h a t zearalenone may cause embryonic m o r t a l i t y by changing the s e c r e t o r y response of the u t e r u s . G i l t s r e c e i v i n g the mycotoxin on days 7-10 had a l t e r e d calcium, t o t a l p r o t e i n , and a c i d phosphatase a c t i v i t y when u t e r i n e horns were f l u s h e d and the f l u i d examined 5 days f o l l o w i n g treatment. So f a r , no study has demonstrated whether zearalenone 37 causes embryonic m o r t a l i t y i n swine i n d i r e c t l y (e.g. s e c r e t o r y changes) or by d i r e c t a c t i o n . 3.2. PRIMARY OBJECTIVE OF STUDIES The primary o b j e c t i v e of the s t u d i e s i n t h i s s e c t i o n was t o determine i f zearalenone causes embryonic m o r t a l i t y through d i r e c t e f f e c t s on embryos. 3.3. PRELIMINARY STUDIES ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO MURINE PRE-IMPLANTATION EMBRYO DEVELOPMENT 3.3.1. OBJECTIVE OF PRELIMINARY STUDIES In o r d e r t o i n v e s t i g a t e p o s s i b l e t o x i c o l o g i c a l e f f e c t s , development of an i n v i t r o c u l t u r e system f o r e x p e r i m e n t a l l y exposing embryos t o zearalenone was necessary. Procedures i n c l u d i n g b l a s t o c y s t r e c o v e r y and b a s i c c u l t u r e methods were adapted from s t u d i e s i n v o l v i n g i n v i t r o murine p r e -i m p l a n t a t i o n embryo development (Rajamahendran e t a l . , 1986; L i n et a l . , 1989). 3.3.2. MATERIALS AND METHODS Animal Environment Experimental mice were obtained from the UBC Animal Care U n i t and housed i n the Department of Animal S c i e n c e mouse colony room. Stud males were kept i n d i v i d u a l l y i n wire-bottomed cages w h i l e females were group-housed i n p l a s t i c cages l i n e d w i t h wood shavings. 38 Room temperature was approximately 21°C and a 12-hour l i g h t / d a r k c y c l e was maintained w i t h a t i m e r mechanism. P e l l e t e d feed (Purina Mouse Chow 5015™) and f r e s h water were p r o v i d e d on an ad l i b i t u m b a s i s . S u p e r o v u l a t i o n Prepuberal (18-21 day old) CD-I mice were su p e r o v u l a t e d through treatment with the hormones pregnant mares 1 serum gonadotropin (PMSG:Equinex™) and human c h o r i o n i c gonadotropin (hCG:A.P.L.™) . I n t r a p e r i t o n e a l PMSG (5 IU) i n j e c t i o n was fo l l o w e d 48 hours l a t e r by hCG (2.5 IU) a d m i n i s t r a t i o n . F o l l o w i n g the hCG i n j e c t i o n , mice were p a i r e d w i t h s t u d males and checked f o r a v a g i n a l p l u g the next morning as evidence o f breedin g . Recovery o f B l a s t o c y s t s On the morning of the t h i r d day a f t e r c o n f i r m a t i o n o f breeding, mice were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n and p l a c e d under a laminar flow hood f o r b l a s t o c y s t r e c o v e r y . T h e i r u t e r i n e horns were c u t f r e e from the ovary, o v i d u c t and body of the u t e r u s , and p l a c e d on a paper towel soaked w i t h D u l b e c c o 1 s phosphate-buffered s a l i n e (PBS) (Gibco, Grand I s l a n d , New York) (Appendix 2). A 26 gauge needle a t t a c h e d t o a 1 ml s y r i n g e was i n s e r t e d i n t o the p o s t e r i o r end of each horn h e l d i n p l a c e by j e w e l l e r ' s f o r c e p s . The contents o f each horn were f l u s h e d with Ham's F-10 medium i n t o a watch g l a s s . A stereomicroscope (30 X m a g n i f i c a t i o n ) was used t o examine the f l u s h i n g of each horn f o r b l a s t o c y s t s . Located h e a l t h y b l a s t o c y s t s were t r a n s f e r r e d i n t o another watch g l a s s c o n t a i n i n g Ham's F-10 medium. T h i s step c l e a n s the embryos of d e b r i s s i n c e by-products of the recovery process such as b l o o d and u r i n e can h i n d e r development (Ackerman e t a l . , 1985; J i n n o e t a l . , 1987; McDowell e t a l . , 1988). From t h i s p o i n t , b l a s t o c y s t s were randomly d i s t r i b u t e d i n t o c u l t u r e . C u l t u r e Medium P r e p a r a t i o n In both p r e l i m i n a r y s t u d i e s , the c u l t u r e medium was a mixture of 80% Ham's F-10 and 20% e s t r o u s cow serum. Ham's F-10 (Gibco, Grand I s l a n d , New York) was prepared and s t e r i l i z e d from powder form (Appendix 3) a c c o r d i n g t o a p r o t o c o l developed by the UBC Human In V i t r o F e r t i l i z a t i o n Program (Appendix 4). Serum was obtained by c e n t r i f u g i n g b l o o d taken from the j u g u l a r v e i n of a H o l s t e i n d a i r y cow i n e s t r u s . Serum was heat-i n a c t i v a t e d by immersing i n a 56.5°C water bath f o r 30 minutes. Zearalenone P r e p a r a t i o n For the f i r s t p r e l i m i n a r y study, pure zearalenone (Sigma Chemical Company, S t . Lo u i s , M i s s o u r i ) was d i s s o l v e d i n 95% eth a n o l and then mixed with v e g e t a b l e o i l (canola oilrCrisco™) . The mixture was heated t o remove the ethanol l e a v i n g a s t o c k s o l u t i o n of 10 M9J zearalenone per 10 ill of o i l . The same steps were f o l l o w e d t o prepare zearalenone f o r the second p r e l i m i n a r y study w i t h the e x c e p t i o n t h a t the f i n a l s t o c k s o l u t i o n c o n t a i n e d 50 /xg zearalenone per 10 jul of o i l . P r e l i m i n a r y Study 1: C u l t u r e Procedure A t o t a l of 38 e a r l y b l a s t o c y s t s were c u l t u r e d i n microscope s l i d e c u l t u r e d i s h e s (Lux™:Miles L a b o r a t o r i e s , N a p e r v i l l e , I l l i n o i s ) . Ten /xl of medium c o n t a i n i n g a randomly s e l e c t e d embryo was added t o e i t h e r c o n t r o l or treatment w e l l s v i a a m i c r o p i p e t . C o n t r o l : 80 /xl of medium + 10 _/xl of v e g e t a b l e o i l Treatment: 80 /xl of medium + 10 /xl of v e g e t a b l e o i l c o n t a i n i n g 10 /xg of zearalenone The t o t a l c u l t u r e volume of a l l w e l l s was 100 /xl. A drop of p a r a f f i n o i l was added t o each w e l l t o prevent e v a p o r a t i o n . Embryos were then c u l t u r e d i n a w a t e r - j a c k e t e d i n c u b a t o r a t 37°C w i t h an atmosphere of 95% a i r and 5% carbon d i o x i d e . Development of embryos was observed every 12 hours from the e a r l y b l a s t o c y s t stage t o the hatched b l a s t o c y s t stage. P r e l i m i n a r y Study 2: C u l t u r e Procedure A t o t a l of 2 0 embryos were c u l t u r e d i n round-bottomed w e l l s (Falcon™: Becton D i c k i n s o n , L i n c o l n Park, New Jersey) . Ten /xl of medium c o n t a i n i n g a randomly s e l e c t e d embryo was added t o e i t h e r c o n t r o l or treatment w e l l s v i a a m i c r o p i p e t . C o n t r o l : 80 /xl of medium + 10 /xl of v e g e t a b l e o i l Treatment: 80 /xl of medium + 10 /xl of v e g e t a b l e o i l c o n t a i n i n g 41 50 /xg of zearalenone The t o t a l c u l t u r e volume of a l l w e l l s was 100 /xl. A drop of p a r a f f i n o i l was added t o each w e l l t o prevent e v a p o r a t i o n . Embryos were then c u l t u r e d and development noted as d e s c r i b e d i n p r e l i m i n a r y Study 1 above. 42 3.3.3. RESULTS Tables 2 and 3 show the number of c o n t r o l and treatment embryos which developed t o each stage i n both p r e l i m i n a r y s t u d i e s . Table 2. P r e l i m i n a r y Study 1 e f f e c t o f zearalenone on i n v i t r o murine b l a s t o c y s t development Embryos which developed i n t o : B l a s t o c y s t (n) Expanded B l a s t o c y s t n (%) Hatching B l a s t o c y s t n (%) Hatched B l a s t o c y s t n (%) Treatment 8 8 (100) 4 (50) 4 (50) C o n t r o l 30 29 (97) 22 (73) 22 (73) Table 3. P r e l i m i n a r y Study murine b l a s t o c y s t 2 e f f e c t of development zearalenone on i n v i t r o Embryos which developed i n t o : B l a s t o c y s t (n) Expanded B l a s t o c y s t n (%) Hatching B l a s t o c y s t n (%) Hatched B l a s t o c y s t n (%) Treatment 5 5 (100) 5 (100) 5 (100) C o n t r o l 15 15 (100) 15 (100) 15 (100) 43 3.3.4. DISCUSSION The r e s u l t s i n d i c a t e t h a t a t the l e v e l s of zearalenone s t u d i e d , development of murine embryos from the e a r l y b l a s t o c y s t t o the hatched b l a s t o c y s t stage i s not a f f e c t e d by the mycotoxin. The low s o l u b i l i t y o f zearalenone i n water n e c e s s i t a t e d the use of an i n e r t v e h i c l e i n order f o r the mycotoxin t o be s o l u b l e f o r i n t r o d u c t i o n i n t o water-based media. P o s s i b l y , i n c o m p a t i b i l i t y between the v e g e t a b l e o i l s o l v e n t and the c u l t u r e medium r e s u l t e d i n minimal exposure of embryos t o mycotoxin. As a r e s u l t , a d e c i s i o n was made t o use a s o l v e n t i m m i s c i b l e i n water f o r f u r t h e r s t u d i e s . Although these p r e l i m i n a r y s t u d i e s d i d not f u l f i l l the o b j e c t i v e of d e v e l o p i n g an e f f e c t i v e method of i n t r o d u c i n g zearalenone i n t o c u l t u r e , the c u l t u r e system i t s e l f was s u c c e s s f u l . T h e r e f o r e , a s i m i l a r system was employed i n subsequent i n v e s t i g a t i o n s . 3.4. STUDIES ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO MURINE PRE-IMPLANTATION EMBRYO DEVELOPMENT 3.4.1. INTRODUCTION The p r e l i m i n a r y s t u d i e s p r o v i d e d a s u i t a b l e system f o r o b t a i n i n g and c u l t u r i n g murine e a r l y b l a s t o c y s t s i n v i t r o . However, a s u i t a b l e s o l v e n t f o r i n t r o d u c i n g the mycotoxin i n t o medium was not determined. The low s o l u b i l i t y of zearalenone i n the water based medium meant t h a t a s u i t a b l e s o l v e n t was r e q u i r e d b e f o r e t o x i c o l o g i c a l s t u d i e s c o u l d be done. A paper p u b l i s h e d by Long and Turek (1989) p r o v i d e d the answer t o t h i s problem. T h e i r r e s u l t s showed t h a t 5 /xl 95% eth a n o l per ml medium can be used as a v e h i c l e t o i n t r o d u c e zearalenone i n t o c u l t u r e without a f f e c t i n g murine embryo development. With t h i s i n f o r m a t i o n , t o x i c o l o g i c a l s t u d i e s were made p o s s i b l e . 3.4.2. MATERIALS AND METHODS The animal environment, s u p e r o v u l a t i o n method, b l a s t o c y s t r e c o v e r y procedure, and c u l t u r e medium p r e p a r a t i o n were as d e s c r i b e d f o r the p r e l i m i n a r y i n v e s t i g a t i o n s . P r e p a r a t i o n of Zearalenone Stock S o l u t i o n s Stock s o l u t i o n #1: A 25 mg v i a l of zearalenone (Sigma Chemical Company St. L o u i s , M i s s o u r i ) was d i s s o l v e d with 500 /xl of 95% e t h a n o l . Stock s o l u t i o n #2: A 25 mg v i a l of zearalenone was d i s s o l v e d with 250 /xl of 95% e t h a n o l . Stock s o l u t i o n #3: The contents of two 25 mg zearalenone v i a l s were d i s s o l v e d with 300 /xl o f 95% e t h a n o l . Treatment P r e p a r a t i o n Study 1 Treatment 1: From the zearalenone s t o c k s o l u t i o n #1, 5 /xl was removed and mixed wi t h 995 /xl o f prepared c u l t u r e medium (80% Ham's F-10 + 20% e s t r o u s cow serum). 45 T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 250 /ig/ml o f medium or 25 /ig per w e l l . Treatment 2: From the zearalenone s t o c k s o l u t i o n #2, 5 / i l was removed and mixed wi t h 995 / i l o f prepared c u l t u r e medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 500 /ng/ml of medium or 50 /ng per w e l l . Treatment 3: From the zearalenone s t o c k s o l u t i o n #3, 5 /nl was removed and mixed with 995 /nl of prepared c u l t u r e medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 833 /ig/ml of medium or 83.3 ng per w e l l . C o n t r o l : To 995 /nl of c u l t u r e medium, 5 /nl of 95% e t h a n o l was added. Study 2 From the zearalenone stock s o l u t i o n #1, 5 / i l was removed and mixed with 995 / i l of c u l t u r e medium. From t h i s mixture A treatments 1 and 2 were d e r i v e d . Treatment 1: From the prepared mixture A, 12 0 /nl was removed and mixed with 880 /nl of c u l t u r e medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 3 0 /ng/ml medium or 3.0 /tg per w e l l . Treatment 2: From the prepared mixture A, 280 / i l was removed and mixed with 720 / i l of medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n o f 70 /ig/ml medium or 7.0 /tg per w e l l . Treatment 3: From the zearalenone s t o c k s o l u t i o n #1, 5 / i l was removed and mixed wi t h 995 / i l of c u l t u r e medium. 46 T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 250 /ig/ml medium or 25 /ig per w e l l . C o n t r o l : To 995 / i l of c u l t u r e medium, 5 / i l o f 95% et h a n o l was added. Study 3 From the zearalenone s t o c k s o l u t i o n #1, 5 / i l was removed and mixed wi t h 995 / i l o f medium. From t h i s mixture B treatments 1 and 2 were d e r i v e d . Treatment 1: From the prepared mixture B, 280 / i l was removed and mixed with 720 / i l o f c u l t u r e medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n o f 7 0 /ig/ml medium or 7.0 /tg per w e l l . Treatment 2: From the prepared mixture B, 64 0 / i l was removed and mixed with 360 / i l o f medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n of 160 /ig/ml medium or 16 /ig per w e l l . Treatment 3: From the zearalenone s t o c k s o l u t i o n #1, 5 / i l was removed and mixed wi t h 995 / i l o f prepared medium. T h i s y i e l d e d a zearalenone c o n c e n t r a t i o n o f 250 /ig/ml medium or 25 /ig per w e l l . C o n t r o l : To 995 / i l of medium, 5 / i l of 95% et h a n o l was added. Note: A summary of zearalenone c o n c e n t r a t i o n s used f o r a l l t h r e e s t u d i e s i s p r o v i d e d i n Table 4. C u l t u r e Procedure Healthy e a r l y b l a s t o c y s t stage embryos were i n d i v i d u a l l y 47 Table 4. Summary of zearalenone c o n c e n t r a t i o n s f o r a l l t h r e e s t u d i e s ZEARALENONE CONCENTRATION (jug/ml medium) STUDY 1 C o n t r o l 0 Treatment 1 250 Treatment 2 500 Treatment 3 833 STUDY 2 C o n t r o l 0 Treatment 1 30 Treatment 2 70 Treatment 3 250 STUDY 3 C o n t r o l 0 Treatment 1 70 Treatment 2 160 Treatment 3 250 48 t r a n s f e r r e d i n 10 /xl of c u l t u r e medium t o a U-bottomed c u l t u r e w e l l (Falcon™:Becton D i c k i n s o n , L i n c o l n Park, New Jersey) c o n t a i n i n g 90 /xl of treatment or c o n t r o l . In a l l t h r e e s t u d i e s , a drop of p a r a f f i n o i l was added t o each c u l t u r e w e l l t o prevent e v a p o r a t i o n . C u l t u r e t r a y s were p l a c e d i n a wat e r - j a c k e t e d i n c u b a t o r s e t a t 3 7°C and an atmosphere of 95% a i r and 5% carbon d i o x i d e . Embryo E v a l u a t i o n Embryos were observed every 6-10 hours f o r a t o t a l o f 44 hours and t h e i r development stage recorded by an a l p h a b e t i c a l s c o r e (Table 5) . A l s o , embryo development was photographed under 400-1000 X m a g n i f i c a t i o n . T a b l e 5. Development stage a l p h a b e t i c a l s c o r i n g A l p h a b e t i c a l Score Development Stage A e a r l y b l a s t o c y s t B mature b l a s t o c y s t C expanded b l a s t o c y s t D ha t c h i n g b l a s t o c y s t E hatched b l a s t o c y s t F degenerating b l a s t o c y s t G degenerated b l a s t o c y s t S t a t i s t i c a l A n a l y s i s The embryo development r e s u l t s o b t a i n e d i n Study 3 were analyzed by the Chi-square t e s t f o r independence f o r treatment e f f e c t s (p > 0.05) ( L i t t l e and Jackson, 1978). 49 3.4.3. RESULTS The a l p h a b e t i c a l s c o r e s f o r b l a s t o c y s t development under c o n t r o l and treatment c o n d i t i o n s f o r s t u d i e s 1, 2, and 3 are recorded i n Appendices 5, 6, and 7 r e s p e c t i v e l y . G r a p h i c a l i l l u s t r a t i o n s of the e f f e c t o f zearalenone c o n c e n t r a t i o n on b l a s t o c y s t development are p r o v i d e d i n F i g u r e s 2 (A-F) (Study 1), 3 (A-F) (Study 2), and 4 (A-F) (Study 3). A l s o , summaries of b l a s t o c y s t development are g i v e n i n Ta b l e s 6, 7 and 8. Study 1 A f t e r 6 hours c u l t u r e , a l l " b l a s t o c y s t s exposed t o zearalenone at c o n c e n t r a t i o n s of 500 (Treatment 2) and 833 (Treatment 3) /ig/ml medium were degenerating. At 12 hours c u l t u r e , 50% of b l a s t o c y s t s were degenerating i n medium c o n t a i n i n g the mycotoxin a t 2 50 /tg/ml (Treatment 1) . The number of Treatment 1 embryos degenerating i n c r e a s e d throughout the c u l t u r e p e r i o d u n t i l e v e n t u a l l y a l l were d e t r i m e n t a l l y a f f e c t e d . Study 2 A f t e r 6 hours c u l t u r e , a l l b l a s t o c y s t s i n medium c o n t a i n i n g zearalenone a t a c o n c e n t r a t i o n of 250 /tg/ml (Treatment 3) were degenerating. For the m a j o r i t y of b l a s t o c y s t s a s s i g n e d t o Treatment 1 (30 /tg/ml) and Treatment 2 (70 /ig/ml) , development was not a f f e c t e d by the mycotoxin. Study 3 At 12 hours c u l t u r e , the m a j o r i t y of b l a s t o c y s t s exposed t o a zearalenone c o n c e n t r a t i o n of 250 /tg/ml medium (Treatment 3) 50 were degenerating. For those b l a s t o c y s t s a s s i g n e d t o Treatment 2 (160 /xg zearalenone/ml) , development was not markedly a f f e c t e d u n t i l 28 hours c u l t u r e when 14/30 were degenerating. As f o r Treatment 1 b l a s t o c y s t s (70 /xg zearalenone/ml) , the m a j o r i t y of them developed normally. S t a t i s t i c a l a n a l y s i s of Study 3 r e s u l t s (Table 8) demonstrates t h a t zearalenone a t a c o n c e n t r a t i o n of 250 /xg/ml medium s i g n i f i c a n t l y a f f e c t e d embryo development t o the mature, expanded, h a t c h i n g , and hatched stages. However, a t a c o n c e n t r a t i o n l e v e l of 160 /ig/ml medium, development of embryos to the expanded, h a t c h i n g , and hatched stages was s i g n i f i c a n t l y a f f e c t e d , but not t o the mature stage. F i g u r e s 5-9 show the developmental stages observed d u r i n g the c u l t u r e p e r i o d . A d d i t i o n a l l y , F i g u r e 10 d e p i c t s a t y p i c a l d e g enerating b l a s t o c y s t . 51 F i g u r e 2 (A-F). Study 1: e f f e c t o f zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. 6 hours culture B. 12 hours culture Number of embryos 12-i A B C D E F G Stage of development control UMi treat 1 111 treat 2 I U treat 3 52 F i g u r e 2 (A-F). Continued C. 22 hours culture Number of embryos 12-i A B C D E F G Stage of development control K M treat 1 H H treat 2 H H treat 3 D. 28 hours culture Number of embryos 12 -i A B C D E F G Stage of development control i l treat 1 mm treat 2 Wm. treat 3 53 F i g u r e 2 (A-F). Continued E. 34 hours culture Number of embryos 12 i A B C D E F G Stage of development control treat 1 CZD treat 2 H H treat 3 F. 44 hours culture Number of embryos A B C D E F G Stage of development control treat 1 111 treat 2 H H treat 3 54 Table 6. Study 1: Summary of murine b l a s t o c y s t development B l a s t o c y s t Stage E a r l y n Mature n (%) Expanded n (%) Hatching n (%) Hatched n (%) C o n t r o l 10 10 (100) 10 (100) 9 (90) 9 (90) Treatment 1 10 7 (70) 5 (50) 2 (20) 0 Treatment 2 9 0 0 0 0 Treatment 3 10 0 0 0 0 55 F i g u r e 3 (A-F). Study 2: e f f e c t o f zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. 6 hours culture Number of embryos 12 n A B C D E F G Stage of development control treat 1 111 treat 2 treat 3 B. 12 hours culture 56 gure 3 (A-F). Continued C. 22 hours culture Number of embryos 12 - i A B C D E F G Stage of development control treat 1 MM treat 2 ! • treat 3 D. 28 hours culture Number of embryos 12 -i A B C D E F G Stage of development 57 F i g u r e 3 (A-F). Continued E. 34 hours culture Number of embryos 12 -i A B C D E F G Stage of development control treat 1 H H treat 2 i H H treat 3 F. 44 hours culture Number of embryos 12 1 : A B C D E F G Stage of development control S I treat 1 111 treat 2 I U treat 3 58 Ta b l e 7. Study 2: Summary of murine b l a s t o c y s t development B l a s t o c y s t Stage E a r l y n Mature n (%) Expanded n (%) Hatching n (%) Hatched n (%) C o n t r o l 10 9 (90) 9 (90) 9 (90) 9 (90) Treatment 1 10 9 (90) 9 (90) 9 (90) 9 (90) Treatment 2 9 9 (100) 9 (100) 5 (56) 4 (44) Treatment 3 10 0 0 0 0 59 F i g u r e 4 (A-F). Study 3: e f f e c t o f zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. 6 hours culture Number of embryos A B C D E F G Stage of development control treat 1 I I treat 2 iHH treat 3 B. 1 2 hours culture Number of embryos 30 -i A B C D E F G Stage of development control tMXsl treat 1 mm treat 2 Wm. treat 3 6 0 F i g u r e 4 (A-F). Continued C. 22 hours culture Number of embryos 35 A B C D E F G Stage of development control IM^i treat 1 IS^l treat 2 H H treat 3 D. 28 hours culture Number of embryos 35 -i 61 F i g u r e 4 (A-F). Continued E. 34 hours culture Number of embryos 35 - i A B C D E F G Stage of development H i control KM treat 1 I I treat 2 WM treat 3 F. 44 hours culture Number of embryos A B C D E F G Stage of development 62 Table 8. Study 3: Summary of murine b l a s t o c y s t development B l a s t o c y s t Stage E a r l y n Mature n (%) Expanded n (%) Hatching n (%) Hatched n (%) C o n t r o l 30 30 a(100) 28 a(93) 26 a(87) 26 a(87) Treatment 1 30 29 a (97) 29 a(97) 26 a(87) 26 a(87) Treatment 2 30 27 a (90) 19 b(63) 5 b(17) 5 b(17) Treatment 3 30 13 b (43) 2 C (7) 0 b 0 b v a l u e s i n the same column wi t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (p < 0.05). 64 F i g u r e 7 . Expanded murine b l a s t o c y s t 66 3.4.4. DISCUSSION Based on the r e s u l t s presented, murine b l a s t o c y s t s c u l t u r e d i n v i t r o can t o l e r a t e zearalenone c o n c e n t r a t i o n l e v e l s as h i g h as 70 /xg/ml medium without development being a f f e c t e d . However, a c o n c e n t r a t i o n of 160 /tg/ml w i l l cause b l a s t o c y s t d egeneration. T h e r e f o r e , the c r i t i c a l l e v e l f o r the mycotoxin t o have d e l e t e r i o u s e f f e c t s occurs i n the c o n c e n t r a t i o n range of 70-160 /tg zearalenone/ml medium. A d d i t i o n a l l y , the r e s u l t s demonstrate a c o n c e n t r a t i o n e f f e c t on the l e n g t h of time r e q u i r e d f o r zearalenone t o e x e r t i t s d e t r i m e n t a l a c t i o n s . At mycotoxin c o n c e n t r a t i o n s of 500 and 833 /tg/ml medium, b l a s t o c y s t s degenerate a f t e r 6 hours of c u l t u r e . While a t a lower l e v e l of 160 /tg/ml, b l a s t o c y s t s are not a f f e c t e d u n t i l 28 hours c u l t u r e . The r e s u l t s support Long and Turek's (1989) s t u d i e s which demonstrate t h a t zearalenone n e g a t i v e l y a f f e c t s i n v i t r o murine embryo development. However, h i g h e r zearalenone t o l e r a n c e by murine b l a s t o c y s t s was found i n t h i s t h e s i s than r e p o r t e d i n t h e i r i n v e s t i g a t i o n s . In Long and Turek's (1989) s t u d i e s , the number of embryos d e v e l o p i n g from the b l a s t o c y s t t o the egg c y l i n d e r stage decreased as the c o n c e n t r a t i o n of mycotoxin was i n c r e a s e d from 8.5 t o 68 /tg/ml of c u l t u r e medium. At a c o n c e n t r a t i o n of 32 /tg/ml, zearalenone a f f e c t e d growth i n 50% of the embryos. 67 C l o s e r examination showed t h a t t r e a t e d embryos were s m a l l e r , had l e s s c e l l u l a r d i f f e r e n t i a t i o n , and had more p h a g o c y t i c v a c u o l e s than c o n t r o l embryos (Long and Turek, 1989). The s m a l l e r s i z e was determined t o be the r e s u l t of decreased numbers of c e l l s o r i g i n a t i n g from the i n n e r c e l l mass. Based on these f i n d i n g s , a decreased c e l l d i v i s i o n r a t e combined wi t h an i n c r e a s e d r a t e of c e l l death l i k e l y were the f a c t o r s a f f e c t i n g growth. S t u d i e s with agents proven t o a f f e c t b l a s t o c y s t growth have demonstrated harmful e f f e c t s on the i n n e r c e l l mass (Sp i n d l e and Wu, 1985). In a d d i t i o n , Long and Turek (1989) exposed b l a s t o c y s t s t o 1 7 6 - e s t r a d i o l . No harmful e f f e c t s of the hormone on b l a s t o c y s t development were found. T h e r e f o r e , they concluded t h a t the d e t r i m e n t a l e f f e c t s of zearalenone on murine embryo development are not the r e s u l t of an e s t r o g e n i c e f f e c t . In f a c t , t h e r e i s evidence t o suggest t h a t i n mice e s t r a d i o l i s necessary f o r i m p l a n t a t i o n t o occur, and t h a t embryos may even s e c r e t e t h e i r own estrogens. For i n s t a n c e , Gupta e t a l . (1977) showed t h a t the i n v i t r o development of murine 4-8 c e l l embryos to the b l a s t o c y s t stage i s i n h i b i t e d by the a d d i t i o n of a n t i - e s t r o g e n to the media. A l a t e r study by Sengupta e t a_l. (1982) demonstrated t h a t development of murine 4-8 c e l l embryos i s blocked by the presence of estrogen s y n t h e s i s i n h i b i t o r . When 1 7 6 - e s t r a d i o l was added t o c u l t u r e , embryos developed normally, s u g g e s t i n g t h a t blastomere estrogen 68 s y n t h e s i s i s e s s e n t i a l f o r t r a n s i t i o n t o the b l a s t o c y s t stage. L i k e zearalenone, other chemicals i n c l u d i n g c a f f e i n e and gossypol have been found t o i n t e r f e r e with i n v i t r o murine b l a s t o c y s t development (Spi n d l e and Wu, 1985 and L i n e t a l . , 1989). In order f o r these substances t o a f f e c t development i n v i v o , they must e n t e r the u t e r i n e environment. In the case of zearalenone, a u t o r a d i o g r a p h i c s t u d i e s w i t h pregnant mice by Appelgren e t a l . (1982) found accumulation of the mycotoxin i n f e t u s e s , p l a c e n t a l t i s s u e s and u t e r i n e f l u i d . During l a t e pregnancy, zearalenone c o n c e n t r a t i o n i n the u t e r i n e f l u i d and y o l k sac p l a c e n t a was almost as h i g h as t h a t i n maternal u r i n e . T h i s demonstrate 1s z e a r a l e n o n e 1 s a b i l i t y t o c r o s s the p l a c e n t a l b a r r i e r . Although the mechanism(s) by which zearalenone a f f e c t s development have ye t to be f u l l y e x p l a i n e d , the mycotoxin can a c t d i r e c t l y on murine embryos as r e v e a l e d by i n v i t r o s t u d i e s . 69 3.5. DEVELOPMENT OF A PORCINE PRE-IMPLANTATION EMBRYO CULTURE SYSTEM 3.5.1. INTRODUCTION In order t o study the t o x i c o l o g i c a l e f f e c t s o f zearalenone on i n v i t r o p o r c i n e p r e - i m p l a n t a t i o n embryo development, a proven c u l t u r e system was necessary. T h e r e f o r e , the i n i t i a l o b j e c t i v e was t o s u c c e s s f u l l y c u l t u r e p o r c i n e morulae t o the hatched b l a s t o c y s t stage. 3.5.2. MATERIALS AND METHODS Note: P r i o r t o the experimental study, p r e l i m i n a r y t r i a l s were conducted i n order t o l e a r n techniques such as hormonal puberty i n d u c t i o n , s u p e r o v u l a t i o n , a r t i f i c i a l i n s e m i n a t i o n , as w e l l as embryo re c o v e r y d e s c r i b e d below. Experimental Animals Prepuberal Large White X Landrace g i l t s (8 0-90 kg) from the UBC swine herd were used. Media P r e p a r a t i o n Ham's F-10 (Gibco, Grand I s l a n d , New York) was prepared from powder form (Appendix 3) a c c o r d i n g t o a p r o t o c o l developed by the UBC Human In V i t r o F e r t i l i z a t i o n program (Appendix 4). Cow serum was obtained from the bloo d o f UBC d a i r y herd H o l s t e i n s i n e s t r u s . Blood was c e n t r i f u g e d t o o b t a i n serum which was immediately h e a t - i n a c t i v a t e d by i n c u b a t i n g i n a 56.5°C water bath f o r 3 0 minutes. Serum was f r o z e n a t -2 0°C u n t i l use. 70 Minimal E s s e n t i a l Medium (MEM) (Gibco, Grand I s l a n d , New York) was obtained i n l i q u i d form (Appendix 8 ) . To the prepared medium, an a n t i b i o t i c / a n t i m y c o t i c s o l u t i o n (Gibco, Grand I s l a n d , New York) was added t o p r o v i d e 10,000 u n i t s p e n i c i l l i n , 10,000 jug s t r e p t o m y c i n and 25 /ng amphotericin per ml medium. H e a t - i n a c t i v a t e d f e t a l c a l f serum (HIFCS) was ob t a i n e d from Sigma Chemical Company (St. L o u i s , M i s s o u r i ) and was s t o r e d a t -2 0°C u n t i l use. Hormonal Puberty I n d u c t i o n and S u p e r o v u l a t i o n A t o t a l of 5 pre p u b e r a l g i l t s which had reached s l a u g h t e r weight (80-90 kg) were t r e a t e d with a simultaneous i n t r a m u s c u l a r i n j e c t i o n of 400 IU PMSG (Equinex™) and 200 IU hCG (A.P.L.™) t o induce e s t r u s and o v u l a t i o n . Three days f o l l o w i n g the hormonal treatment, g i l t s observed t o be i n e s t r u s were bred a r t i f i c i a l l y w ith p r e s e r v e d semen u s i n g a s p i r e t t e . Boar semen was ob t a i n e d from the A l b e r t a Swine Breeding Centre, Leduc, A l b e r t a . Embryo Recovery Procedure F i v e days pos t - b r e e d i n g , g i l t s were s l a u g h t e r e d , exsanguinated, and s c a l d e d by I n t e r c o n t i n e n t a l Packers, Vancouver, B.C.. Reproductive t r a c t s were then c o l l e c t e d and t r a n s p o r t e d t o the l a b o r a t o r y approximately 1.5 hours f o l l o w i n g donor death. P r i o r t o embryo recovery, o v a r i e s were examined and s i z e , 71 number of f o l l i c l e s , and number of co r p o r a l u t e a recorded. The upper t h i r d of each u t e r i n e horn was f l u s h e d w i t h p r e -warmed (37°C) D u l b e c c o 1 s PBS (Appendix 2) u s i n g an 18 gauge needle a t t a c h e d t o a 60 ml s y r i n g e . Flushed s a l i n e , p r e -i m p l a n t a t i o n embryos and u n f e r t i l i z e d oocytes were c o l l e c t e d i n a 9 X 1.5 cm s t e r i l e p e t r i d i s h . Recovered morulae and u n f e r t i l i z e d oocytes i n the p e t r i d i s h were l o c a t e d , e v a l u a t e d and recorded w i t h the a i d of a s t e r e o s c o p i c microscope (30 X m a g n i f i c a t i o n ) . Morula stage embryos were washed i n D u l b e c c o 1 s PBS t o remove d e b r i s and then were randomly a s s i g n e d t o organ c u l t u r e w e l l s (Falcon™:Becton Di c k i n s o n , L i n c o l n Park, New Jersey) c o n t a i n i n g one of two treatment media: Treatment 1: 80% Ham's F-10 + 20% h e a t - i n a c t i v a t e d e s t r o u s cow serum Treatment 2: 90% Minimum E s s e n t i a l Medium + 10% h e a t - i n a c t i v a t e d f e t a l c a l f serum A t o t a l of 31 h e a l t h y morula stage embryos were recovered with 15 a s s i g n e d to Treatment 1 and 16 a s s i g n e d t o Treatment 2. Organ c u l t u r e w e l l s were p l a c e d i n a w a t e r - j a c k e t e d i n c u b a t o r s e t a t 37°C and a t an atmosphere of 95% a i r and 5% carbon d i o x i d e . Development of embryos was observed and recorded 6, 12, 18, and 24 hours a f t e r the s t a r t of i n c u b a t i o n . A d d i t i o n a l l y , photographs were taken under 400 X m a g n i f i c a t i o n . 72 Note: As p a r t of a t e a c h i n g e x e r c i s e , undergraduate r e p r o d u c t i v e p h y s i o l o g y students p a r t i c i p a t e d i n g i l t a r t i f i c i a l i n s e m i n a t i o n and morula recovery procedures. 3.5.3. RESULTS Based on ovary corpora l u t e a counts (Table 9) , the average number of o v u l a t i o n s per hormonally t r e a t e d g i l t was 30 w i t h the range being 8-39. Average recovery percentage (number of morulae + number of u n f e r t i l i z e d oocytes/number of o v u l a t i o n s X 100) was 48% w i t h the range being 0-63% (Table 10). The average number of morulae recovered per g i l t was 6 wh i l e the average f o r u n f e r t i l i z e d oocytes was 11. The range of rec o v e r y was 0-15 and 0-21 f o r morulae and oocytes r e s p e c t i v e l y . In both c u l t u r e media, none .of the morulae developed (Table 11). Approximately 24 hours f o l l o w i n g c u l t u r e , morulae had begun t o form v e s i c l e s and degenerate ( F i g u r e 11). 73 Tab l e 9. Reproductive t r a c t e v a l u a t i o n o f g i l t s LEFT OVARY RIGHT OVARY GILT 1 ovary s i z e (cm) 3.5 3.5 No. c o r p o r a l u t e a 18 21 No. f o l l i c l e s .0 0 GILT 2 ovary s i z e (cm) 4 . 0 3.0 No. corp o r a l u t e a 4 4 No. f o l l i c l e s 12 8 GILT 3 ovary s i z e (cm) 3 . 0 5.0 No. corp o r a l u t e a 17 18 No. f o l l i c l e s 1 2 GILT 4 ovary s i z e (cm) 3 . 0 3 . 0 No. corp o r a l u t e a 24 14 No. f o l l i c l e s 3 0 GILT 5 ovary s i z e (cm) 3 . 5 3 . 5 No. corp o r a l u t e a 14 16 No. f o l l i c l e s 2 4 74 Table 10. Oocyte and embryo r e c o v e r y from g i l t r e p r o d u c t i v e t r a c t s LEFT HORN RIGHT HORN GILT 1 No. CL*=18 No. CL= 21 No. morulae 3 5 No. oocytes 5 9 GILT 2 No. CL =4 No. CL= 4 No. morulae 0 0 No. oocytes 0 0 GILT 3 No. CL =17 No. CL= 18 No. morulae 0 1 No. oocytes 4 17 GILT 4 No. CL =24 No. CL= 14 No. morulae 5 2 No. oocytes 8 9 GILT 5 No. CL =14 No. CL= 16 No. morulae 9 6 No. oocytes 0 2 *CL = corpora l u t e a 75 Table 11. P o r c i n e morulae c u l t u r e r e s u l t s CULTURE MEDIA No. morulae c u l t u r e d No. morulae deg e n e r a t i n g a f t e r 24 h Ham's F-10 15 15 MEM 16 16 76 F i g u r e 11. Degenerating p o r c i n e morulae 77 3.5.4. DISCUSSION Although the o v u l a t i o n r a t e i n d i c a t e s a good response t o hormonal puberty i n d u c t i o n and s u p e r o v u l a t i o n , f e r t i l i z a t i o n and r e c o v e r y r e s u l t s were poor. Perhaps, the i n e x p e r i e n c e of students i n v o l v e d with a r t i f i c i a l i n s e m i n a t i o n techniques and f l u s h i n g procedures was r e s p o n s i b l e f o r poor f e r t i l i z a t i o n and r e c o v e r y r a t e s . S i n c e none of the morulae developed i n c u l t u r e , the r e s u l t s c o n f i r m the c o n c l u s i o n of Meyen e t al,. (1989) t h a t MEM amino a c i d s and v i t a m i n s are d e t r i m e n t a l t o p o r c i n e p r e - i m p l a n t a t i o n embryo development. MEM was chosen as a c u l t u r e medium based on the c l a i m s of Kuzan and Wright (1982) and Stone e t a l . (1984) t h a t i t supports both p o r c i n e morula and b l a s t o c y s t development. F e t a l c a l f serum was i n c l u d e d s i n c e t h i s p r o t e i n source i s a l s o r e p o r t e d t o support development (Robl and Davis, 1981; Kuzan e t a l . , 1982; Stone e t a l . , 1984). Disagreement among r e s e a r c h f i n d i n g s c o n c e r n i n g the s u i t a b i l i t y of MEM i l l u s t r a t e s the l a c k of i n f o r m a t i o n r e g a r d i n g the i n v i t r o requirements of p o r c i n e morulae and b l a s t o c y s t s (Robl and Davis, 1981). However, u n s u c c e s s f u l p o r c i n e i n v i t r o c u l t u r e can not be blamed e n t i r e l y on inadequate n u t r i e n t s and c o n d i t i o n s . Recovery procedures can harm p r e - i m p l a n t a t i o n embryos and a f f e c t t h e i r 78 v i a b i l i t y . Before a r e p r o d u c t i v e t r a c t i s c o l l e c t e d from a commercial s l a u g h t e r f a c i l i t y , the donor animal has been k i l l e d and the body exsanguinated as w e l l as s u b j e c t e d t o h i g h temperature (63°C) s c a l d i n g and s c r u b b i n g . S c h l i e p e r and H o l t z (1986) demonstrated t h a t embryos s u b j e c t e d t o the l a t t e r two s t r e s s e s are l e s s v i a b l e than those c o l l e c t e d by laparotomy, or immediately a f t e r s l a u g h t e r . A l s o , d e l a y i n g the f l u s h i n g of t r a c t s a f t e r s l a u g h t e r can harm the embryos even i f the temperature i s kept a t 37°C (Wollenberg e t a l . , 1990). P o s s i b l y , u n f a v o r a b l e c o n d i t i o n s are c r e a t e d by postmortem changes t o the u t e r u s . Since i n our study the recovery of morulae was not p r a c t i c a l a t the s l a u g h t e r s i t e ( I n t e r c o n t i n e n t a l P a c k e r s ) , t r a c t s were t r a n s f e r r e d t o the l a b o r a t o r y and f l u s h e d approximately 2 hours a f t e r s l a u g h t e r . S t r e s s e s i n f l i c t e d upon morulae at I n t e r c o n t i n e n t a l Packers and d u r i n g the unavoidable r e c o v e r y d e l a y most l i k e l y a f f e c t e d v i a b i l i t y . T h e r e f o r e , our u n s u c c e s s f u l r e s u l t s can be a t t r i b u t e d t o both harmful embryo recover y methods and inadequate c u l t u r e c o n d i t i o n s . Because a s u c c e s s f u l c u l t u r e system c o u l d not be e s t a b l i s h e d , no t o x i c o l o g i c a l s t u d i e s concerning zearalenone and p o r c i n e p r e - i m p l a n t a t i o n development were conducted. 79 4.0 STUDY ON THE DIRECT TOXICOLOGICAL EFFECTS OF ZEARALENONE ON IN VITRO BOVINE OOCYTE MATURATION 4.1. INTRODUCTION The u t i l i z a t i o n of bovine oocytes r e c o v e r e d from o v a r i e s o b t a i n e d from slaughterhouses p r o v i d e s an economical way of maximizing female r e p r o d u c t i v e p o t e n t i a l . Recovered oocytes can be matured and f e r t i l i z e d i n v i t r o t o produce a p l e n t i f u l supply of embryos f o r r e s e a r c h as w e l l as embryo t r a n s f e r programs. In v i t r o n u c l e a r maturation (resumption and completion of m e i o s i s I) of bovine oocytes taken from non-ovulatory stage f o l l i c l e s was f i r s t accomplished by Edwards (1965). However, not u n t i l s e v e r a l years l a t e r was the p o t e n t i a l of i n v i t r o matured oocytes r e a l i z e d . Newcomb et a l . (1978) r e p o r t e d the b i r t h of c a l v e s from the s u c c e s s f u l t r a n s f e r of c u l t u r e d oocytes t o o v i d u c t s of r e c i p i e n t cows f o r f e r t i l i z a t i o n and g e s t a t i o n . L a t e r , C r i t s e r e t a l . (1986) r e p o r t e d the f i r s t pregnancy a f t e r embryo t r a n s f e r of b l a s t o c y s t s produced by i n v i t r o m aturation and f e r t i l i z a t i o n of oocytes obtained from slaughterhouse o v a r i e s . The b e s t i n d i c a t o r of bovine oocyte a b i l i t y t o mature i n v i t r o i s the q u a l i t y of i t s c e l l u l a r investments. Those oocytes which are completely surrounded by s e v e r a l l a y e r s of cumulus have the b e s t chance f o r s u c c e s s f u l i n v i t r o development ( L e i b f r i e d and F i r s t , 1979; Fukai and Sakuma, 1980; Suss e t a l . , 1988) . 80 A c c o r d i n g t o L e i b f r i e d and F i r s t (1979), n e i t h e r s i z e of f o l l i c l e s g r e a t e r than or equal t o 1 mm or e s t r o u s stage of the donor cow i n f l u e n c e s oocyte maturation a b i l i t y . In o r d e r f o r oocytes t o mature v i a completion of the f i r s t r e d u c t i o n d i v i s i o n , the c u l t u r e system must p r o v i d e a p p r o p r i a t e c o n d i t i o n s . T i s s u e c u l t u r e medium (TCM) 199 has been demonstrated t o support oocyte maturation ( L e i b f r i e d and F i r s t , 1979; C r i t s e r e t a l . , 1986; L e i b f r i e d - R u t l e d g e e t a l . , 1986). A l s o , f e t a l c a l f serum has been shown t o be an a p p r o p r i a t e p r o t e i n source ( C r i t s e r e t a l . , 1986; Fukui and Ono, 1989). A d d i t i o n a l l y , g ranulosa c e l l s have been i n c l u d e d i n c u l t u r e t o promote the c e l l u l a r i n t e r a c t i o n s o c c u r r i n g d u r i n g f i n a l oocyte maturation ( C r i t s e r e t a l . , 1986; Fukui and Ono, 1988; Fukui and Ono, 1989). 4.2. OBJECTIVE OF STUDY The o b j e c t i v e of t h i s study was t o determine i f a zearalenone c o n c e n t r a t i o n l e v e l known t o have a t o x i c o l o g i c a l e f f e c t on murine p r e - i m p l a n t a t i o n embryo development would a f f e c t bovine oocyte maturation. 4.3. MATERIALS AND METHODS 4.3.1. C u l t u r e Medium P r e p a r a t i o n A mixture of 90% T i s s u e C u l t u r e Medium (TCM) 199 (Gibco, 81 Grand I s l a n d , New York) (Appendix 9) and 10% h e a t - i n a c t i v a t e d f e t a l c a l f serum (Sigma Chemical Company, St . L o u i s , M i s s o u r i ) was used f o r oocyte washing. For oocyte c u l t u r e , zearalenone (Sigma Chemical Company, St . L o u i s , M i s s o u r i ) was added t o the medium a t a c o n c e n t r a t i o n of 250 jug/ml. 4.3.2. Ovary R e t r i e v a l from the slaughterhouse O v a r i e s from cows and h e i f e r s were c o l l e c t e d w i t h i n 4 5 min a f t e r s l a u g h t e r a t P i t t Meadows A b a t t o i r . They were p l a c e d i n a Whirlpack bag and immersed i n a Dewar f l a s k c o n t a i n i n g heated (25-30°C) s a l i n e b u f f e r (0.9%). In the s a l i n e b u f f e r was bovine serum albumin (0.3%) (BSA F r a c t i o n V:Sigma Chemical Company, St. L o u i s , M i s s o u r i ) , and 0.5 ml a n t i b i o t i c / a n t i m y c o t i c s o l u t i o n ( p e n i c i l l i n , s t r e p t o m y c i n and amphotericin) (Gibco, Grand I s l a n d , New York) per l i t r e s o l u t i o n . O v a r i e s were then taken t o the l a b o r a t o r y w i t h an approximate t r a v e l time of 1 h. 4.3.3. Oocyte Recovery Immature oocytes from v e s i c u l a r f o l l i c l e s (2-6 mm diameter) were a s p i r a t e d u s i n g an 18 gauge needle a t t a c h e d t o a 10 ml s y r i n g e . 4.3.4. Washing of Oocytes Oocytes were washed twice i n medium c o n t a i n i n g 90% TCM-199 and 10% h e a t - i n a c t i v a t e d f e t a l c a l f serum. 4.3.5. I n c u b a t i o n and Oocyte M a t u r a t i o n 82 A t o t a l of 14 oocytes with good c e l l u l a r investments were then t r a n s f e r r e d t o a 35 X 10 mm c u l t u r e d i s h (10-20 oo c y t e s / d i s h ) c o n t a i n i n g 2 ml of zearalenone c o n t a i n i n g c u l t u r e medium and 5-7 m i l l i o n / m l g r a n u l o s a c e l l s . C u l t u r e d i s h e s (Falcon™, Becton D i c k i n s o n , L i n c o l n Park, New Jersey) were p l a c e d i n a w a t e r - j a c k e t e d i n c u b a t o r (39°C and a gas composition of 95% a i r and 5% carbon d i o x i d e ) f o r 24 h. Note: Granulosa c e l l s were obtained by s c r a p i n g the i n s i d e of a medium-sized f o l l i c l e w ith an 18 gauge hypodermic needle. Then, the granulosa c e l l s were c e n t r i f u g e d a t 400 G f o r 10 minutes. C o n c e n t r a t i o n was a d j u s t e d v i a haemocytometer counts. Approximately 100 /xl of the c e n t r i f u g e d g r a n u l o s a c e l l s were t r a n s f e r r e d t o g i v e a c o n c e n t r a t i o n of 5-7 m i l l i o n per ml of c u l t u r e medium. 4.3.6. S t a i n i n g Procedure Before the s t a i n i n g p r e p a r a t i o n procedure, oocytes were examined f o r cumulus c e l l expansion (Figure 12: 400 X m a g n i f i c a t i o n ) as a s i g n of maturation. Then, the cumulus l a y e r was removed from oocytes by p l a c i n g them i n v i a l s c o n t a i n i n g 0.5-1.0 ml TCM-199 and c e n t r i f u g i n g f o r 5 minutes at 4 00 G. The next step i n v o l v e d washing the naked oocytes ( F i g u r e 13: 400 X m a g n i f i c a t i o n ) with a 1.0% sodium c i t r a t e s o l u t i o n . Oocytes were kept i n the washing s o l u t i o n f o r 10 min t o cause c e l l s w e l l i n g b e f o r e being t r a n s f e r r e d t o a 83 c l e a n microscope s l i d e . A f t e r a i r d r y i n g o f s l i d e s , oocytes were f i x e d w i t h a 3:1 ethanol and a c e t i c a c i d s o l u t i o n . F i x e d s l i d e s were then s t a i n e d with 2% a c e t o - o r c e i n f o r 1 min and examined under a compound microscope (200 X m a g n i f i c a t i o n ) . 4.3.7. Ma t u r a t i o n E v a l u a t i o n S t a i n e d oocytes were examined f o r metaphase I I chromosome c o n f i g u r a t i o n as c o n c l u s i v e evidence t h a t m e i o s i s had resumed and the oocyte had matured. 84 F i g u r e 12. Cumulus c e l l expansion of bovine oocyte 85 4.4. RESULTS A l l 14 oocytes appeared t o have undergone cumulus expansion when examined a f t e r i n c u b a t i o n . Chromosome c o n f i g u r a t i o n e v a l u a t i o n demonstrated t h a t 12 of 14 oocytes had a c t u a l l y matured ( F i g u r e 14:400 X m a g n i f i c a t i o n ) . 8 6 F i g u r e 14. Metaphase II chromosome c o n f i g u r a t i o n of a mature bovine oocyte 4.5. DISCUSSION 87 S i n c e a h i g h percentage of oocytes matured i n c u l t u r e , zearalenone exposure d i d not appear t o have a d e t r i m e n t a l e f f e c t . However, the same c o n c e n t r a t i o n of mycotoxin prevented i n v i t r o development of murine b l a s t o c y s t s as d i s c u s s e d i n s e c t i o n 3.4 of t h i s paper. P o s s i b l y , the surrounding cumulus l a y e r a c t s as a b a r r i e r t o prevent zearalenone from d i r e c t l y a c t i n g on the oocyte. 88 5.0 GENERAL DISCUSSION The b a r l e y survey f o r zearalenone contamination demonstrated a l l samples t o be f r e e of the mycotoxin a t a d e t e c t i o n l e v e l o f 500 ppb. T h e r e f o r e , l i v e s t o c k r e c e i v i n g feeds formulated from the sources of these samples would not l i k e l y e x p e r i e n c e zearalenone t o x i c o s i s problems. In a d d i t i o n , A g r i -Screen™ by Neogen C o r p o r a t i o n (Lansing, Michigan) i s recommended as a simple and economical method f o r p r e - s c r e e n i n g of commodities i n the f i e l d . In v i t r o c u l t u r e s t u d i e s with murine b l a s t o c y s t s demonstrated d i r e c t t o x i c o l o g i c a l e f f e c t s of zearalenone on development. The c r i t i c a l c o n c e n t r a t i o n range f o r the mycotoxin to cause embryo degeneration was determined t o be between 70-160 Mg/ml c u l t u r e medium. The r e s u l t s i n d i c a t e a h i g h e r zearalenone t o l e r a n c e l e v e l than r e p o r t e d by the i n v e s t i g a t i o n s of Long and Turek (1989) which show the c r i t i c a l l e v e l t o be i n the 8.5-68 Mg/ml range. Furthermore, a c o n c e n t r a t i o n e f f e c t on the l e n g t h of time r e q u i r e d t o cause d e t r i m e n t a l e f f e c t s was demonstrated. At mycotoxin c o n c e n t r a t i o n s of 500 Mg/ ml medium and above, b l a s t o c y s t s degenerate a f t e r 6 hours of c u l t u r e . At a lower l e v e l of 160 nq/ml, b l a s t o c y s t s are not a f f e c t e d u n t i l 28 hours of c u l t u r e . S i n c e d e t r i m e n t a l e f f e c t s of i n v i t r o exposure of murine p r e - i m p l a n t a t i o n embryos t o zearalenone have been demonstrated, i n v i v o i n v e s t i g a t i o n s should be conducted. 89 Zearalenone c o u l d be a d m i n i s t e r e d by gavage t o e a r l y g e s t a t i o n mice. In order t o study p o s s i b l e e f f e c t s on p r e -i m p l a n t a t i o n embryos, the o v i d u c t s and u t e r i n e horns c o u l d be f l u s h e d and the contents examined. F a c t o r s such as embryo appearance and l o c a t i o n i n the r e p r o d u c t i v e t r a c t c o u l d be compared with c o n t r o l animals. Gavage a d m i n i s t r a t i o n i s p r e f e r a b l e s i n c e i t i s a r e a l i s t i c way f o r a s s e s s i n g d e t r i m e n t a l e f f e c t s caused by mycotoxins i n feed. A l s o , l i k e other phytoestrogens zearalenone d i f f e r s from mammalian estrogens which are more e f f e c t i v e when i n j e c t e d (Schoop, 1956). In the case of z e a r a n o l , a zearalenone d e r i v a t i v e , a d m i n i s t r a t i o n t o pregnant mice has been shown t o a f f e c t f e c u n d i t y i n terms of number of l i v e l i t t e r s (number of females inseminated/number of l i v e b i r t h s ) and l i t t e r s i z e (Davis e t a l . , 1977). A d d i t i o n a l l y , Brooks et a l . (1971) demonstrated a n t i -i m p l a n t a t i o n a c t i v i t y of r e s o r c y l i c a c i d l a c t o n e d e r i v a t i v e s o r a l l y a d m i n i s t e r e d t o r a t s on day 2 of pregnancy. A n t i -i m p l a n t a t i o n was a t t r i b u t e d t o a c c e l e r a t e d passage of embryos through the o v i d u c t s s i n c e t h i s i s known t o occur w i t h a d m i n i s t r a t i o n of estrogens t o pregnant r a t s (Greenwald, 1961). Because the embryo passage has been a c c e l e r a t e d , they a r r i v e i n an asynchronous uterus and are not a b l e t o implant (Dickmann and Noyes, 1960). 90 U n s u c c e s s f u l i n v i t r o p o r c i n e morula c u l t u r e attempts prevented t o x i c o l o g i c a l s t u d i e s on the e f f e c t ( s ) o f zearalenone on p r e - i m p l a n t a t i o n development. S i n c e the morula r e c o v e r y methods employed probably i n c u r r e d d e t r i m e n t a l e f f e c t s on embryos, a l t e r n a t i v e procedures c o u l d be used. For example, e a r l y b l a s t o c y s t s c o l l e c t e d s u r g i c a l l y from donors and immediately p l a c e d i n c u l t u r e might have a b e t t e r chance f o r development than those c o l l e c t e d from slaughterhouse animals. 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In "Proceedings of the I n t e r n a t i o n a l Symposium on Mycotoxins, September 6-8, 1981, C a i r o , Egypt", pp. 311-315. 106 Ware, G.M. and C.W. Thorpe, (1978). Determination of zearalenone i n c o r n by h i g h p r e s s u r e l i q u i d chromatography and f l u o r e s c e n c e d e t e c t i o n . J o u r n a l of the A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists 61:1058-1062. Warner, R., B.P. Ram, L.P. Hart, and J . J . Pestka, (1986). S c r e e n i n g f o r zearalenone i n corn by c o m p e t i t i v e d i r e c t enzyme-linked immunosorbent assay. J o u r n a l of A g r i c u l t u r a l and Food Chemistry 34:714-717. Weaver, G.A., H.J. Kurtz, J.C. Behrens, T.S. Robison, B.E. Seguin, F.Y. Bates, and C.J. Mirocha, (1986). E f f e c t of zearalenone on the f e r t i l i t y o f v i r g i n d a i r y h e i f e r s . American J o u r n a l of V e t e r i n a r y Research 47:1395-1397. Wicklow, D.T., G.A. Bennett, O.L. S h o t w e l l , (1987). Secondary i n v a s i o n of soybeans by Fusarium graminearum and r e s u l t i n g mycotoxin contamination. P l a n t Disease 71:1146 ( A b s t r a c t ) . Wilson, R.F., V.D. Sharma, L.E. W i l l i a m s , D.P. Sharda, and H.S. Teague, (1967). E f f e c t s of f e e d i n g Fusarium roseum mold t o r a t s and hogs. J o u r n a l of Animal Science 26:1479-1480 ( A b s t r a c t ) . Wilson, D.M., W.H. Tabor, and M.W. Trucksess, (1976). S c r e e n i n g method f o r the d e t e c t i o n of a f l a t o x i n , o c h r a t o x i n , zearalenone, p e n i c i l l i c a c i d , and c i t r i n i n . J o u r n a l o f t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists 59:125-127. W i t t l i f f , J.L., R.G. Mehta, P.A. Boyd, and J.E. G o r a l , (1976). S t e r o i d - b i n d i n g p r o t e i n s of the mammary gland and t h e i r c l i n i c a l s i g n i f i c a n c e i n b r e a s t cancer. J o u r n a l of T o x i c o l o g y and Environmental H e a l t h . Supplement 1:231-256. Wolf, J . C , J.R. Lieberman, and C.J. Mirocha, (1972). I n h i b i t i o n of F-2 (zearalenone), b i o s y n t h e s i s and p e r i t h e c i u m p r o d u c t i o n i n Fusarium roseum 'Graminearum'. Phytopathology 62:937-939. Wolf, J.C. and C.J. Mirocha, (1977). C o n t r o l of s e x u a l r e p r o d u c t i o n i n G i b b e r e l l a zeae (Fusarium roseum "Graminearum"). A p p l i e d and Environmental M i c r o b i o l o g y 33:546-550. Wollenberg, C , I. Wentz, B. Blum, and W. H o l t z , (1990). S u r v i v a l of p i g embryos f l u s h e d from the r e p r o d u c t i v e t r a c t immediately or two hours a f t e r s l a u g h t e r of donors. J o u r n a l of Animal Science 68:2023-2026. Young, L.G., G.J. King, B. Wilcock, and J.C. Sutton, (1979). Mold contaminated corn i n d i e t s of young p i g s . J o u r n a l of Animal Science Supplement 1 49:86 ( A b s t r a c t ) . 107 Young, L.G. and G.J. King, (1983). Feeding of low l e v e l s o f zearalenone i n d i e t s of pu b e r a l g i l t s . J o u r n a l of Animal S c i e n c e 57 Supplement 1:313-314 ( A b s t r a c t ) . Young, L.G., H. Ping, and G.J. King, (1990). E f f e c t s o f f e e d i n g zearalenone t o sows on r e b r e e d i n g and pregnancy. J o u r n a l of Animal S c i e n c e 68:15-20. 108 Appendix 1. B a r l e y zearalenone contamination survey r e s u l t s ELEVATOR LOCATION SUBSAMPLE COLOR AWP Bashaw, 1 medium b l u e A l b e r t a 2 medium b l u e AWP Chipman, 1 medium b l u e A l b e r t a 2 medium blu e AWP E l n o r a , 1 medium b l u e A l b e r t a 2 medium b l u e AWP I r r i c a n a , 1 medium b l u e A l b e r t a 2 medium b l u e AWP Kathyrn, 1 medium b l u e A l b e r t a 2 medium b l u e AWP Spruce Grove, 1 medium b l u e A l b e r t a 2 medium b l u e AWP Stony P l a i n , 1 medium b l u e A l b e r t a 2 medium b l u e AWP Three H i l l s , 1 medium b l u e A l b e r t a 2 medium b l u e AWP Trochu, 1 medium b l u e A l b e r t a 2 medium b l u e CARGILL Beiseker, 1 medium b l u e A l b e r t a 2 medium b l u e CARGILL T a y l o r , 1 medium b l u e A l b e r t a 2 medium b l u e UGG Beis e k e r , 1 medium blu e A l b e r t a 2 medium blu e 109 Appendix 1. Continued UGG Camrose, A l b e r t a 1 medium b l u e 2 medium b l u e UGG Gaudin, A l b e r t a 1 medium b l u e 2 medium b l u e UGG Gibbons, A l b e r t a 1 medium blu e 2 medium b l u e UGG Huxley, A l b e r t a 1 medium b l u e 2 medium b l u e UGG Lamont, A l b e r t a 1 medium b l u e 2 medium b l u e UGG M o r i n v i l l e , A l b e r t a 1 medium b l u e 2 medium b l u e UGG Olds, A l b e r t a 1 medium b l u e 2 medium b l u e AWP Dawson Creek, B r i t i s h Columbia 1 medium b l u e 2 medium blu e AWP F o r t St. John, B r i t i s h Columbia 1 medium b l u e 2 medium b l u e CARGILL F o r t St. John, B r i t i s h Columbia 1 medium b l u e 2 medium blu e UGG Dawson Creek, B r i t i s h Columbia 1 medium b l u e 2 medium b l u e UGG F o r t S t . John, B r i t i s h Columbia 1 medium b l u e 2 medium b l u e AWP = A l b e r t a Wheat Pool UGG = U n i t e d G r a i n Growers 110 Appendix 2. Dulbecco's phosphate-buffered s a l i n e (PBS) composition COMPONENT g/L C a C l ? (anhydrous) 0.10 KCl 0.20 KHPPO, 0.20 MgCl ?-6H ?0 0. 10 NaCl 8.00 Na,HPO,"7H,0 2 4 2 2.16 I l l Appendix 3. Ham's F-10 n u t r i e n t mixture - Powder form composition COMPONENT mg/L Ino r g a n i c s a l t s C a C l ? (anhydrous) 33 .29 CuSO^'SHpO 0.0025 FeS0 4'7H ?0 0.834 KCl 285.00 KH?POA 83 . 00 MgSO^ (anhydrous) 74.64 NaCl 7400.00 Na ?HP0 4 (anhydrous) 153.70 ZnS0^7H ?0 0.0288 Other components D-Glucose 1100.00 Hypoxanthine (sodium s a l t ) 4 . 68 L i p o i c a c i d 0.20 Phenol red 1.20 Sodium pyruvate 110.00 Thymidine 0.70 Amino a c i d s L - A l a n i n e 9 . 00 L - A r g i n i n e HC1 211.00 L-Asparagine -H ?0 15.01 L - A s p a r t i c a c i d 13.00 L-C y s t e i n e 25.00 L-Glutamic a c i d 14.70 112 Appendix 3. Continued L-Glutainine 146.00 G l y c i n e 7.51 L - H i s t i d i n e HCI'H.,0 23.00 L - I s o l e u c i n e 2 . 60 L-Leucine 13.00 L- L y s i n e HCl 29. 00 L-Methionine 4.48 L-P h e n y l a l a n i n e 5. 00 L - P r o l i n e 11.50 L-Se r i n e 10.50 L-Threonine 3 . 57 L-Tryptophan 0. 60 L - T y r o s i n e (disodium s a l t ) 2 . 61 L - V a l i n e 3 . 50 VITAMINS B i o t i n 0. 024 D-Calcium pantothenate 0.715 C h o l i n e c h l o r i d e 0. 698 F o l i c a c i d 1. 32 i - I n o s i t o l 0.541 Niacinamide 0. 615 P y r i d o x i n e HCl 0.206 R i b o f l a v i n 0. 376 Thiamine HCl 1. 00 V i t a m i n B 1 2 1.36 Appendix 4. UBC human i n v i t r o f e r t i l i z a t i o n program Ham's F-10 p r e p a r a t i o n p r o t o c o l Procedure: a) Add 9.0 g powdered Ham's F-10 n u t r i e n t mixture (Appendix 2) to a 1 L v o l u m e t r i c f l a s k and d i s s o l v e i n p u r i f i e d water (e.g. M i l l i p o r e C o r p o r a t i o n p u r i f i c a t i o n system) b) Add 50.0 mg p e n i c i l l i n G, add more water and shake f l a s k t o d i s s o l v e . c) D i s s o l v e 1.68 g NaHC03 i n approximately 50 ml of p u r i f i e d water i n a 250 ml Erlenmeyer " f l a s k . d) Add 507 mg KHC03 t o the NaHC03 s o l u t i o n , add more p u r i f i e d water and s w i r l t o d i s s o l v e . e) D i s s o l v e 245 mg ca l c i u m l a c t a t e i n approximately 50 ml of p u r i f i e d water i n a second Erlenmeyer f l a s k . f) Add the HC03" s o l u t i o n t o the 1 L v o l u m e t r i c f l a s k , and mix w e l l . g) Slowly add the ca l c i u m l a c t a t e s o l u t i o n t o the 1 L v o l u m e t r i c f l a s k , and mix w e l l . h) Make up t o 1 L volume by adding p u r i f i e d water. i ) Check the o s m o l a r i t y t o read 278-280 mOsmol/kg. j) The pH should be 7.4-7.6. k) S t e r i l i z e the media through a f i l t r a t i o n u n i t w i t h a 0.2 2 micron membrane. 1) Media can be s t o r e d f o r 4 weeks a t 4 °C. 114 Appendix 5 (A-F). Study 1: e f f e c t o f zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. Embryo Developmental Stage (6 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 10 Treatment 1 10 7 3 Treatment 2 9 9 Treatment 3 10 10 B . Embryo Developmental Stage (12 Hours C u l t u r e ) n A B . C D E F G C o n t r o l 10 10 Treatment 1 10 5 5 Treatment 2 9 9 Treatment 3 10 10 C. Embryo Developmental Stage (22 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 7 3 Treatment 1 10 4 5 1 Treatment 2 9 6 3 Treatment 3 10 10 115 Appendix 5 (A-F). Continued D. Embryo Developmental Stage (28 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 2 5 3 Treatment 1 10 2 1 6 1 Treatment 2 9 7 2 Treatment 3 10 - 10 E . Embryo Developmental Stage (34 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 1 2 7 Treatment 1 10 1 2 6 1 Treatment 2 9 5 4 Treatment 3 10 10 F. Embryo Developmental Stage (44 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 1 9 Treatment 1 10 8 2 Treatment 2 9 5 4 Treatment 3 10 10 116 Appendix 6 (A-F). Study 2: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. Embryo Developmental Stage (6 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 9 1 Treatment 1 10 9 1 Treatment 2 9 1 8 Treatment 3 10 10 B. Embryo Developmental Stage (12 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 9 1 Treatment 1 10 9 1 Treatment 2 9 8 1 Treatment 3 10 10 C. Embryo Developmental Stage (22 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 2 6 1 1 Treatment 1 10 6 2 1 1 Treatment 2 9 5 4 Treatment 3 10 10 117 Appendix 6 (A-F). Continued D. Embryo Developmental Stage (28 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 2 7 1 Treatment 1 10 3 1 5 1 Treatment 2 9 4 2 3 Treatment 3 10 10 E. Embryo Developmental Stage (34 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 1 8 1 Treatment 1 10 1 2 6 1 Treatment 2 9 3 1 4 1 Treatment 3 10 10 F. Embryo Developmental Stage (44 Hours C u l t u r e ) n A B C D E F G C o n t r o l 10 9 1 Treatment 1 10 9 1 Treatment 2 9 2 4 3 Treatment 3 10 10 118 Appendix 7 (A-F). Study 3: e f f e c t of zearalenone c o n c e n t r a t i o n on i n v i t r o murine b l a s t o c y s t development A. Embryo Developmental Stage (6 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 30 Treatment 1 30 1 29 Treatment 2 30 1 27 2 Treatment 3 30 7 13 10 B. Embryo Developmental Stage (12 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 5 23 2 Treatment 1 30 1 27 1 1 Treatment 2 30 5 20 2 3 Treatment 3 30 2 2 26 C. Embryo Developmental Stage (22 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 11 11 6 2 Treatment 1 30 15 7 7 1 Treatment 2 30 4 17 1 1 7 Treatment 3 30 30 119 Appendix 7 (A-F). Continued D. Embryo Developmental Stage (28 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 3 8 16 3 Treatment 1 30 5 7 17 1 Treatment 2 30 2 9 2 3 14 Treatment 3 30 30 E. Embryo Developmental Stage (34 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 2 25 3 Treatment 1 30 3 1 25 1 Treatment 2 30 2 5 5 18 Treatment 3 30 30 F. Embryo Developmental Stage (44 Hours C u l t u r e ) n A B C D E F G C o n t r o l 30 1 26 3 Treatment 1 30 3 26 1 Treatment 2 30 2 5 23 Treatment 3 30 29 1 Appendix 8. 120 Minimum E s s e n t i a l Medium (MEM) composit COMPONENT mg/L INORGANIC SALTS C a C l ? (anhydrous) 200.00 KCl 400.00 MgS 0^ -711,0 200.00 NaCl 6800.00 NaHCO^ 2200.00 NaH?P04-H70 140.00 OTHER COMPONENTS D-Glucose 1000.00 Phenol r e d 10. 00 AMINO ACIDS L-Arginine-HCl 126.00 L - C y s t i n e 24.00 L-Glutamine 292.00 L - H i s t i d i n e HC1-H?0 42.00 L - I s o l e u c i n e 52 . 00 L-Leucine 52 . 00 L-L y s i n e HCl 72.50 L-Methionine 15. 00 L-Ph e n y l a l a n i n e 32.00 L-Threonine 48.00 L-Tryptophan 10. 00 L- T y r o s i n e 36. 00 L - V a l i n e 46. 00 121 Appendix 8. Continued VITAMINS L - A s c o r b i c a c i d 50.00 B i o t i n 0.10 D-Ca pantothenate 1.00 C h o l i n e c h l o r i d e 1. 00 F o l i c a c i d 1. 00 i - I n o s i t o l 2 . 00 Nicotinamide 1.00 P y r i d o x a l HC1 1. 00 R i b o f l a v i n 0.10 Thiamine HC1 1.00 Vi t a m i n B 1 2 1.36 122 Appendix 9. T i s s u e C u l t u r e Medium (TCM) 199 composition COMPONENT mg/L INORGANIC SALTS C a C l ? 200.00 Fe(NO,),-9H?0 0.72 KCl 400.00 MgSOA-7H?0 200.00 NaCl 6100.00 NaHCO, 2200.00 NaHpPO^-HpO 140.00 OTHER COMPONENTS Adenine s u l f a t e 10.00 Adenosinetriphosphate (disodium s a l t ) 1. 00 A d e n y l i c a c i d 0.20 C h o l e s t e r o l 0.20 Deoxyribose 0.50 D-Glucose 1000.00 G l u t a t h i o n e (reduced) 0.50 Guanine HC1 0.30 HE PES 5958.00 Hypoxanthine 0.30 Phenol red 20. 00 Ribose 0.50 Sodium a c e t a t e 50.00 Thymine 0.30 123 Appendix 9. Continued Tween 80™ 20.00 U r a c i l 0.30 Xanthine 0.30 AMINO ACIDS DL-Alanine 50.00 L - A r g i n i n e HC1 70.00 DL - A s p a r t i c a c i d 60. 00 L-C y s t e i n e HC1"H?0 0. 11 L- C y s t i n e 20. 00 DL-Glutamic acid"H ?0 150.00 L-Glutamine 100.00 G l y c i n e 50. 00 L - H i s t i d i n e HC1"HP0 21. 88 L-Hydroxyproline 10. 00 DL- I s o l e u c i n e 40. 00 DL-Leucine 120.00 L- L y s i n e HC1 70.00 DL-Methionine 30. 00 DL-Phenylalanine 50. 00 L - P r o l i n e 40. 00 DL-Serine 50. 00 DL-Threonine 60. 00 DL-Tryptophan 20. 00 L- T y r o s i n e 40.00 DL-Valine 50.00 124 Appendix 9. Continued VITAMINS A s c o r b i c a c i d 0. 05 a-Tocopherol phosphate (disodium s a l t ) 0. 01 d - B i o t i n 0. 01 C a l c i f e r o l 0.10 D-Ca pantothenate 0. 01 C h o l i n e c h l o r i d e 0.50 F o l i c a c i d 0. 01 i - I n o s i t o l 0. 05 Menadione 0. 01 N i a c i n 0. 025 Niacinamide 0. 025 Para-aminobenzoic a c i d 0. 05 P y r i d o x a l HCl 0.025 P y r i d o x i n e HCl 0.025 R i b o f l a v i n 0. 01 Thiamine HCl 0. 01 Vi t a m i n A (acetate) 0.14 

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