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ZEARALENONE IN PASTURE AND ITS EFFECTS ON 
REPRODUCTION IN EWES 
A thesis presented in partial fulfilment for the degree of 
Master of Applied Science in Animal Science at Massey 
University 
RICHARD KRAMER 
1997 
ABSTRACT 
Kramer, R. 1997. Zearalenone in pasture and its effects on reproduction in ewes. MApplSc 
thesis, Massey University, Palmerston North, New Zealand, 76pp. 
ii 
Zearalenone is an oestrogenic mycotoxin which has the potential to cause reproductive 
disorders in sheep. Zearalenone-producing Fusarium species are present in New Zealand 
pasture and it is likely that the amount of zearalenone present during the mating period may be 
sufficient to cause reproductive dysfunction in the grazing sheep. 
This study consisted of three trials which aimed to measure zearalenone levels in the pasture 
and sheep, and determine the subsequent effects on reproductive performance. The first trial 
investigated the levels of zearalenone during April in various components of the ryegrass plant 
at various pasture sites, which included urine-patch, dung-patch and inter-excreta sites. 
Zearalenone taken up by the ryegrass plant was also determined. The second trial comprised 
of 6 groups of ewes (n=lO), and compared levels of zearalenone and related metabolites in the 
blood and urine of ewes grazed on pasture or chicory and either orally (5 mg/ewe)or 
intravenously dosed (2 or 0.5 mg/ewe) daily with zearalenone. The subsequent effects on 
ovulation rate, conception rate, and number of lambs carried was also determined. The third 
trial comprised of 4 groups (n=l lO) of ewes, of which two groups were grazed on grass­
dominant pasture and the remaining 2 groups were grazed on chicory for two weeks prior to 
mating at which time one of the groups on each grazing treatment was interchanged and the 
ram introduced. The levels of free and conjugated zearalenone in the blood and urine were 
determined and the subsequent effects on ovulation rate, conception rate and the number of 
lambs carried were measured. 
In the first trial it was shown that zearalenone concentration within sites was highly variable at 
that time of the year, however, urine-patch and dung patch sites yielded significantly higher 
quantities of zearalenone. Zearalenone appeared to be readily taken up by the ryegrass plant 
through the roots and translocated into the young growing tissue of the plant. The distribution 
of zearalenone in the pasture and the plant are discussed with regards to zearalenone intake by 
the animal. 
The zearalenone dosing trial showed that significant levels of zearalenone, a-and J3-zearalenol, 
zeranol and taleranol were present in the blood and urine of dosed ewes and that levels of all 
compounds analysed were higher in ewes grazed on pasture. Ewes grazing pasture had a 
significantly lower (P<O.OS) ovulation rate than ewes grazed on chicory. 
The third trial showed that chicory was effective in reducing the levels of free zearalenone 
present in the ewe around the time of mating with levels in ewes grazed on chicory being 
significantly lower (P<0.05) in both the urine and blood, than in ewes grazed on grass pasture. 
There were no significant differences in reproductive performance. Zearalenone levels in the 
pasture were generally lower in 1995 than in previous years and might have reduced possible 
differences in reproductive performance between ewes on the different feed types. 
The implications of higher zearalenone concentrations in the pasture are discussed with regards 
to reproductive performance and the use of chicory as a feed prior to mating. 
Further research is required to identify and clarify links with zearalenone and metabolites 
produced in pasture and reproductive dysfunction in ewes. 
111 
ACKNOWLEDGEMENTS 
I would like to thank my supervisors Associate Professor M.F. McDonald (Massey University, 
Pahnerston North) and Mr R.G. Keogh (AgResearch Grasslands, Palmerston North) for their 
continued guidance and patience during my studies and I look for to future work with you. 
I am very grateful to Margaret Mason for help with animal handling and sample taking. 
I wish to thank very much Dr Anton Erasmuson (National Chemical Residues Analytical 
Laboratory, MAF, Wallaceville Animal Research Centre, Upper Hun, New Zealand) and Jan 
Sprosen (AgResearch, Ruakura Agricultural Research Centre, Private Bag 3 123, Hamilton 
New Zealand) for zearalenone analysis on the various and numerous samples. 
Thanks to Professor G. Wickham Massey University, Palmerston North) for help with 
statistical analysis. 
A special thanks to Roger Dalrymple, Bulls, for providing sheep, land, and man/dog power for 
one of the trials. 
Finally I would like to thank my family and friends who are never short of advice, support and 
encouragement 
IV 
TABLE OF CONTENTS 
ABSTRACT ........................... ................................. . ............ ............................................... .. ii 
ACKOWLEDGEMENTS ......... ........... . . ................................................................. .. . ......... iii 
LIST OF FIGURES .... ......................................................................... . ............ ................. vii 
LIST OF TABLES . ... ............... ............. .... ................. .................... .... . ................. ................ X 
LIST OF PLATES ............................................................................................................... xi 
LIST OF ABBREVIATIONS ..................................... ....................................................... xii 
CHAPTER I. Introduction ...................................................................................... . ........... 1 
CHAPTER IT. Review of literature .......... ........................................................................... 3 
1. The mycotoxin zearalenone .............................................................................................. 3 
2. Zearalenone metabolism and chemistry .......................................................................... 4 
3. Effects of zearalenone in laboratory animals ................................................................... 6 
4. Effects of zearalenone in pigs ............................................................................... ............ 6 
5. Effects of zearalenone in poultry ...................................................................................... 8 
6. Effects of zearalenone in cattle ......................................................................................... 9 
7. Effects of zearalenone in sheep .............................. .......................................................... 9 
8. Production of zearalenone ................ . ... . .............. ...................................................... ..... 11 
9. Purpose and scope of the study ............ . ..................................... .................... . ........... ... . 13 
CHAPTER Ill. The distribution of zearalenone in ryegrass pasture and uptake by the 
ryegrass plant ....................... ....... ... .... ......................................... ..... . . . . .... ................... 14 
1. Introduction ........... ........ . .............................. ............................... .... ............ . . ............. . . ..  14 
2. Methods and Materials .......................................................................... ..... ................... 15 
2.1. Distribution of zearalenone in ryegrass pasture ....................................... ......... 15 
.. 
2.1.1. Zearalenone deterniination .................................................................... 16 
2.2. Uptake of zearalenone by the ryegrass plant ................................. . . .................. 16 
2.3. Statistical analysis .............................................................................................. 19 
3. Results ............................................................................................................................. 19  
3.1. Zearalenone concentration in pasture sites ............................................ ........... 19  
3.2. Soil samples . . . . ..................... . . ...................... ....................................... . . . . . . . . . . . . . . . . .  2 1  
,, 
V 
3.3. Zearalenone yield (Total zearalenone in the plant tissue) ................................. 2 1  
3.4. Determination of zearalenone concentration and yield i n  components of 
ryegrass tillers from inter-excreta, urine patch and dung patch sites .............. 23 
3.4.1.Zearalenone concentration ...................................................................... 23 
3.4.2. Zearalenone yield ................................................................................... 25 
3.5. Zearalenone uptake .................................................................................... ........ 27 
3.5.1. Zearalenone yield ............................................................. ......... ............. 27 
3.5.2. Zearalenone concentration in nutrient solutions ........ .......................... 28 
4.Discussion ....................................... .......... .... ................... ................................................ 28 
4.1. Zearalenone distribution in pasture ........... .................. ............................. ........ 28 
4.2. Assessment of the method of estimation of zearalenone distribution ............... 30 
4.3. Zearalenone uptake by the ryegrass plant.. ....................................................... 31 
4.4. Assessment of the method ............................................................................. ..... 31 
4.5. Conclusions ......................................................................................................... 32 
CHAPTER IV. Zearalenone and related compounds in the blood and urine of ewes 
intravenously and orally dosed with zearalenone and the effects on reproductive 
performance ............................................................................... ................................. 33 
1. Introduction .................................................................................................................... 33 
2. Methods and Materials ........................................................ .......................................... 34 
2.1. Animals and treatments .................................................................. ................... 34 
2.2. Determination of zearalenone and its metabolites in blood and urine ............. 35 
2.3. Statistical analysis .............................................................................................. 36 
3. Results ............................................................................................................................. 36 
3.1. Liveweight .......................................................................................................... 36 
3.2. Ovulation rate ..................................................................................................... 37 
3.3. Conception rate .................................................................................................. 37 
3.4. Zearalenone in the forages ................................................................................. 38 
3.5.Zearalenone and its metabolites in blood ........................................................... 38 
3.6. Zearalenone and its metabolites in urine ........................................................... 42 
4. Discussion ....................................................................................................................... 45 
4.1. Animal measurements ........................................................................................ 45 
4.2 Zearalenone in the blood ..................................................................................... 46 
4.3. Zearalenone in the urine .................................................................................... 46 
V1 
4.4. Metabolism of zearalenone ................................................................................. 48 
4.5.  Zearalenone-related metabolites in the urine .................................................... 49 
4.6. Conclusions ......................................................................................................... 49 
CHAPTER V. Zearalenone in ewes grazed either on pasture or chicory and subsequent 
effects on reproductive performance .......................................................................... 50 
1. Introduction .................................................................................................................... 50 
2. Methods and Materials .................................................................................................. 51 
2.1. Animals and treatments ..................................................................................... 51 
2.2. Sampling ............................................................................................................. 52 
2.3. Zearalenone determination in herbage, blood and urine .................................. 52 
2.4. Statistical analysis .............................................................................................. 52 
3. Results ..................................................................................................... ........................ 56 
3.1.  Weight change and reproductive performance ....... . ....... . . . . . ......... ..... ... ............ 56 
3.2. Zearalenone in the herbage ...................................................................... .......... 57 
3.3. Zearalenone in the blood .................................................................................... 57 
3.4. Zearalenone in the urine .................................................................................... 59 
4. Discussion ....................................................................................................................... 60 
4.1. Animal measurements ........................................................................................ 60 
4.2. Zearalenone in the herbage ................................................................................ 60 
4.3. Zearalenone in the blood .................................................................................... 61 
4.4.Zearalenone in the urine ..................................................................................... 62 
4.5. Conclusions ......................................................................................................... 62 
CHAPTER VI. General discussion and conclusions ......................................................... 63 
Appendix 1 .......................................................................................................................... 67 
Appendix 2 ......................................................................................................... ................. 68 
Appendix 3 . . ........ . . .............................................. ................................................................ 69 
References ..... ................................................... . .................................................................. 70 
LIST OF FIGURES 
2.1. Zearalenone and related metabolites and the metabolic pathways which link 
vii 
them .......................................................................................................................... 5 
3.1. Mean zearalenone concentrations in tiller tops and roots between inter-excreta, 
urine patch and dung patch sites ................................. ...... . ......... ..................... ........ 20 
3.2. Mean zearalenone yields in tiller tops and roots between inter-excreta, urine 
patch and dung patch sites ...................................................................................... 2 2  
3.3. Mean zearalenone concentrations in dissected components of ryegrass tillers .......... .24 
3.4. Mean zearalenone yields concentrations in dissected components of ryegrass 
tillers ....................................................................................................................... 26 
3.5. Zearalenone concentration in the leaf blade, leaf sheath, mature blade, mature 
sheath, dead material, daughter tillers, flowering stem, old root and young root 
components of ryegrass tillers grown in solution containing zearalenone .................. 27 
3.6. Zearalenone yield in the leaf blade, leaf sheath, mature blade, mature sheath, 
dead material, daughter tillers, flowering stem, old root and young root 
components of ryegrass tillers grown in solution containing zearalenone ................. 28 
4.1. Zearalenone dosing treatment groups ...................................................................... 35 
4.2. Mean ovulation rate for each treatment group .......................................................... 37 
4.3. Number of returns in each treatment group .............................................................. 37 
4.4. Mean levels of unconjugated (free) zearalenone in the blood of ewes in the 
OD. IVH. IVL and control groups either grazing pasture or chicory at four times 
viii 
during a 24 hour period afetr dosing ............................................................. ........ . .. 39 
4.5. Mean levels of conjugated zearalenone in the blood of ewes in the OD, IVH, 
IVL and control groups either grazing pasture or chicory at four times during 
a 24 hour period after dosing .......... ............ ...... . ...... . . . . . . . . ...... .... . ... . .... . .. . ... . . . . . . .. . ....  .40 
4.6. Levels of alkene zearalenone related metabolites in the blood of 
oral dosed and control ewes grazed on either chicory pasture . . . . . . ... . . . . . ....... . . . . . .... . ..  .4 1 
4.7. Levels of alkane zearalenone related metabolites in the blood of 
oral dosed and control ewes grazed on either chicory or pasture ............ . . ..... . . . . ... . . .  .4 1 
4.8. Free and conjugated zearalenone/creatinine ratios in the urine of ewes either 
grazing pasture or chicory for two days prior to the start of dosing . ..... . . . . ............. . .  .42 
t9. Free and conjugated zearalenone/creatinine ratios on day 6 of dosing in the 
urine of the OD, IVL, IVH and control ewes either grazing pasture or chicory ........ .43 
tlO. Levels of alkene zearalenone metabolites in the urine of zearalenone dosed and 
control ewes grazing either chicory or ryegrass pasture ............................................ 44 
tU. Levels of alkane zearalenone metabolites in the urine of zearalenone dosed and 
control ewes grazing either chicory or rye grass pasture ............................................ 44 
5.1. Grazing treatments for each group of 30 synchronised + 80 non-synchronised 
ewes ........................................................................................................................ 51 
5.2. Conjugated zearalenone in the blood of ewes grazing either chicory or 
pasture .................................................................................................................... 58 
5.3. Free zearalenone in the blood of ewes grazing either chicory or 
IX 
pasture .................................................................................................................... 58 
5.4. Free and conjugated zearalenone in the urine of ewes grazing either chicory 
or pasture for two weeks prior to mating ................................................................. 59 
X 
LIST OF TABLES 
fable Page 
5. 1. Weight change, ovulation rate, returns to service, and nwnber of lambs carried per 
ewe in synchronised ewes in each treatment group . . . . ..... . . . . ........ . . .. . . .......... . . . .. . ... . ... 56 
5.2. Weight change, returns to service, and nwnber of lambs carried per ewe in non-
synchronised ewes in each treatment group . .. . . ...... . . . . . .... . . . .. . . . . .. . .. . . . . ......... . . . . . .........  56 
Xl 
LIST OF PLATES 
Plate Page 
R till . . 1 . J.l. yegrass ers m nutnent so utJ.on .......................................................................... 1 7  
J.2. Ryegrass tillers dissected into components ................... .... . . . . . . . . . ...... .. ... ........... . . ...... 18 
). 1 .  Laparoscopic examination of ovaries ....................................................................... 53 
).2. Ewes grazing grass dominant pasture ............ .......................... ........................ ........ 54 
).3. Ewes grazing chicory ........ . ............................................... . ...... . . ............................. 55 
Xl1 
LIST OF ABBREVIATIONS AND DEFINITIONS 
kg 
g 
mg 
)lg 
ng 
pp m 
ml 
mmol 
7JCr 
CIDR 
GC-MS 
HPLC 
IgG 
LH 
FSH 
N 
Fusarium 
Fusarium 
fusaria 
Kilograms 
Grams 
Milligrams 
Micro grams 
Nanograrns 
Parts Per Million 
Millilitres 
Millimols 
Zearalenone:Creatinine ratio 
Controlled Internal Drug Release 
Degrees Celsius 
Gas chromatography-Mass 
spectroscopy 
High performance liquid 
chromatography 
Irrununoglobulin 
Luteinizing Hormone 
Follicle Stimulating Hormone 
Nitrogen 
Used when describing a particular 
species 
Used when description is non-specific\ 
Used when referred to collectively 
CHAPTER I 
Introduction 
The fertility of sheep flocks in New Zealand is well below their potential (Knight, 1990). Even 
in sheep flocks where ewe condition and management are very good, lambing percentages are 
well below that which would be expected. For example, lambing percentages in Northland are 
often below 100% and very rarely above. With progression from North to South in New 
Zealand there is an upward trend in lambing percentages indicating that the possible 
reproductive problems are greater in northern regions of the country (Quinlivan and Martin, 
1969). 
There are several compounds which have been found in New Zealand pastures, which have 
been shown to exhibit oestrogenic effects in grazing animals. It has long been appreciated that 
many plants and some fungi are able to produce compounds which possess oestrogenic activity 
in animals (Miksicek, 1994).The majority of oestrogens produced by plants appear to be 
secondary metabolites which are nonsteriodal in nature. The main pasture plant constituents 
isolated and found to have oestrogenic activity are isoflavones and coumestans from legume 
species. 
The saprophytic fungi belonging to Fusarium species and including., F. crookwellense, F. 
-::ulmorum, and F. semitectum are common in New Zealand pasture herbage (di Menna et al., 
1991 ). It is known that these fungi are capable of producing zearalenone and its metabolites a­
and J3-zearalenol which belong to a rare class of natural products, the �-resorcyclic acid­
lactones, which are capable of binding to estrogen receptors because of their chemical 
iimilarity to oestradiol (Hurd, 1977). 
The effects of zearalenone have been documented with pigs and include hyperestrogenism 
which was initially termed "vulvo-vaginitis" and symptoms such as swelling of the vulva and 
uterine enlargement (Maryamma, et al. , 1992). 
To date the majority of studies have been concerned with the effects of zearalenone on swine 
fed grain contaminated with fusaria and very few studies have examined the effects of 
zearalenone and its metabolites in sheep grazing pasture where zearalenone-producing species 
of fusaria are present Smith et al., (1986) showed that reproductive performance was 
markedly reduced in ewes treated with 25 mg of zearalenone daily for 10 days prior to mating. 
In a later study by Smith et al., (1990) there was a linear decline in ovulation rate with 
increasing dose of zearalenone and there were reductions in conception rates. The results 
obtained in these trials show that zearalenone, ingested in large enough quantity, will disturb 
reproductive function in sheep. However, the question remains as to whether zearalenone­
producing Fusarium species is pasture is a factor in reducing reproductive performance in 
sheep grazing that pasture. 
Zearalenone, in concentrations of 0.4 - 4.0 mglkg dry weight of herbage, have been found in 
pasture samples collected from January to April at sites near Pukekohe, Wanganui and 
Gisborne (di Menna et al., 1987). The presence of the various Fusarium species in pasture and 
their ability to produce significant quantities of the oestrogenic metabolite zearalenone, could 
mean that grazing animals would ingest potent quantities of zearalenone. 
2 
It seemed possible that fusaria populations, which peak at the time most ewes are mated, might 
produce sufficient zearalenone in some pastures to affect reproduction (Jagusch et al., 1986; di 
Menna et al., 1987). Given that up to 4 mglkg dry maner of zearalenone can be present in the 
pasture around the time of mating and that Smith et al., ( 1990) concluded that intakes of 
zearalenone of 3 mg/ewelday or more during the mating period would be reflected as 
depressed ovulation rates and lower lambing percentages, it is likely that zearalenone and its 
metabolites may be factors in reducing reproductive perfonnance in sheep. 
3 
CHAPTERII 
Review of literature. 
1. The mycotoxin zearalenone 
Zearalenone [ 6-(1 0-hydroxy-6-oxo-trans-1 -undecenyle )-P-resorcylic acid lactone] is a 
naturally occuning mycotoxin synthesised by Fusarium mould species endemic to 
temperate climates. Zearalenone and a- and P-zearalenol, which are metabolites of 
zearalenone, belong to a rare class of natural products, the P-resorcylic acid lactones, which 
have the capability of binding to oestrogen receptors (Hurd, 1977; Fitzpatrick et al., 1990; 
Miksicek, 1994). While these fungal oestrogens are less potent on a molar basis than 17P­
oestradioL they can stimulate the activity of the oestrogen receptor to the same maximal 
extent as the natural hormone (Miksicek, 1994 ). Zearalenone is 200-fold less active on a 
molar basis than 17�-oestradiol in stimulating a transcriptional response through the human 
oestrogen receptor (Miksicek, 1994 ). The relative binding affinity to the oestrogen receptor 
differs between metabolites and the species of animal. Fitzpatrick et al., ( 1990) determined 
the relative binding affinity of zearalenone, a-zearalenol and P-zearalenol for oestrogen 
receptors in the pig, rat and chicken. That study found that a-zearalenol exhibited greater 
binding affinity than zearalenone and �-zearalenol the least binding affinity in all species 
exan1ined. The relative bindi..'lg a.ffmity of a-zearalenol was greater in the pig, than the rat 
and significantly greater than in the chicken. Data obtained by Diekman et al., (1989) 
indicated that zearalenone suppressed concentrations of serum follicle stimulating hormone 
(FSH) and luteinizing hormone (LH) in a similar way to oestradiol benzoate, although the 
biphasic stimulatory affect of oestradiol benzoate for LH was not manifested by 
zearalenone. 
a-zeralenol or zeranol is also a nonsteriodal veterinary drug permitted for use as an 
anabolic agent for cattle and sheep (Bories et al., 1990). 
Fusarium species produce many different types of secondary metabolites, which include 
zearalenone and related alcohols. However, the ability to produce mycotoxins varies 
between species and also between strains of the same species. 
To date, most research on fusaria mycotoxins has been concerned with contamination and 
toxin production in cereal products, and the effects on animals which consume the 
contaminated products (Lauren et al., 1988). 
4 
The feeding of cereal products in the animal production industry is largely confined to pig 
and poultry farming and therefore there is more information on the effects of zearalenone in 
these animals, in particular the pig, than in grazing animals such as sheep and cattle. 
2. Zearalenone chemistry and metabolism 
Laboratory-grown fusarium produces zearalenone as a major metabolite but also produces 
double-bond alkene metabolites a- and �-zearalenol and single-bond alkane analogues 
zearanol (a-zearalanol) and taleranol (�-zearalanol). 
There are reports on the effects of resorcylic lactones such as zearalenone in cattle; 
however, compared to swine, cases of zearalenone mycotoxicoses are limited (Sundolf and 
Stricldand, 1986). In practice zearalenone has only moderate effects on bovine fertility in 
comparison to the profound effects exhibited in swine (Mirocha et al., 1980). The 
difference may be partially caused by the dissimilarity of the feed of pigs and cows and 
partially by the different effects zearalenone has on these animals. The distribution of 
urinary and faecal metabolites is different in cattle and swine which suggests variation in 
metabolism of zearalenone between the two species (Mirocha et al., 1980). 
The most essential difference between the metabolism of the swine and that of the bovine is 
caused by rumination. Kallela and Vasenius, (1982) found that rumen fluid had a 
decreasing effect on the amount of zearalenone. This would suggest that the reason why the 
effects of zearalenone are not as severe as that found in pigs is due to detoxification in the 
rumen. However, Kiessling et al., (1984) showed that the decrease in zearalenone was the 
result of a reduction to zearalenol, mainly a-zearalenol. This product has three to four times 
more oestrogenic activity than the parent compound and the reduction of zearalenone to 
zearalenol also increases the polarity, which may influence not only excretion but also 
uptake from the digestive tract into the blood stream. Therefore, the suggestion by Kallela 
and Vasenius, (1982) that zearalenone degradation in the rumen was a first line defence 
against the toxic compound present in the diet is doubtful and the ruminant may be at a 
disadvantage if substances such as zearalenone become more toxic as a result of the action 
of rumina! microbes. Kiessling et al., (1 984) found that the metabolism of zearalenone in 
the bovine rumen is not significantly different from that in the ovine rumen. 
In addition to transformation of zearalenone in the rumen, recent studies have shown that 
the transformation of zearalenone into its various metabolites is also carried out within the 
animal (Fig. 2.1 .). Miles et al., (1996) found labelled a- and �-zeralenol and a- and �­
zearalanol glucuronides in the urine of sheep orally and intravenously dosed with labelled 
zearalenone. Zeranol and taleranol have also been found in the urine of pasture fed animals 
(Erasmuson et al., 1994). 
Figure 2.1. Zearalenone and related metabolites and the metabolic pathways which link 
them. 
HO 0 Me 
HO 
� zear:lleJtDne 
HO 0 Me HO 0 Me 
HO HO 
5 
[3-zeanleJtDl 
� 
a - zeanle110l 
f Fusarium f Fungus 
HO 0 Me � � HO 0 Me 
HO OH HO {3- zearalaJIOl 
��0 
a-zear:alanol 
zearalaJLo:n.e 
6 
3. Effects of zearalenone in laboratory animals 
The effects of zearalenone have also been examined in laboratory animals such as rats, 
mice, rabbits and guinea pigs. Zearalenone and related metabolites have uterotrophic 
activity in rats (Christensen, 1979.; Brooks et al., 1971 and Mirocha et al., 1978). 
Zearalenone has been shown to prevent pregnancy in rats if given on the second day after 
mating (Brooks et al., 1971 ). Zearalenone inhibited the growth of mouse embryos in vitro 
and induced morphological changes in the endometrium (Long et al., 1989). The major 
effect of exogenous oestrogens, including zearalenone, administered during early 
pregnancy in rats and mice, is the accelerated embryo migration in the uterine tubes, with a 
lesser effect of interfering with implantation (Greenwald, 1967). Zearalenone dosed to 
rabbits resulted in the alteration of several trace elements and amino acids known to be of 
critical importance in early embryonic development although anomalies in embryonic 
development were not observed (Osborn et al., 1988). This study concluded that 
zearalenone or its associated metabolites affect factors that influence fertility during the 
early preimplantation period. 
Guinea pigs responded to the oestrogenic effects of zearalenone in early pregnancy in a 
manner similar to that seen in other rodents although, unlike many other rodents, the guinea 
pig has a protracted oestrous cycle similar to that of larger species and implantation occurs 
during midcycle (Long, & Diekman, 1989). 
4. Effects of zearalenone in pigs 
The reproductive effects of mouldy corn in pig diets were first reported by McNutt et al., 
(1928), and included the symptoms which have since collectively been referred to as the 
"oestrogenic syndrome" or "hyperestrogenisim". The induced hyperestrogenism was 
initially tenned "vulvo-vaginitis" and included symptoms such as swelling of the vulva, 
uterine enlargement, ovarian atrophy, mammary development, and in some cases, vaginal 
or rectal prolapse (Aucock et al., 1982). These clinical effects were largely observed in 
prepubital gilts and they appeared to more susceptible to effects of ingested zearalenone. 
Immature gilts given 1 mg of zearalenone/day developed tumefacation of the vulva, and 5 
mg/day also caused an increase 
7 
in uterine weight (Long et al., 1982). Dietary levels of 1-5 mg/kg zearalenone are sufficient 
to produce clinical signs of hyperoestrogenisim in young gilts (Aucock et al. ,  1982 ; Ruhr 
et al., 1983 ). 
Large amounts of zearalenone in the feed (100 mglk:g of feed) have profound effects on 
cycling sows, including nymphomania, pseudopregnancy, ovarian atrophy, and 
morphological changes in the endometrium (Long et al., 1982). Mouldy feed in the diet of 
pregnant sows has been associated with abortion, weak pigs, stillbirths, decreased litter 
size, and foetal mummification, although concentrations of zearalenone in the feed were not 
determined in most of these cases (Long et al., 1982). 
Aside from the obvious clinical effects of ingested zearalenone in swine, the effects on 
various honnone profiles, luteal function and early pregnancy have also been documented 
in the pig. It was shown that feeding zearalenone at concentrations of 5-10 ppm from day 5 
to 20 of the oestrous cycle causes luteal maintenance and extended inter -oestrous intervals 
(Edwards et al., 1987). This could be explained by the fact that oestrogens, given at the 
appropriate time during the oestrous cycle, are luteotrophic in the swine (Kidder et al., 
1955). 
Diekman and Long, (1989) found that zearalenone administered on days 7 to 10 after 
breeding altered secretory patterns of serum LH during days 10 and 14 after breeding. 
Studies by Long et al., (1982) and Diekman and Long, (1989) have shown that ingesting 
zearalenone during early pregnancy has adverse effects on blastocyst development and 
embryo survival in the swine. Diekman and Long, (1989) found that blastocysts from sows 
given a diet containing purified zearalenone (1 mglk:g of body weight) from days 7 to 10 
after breeding, were fragmented and contained foci of necrosis, whereas blastocysts from 
control sows were normal. In an earlier study by Long et al., (1982), gilts that were fed 
Fusarium-contaminated feed or purified zearalenone from days 2 to 15 after breeding lost 
their embryos and retained their corpora lutea. 
Ruhr et al., (1983) examined the effect of zearalenone on the reproductive potential in the 
boar. This study found that zearalenone, administered at 200 mg per kg of the feed ration, 
did not permanently block or adversely affect spermatogenesis in the mature boar nor was 
the reproductive potential of the boar reduced. Berger et al., (1981) found a reduction in 
plasma testosterone concentration and a subsequent reduction in libido after prepubital 
boars were given 40 mglkg of feed from 14 to 18 weeks of age. The results of Ruhr et al., 
( 1983) and Berger et al., ( 1981) would suggest that the effects of zearalenone in boars are 
8 
similar to sows in that prepubital animals appear to be more susceptible. However, it would 
also appear that zearalenone has minimal effects on boars at levels where clinical signs 
would be observed in sows. 
5. Effects of zearalenone in poultry 
The effects of zearalenone have also been documented in production birds such as chickens 
and turkeys. Chickens given zearalenone at lOmg/kg body weight, had cystic changes in 
the oviducts of the females and atrophy of the seminiferous tubules and an increase in 
connective tissue proliferation in the testis in the males (Maryamma, et al., 1992).Allen et 
al., (1981) showed that the weight of male broiler comb and testes were reduced by high 
levels of dietary zearalenone. Chji et al., (1980b) found that zearalenone administered 
orally or intrarnuscularly increased the oviduct weight of growing female White Leghorn 
chickens. However, Marks and Bacon, (1976) fed Fusarium-infected corn to provide 25 
and 100 ppm of zearalenone or purified zearalenone at these levels and did not adversely 
influence the reproductive performance of laying hens. 
Female turkeys fed pure zearalenone (100 ppm) produced less eggs which also weighed 
less than eggs from turkeys with no zearalenone in the diet (Alien et al., 1982). Although 
these observations would suggest that zearalenone was responsible for the significant drop 
in egg production and weight, it is likely that other mycotoxins present in the Fusarium 
infected feed were responsible for the reduction. Chi et al., (1980) and Alien et a/.,(1981) 
concluded that the effects of zearalenone in growing broiler chicks and turkey poults is 
minimal. Alien et al ., ( 1981) showed that male turkey poults fed 400 and 800 mg 
zearalenone/kg of diet had increased development of dewlaps and caruncles and exhibited 
pronounced strutting behaviour. It would appear that turkeys are more susceptible to the 
effects of zearalenone then chickens. A possible reason for this is that turkeys metabolise 
zearalenone mainly to o.-zearalenol which is more oestrogenically active, whereas chickens 
produce roughly equal amounts ofo.-and �-zearalenol (Olsen et al. , 1985) 
The effects of zearalenone on some other species of birds have also been documented. 
Palyusik and Koplic-Kovacs, (1975) reported that dietary F. culmorum-contaminated corn 
produced in female geese a non-significant reduction of egg production and fertility. Vanyi 
and Szeky, (1980) noted a cessation of spermatogenesis in Guinea-cocks fed for 2 to 3 
weeks with grain containing 30 to 40 mg/kg zearalenone. 
9 
The effects of zearalenone in poultry are in many cases unclear due to confli cting results 
however, the incidence of some reported oestrogenic effects means that zearalenone cannot 
be d iscounted as an oestrogenic agent in poultry. 
6. Effects of zearalenone in cattle 
Fusaria fungi and the mycotoxin zearalenone were id entified in suspect mouldy feed 
implicated in d airy herd health problems, includ ing d ecreased fertility. Zearalenone fed at 
dosages of 14 to 75 mglkg of ration may have caused a high artificial insemination index, 
vaginitis, prolonged oestrus, swollen vulvas, d ecreased milk production, and precocious 
mammary gland development in dairy cows and heifers (Bugeac and Berbinsch, 1967; 
Danko and Ald say, 1969; Miller et al., 1973; Shreeve and Patterson, 1975). Mirocha et al., 
(1968) reported d ecreased fertility in a 150-cow d airy herd fed mould y hay with a 
zearalenone concentration of 14 mg/kg of feed . The artificial insemination ind ex returned to 
an acceptable value after the feed ing of the heavily fungus-infected hay was stopped. A 
dairy herd being fed between 5 and 75 mg of zearalenone/kg were affected with 
unexplained swollen vulvas, d ecreased milk production and partial anorexia (Vanyi et al., 
1973 ). Kallela and Etta.Ia. (1984) found that zearalenone content of the hay fed to cows was 
related to the occurrence of abortions. 
In dairy heifers given zearalenone at 250 mg d aily,there was a reduced conception rate 
Weaver et al., (1986a) although it was conclud ed that zearalenone, by itself, was not a 
major factor in bovine infertility. Weaver et al., (1986b) found that d airy cows which were 
administered zearalenone had corpora lutea which were reduced in size but there was no 
change in serum progesterone concentration, red and white blood cell count, haemoglobin, 
and in oestrous cycle length. This study also concluded that zearalenone by itself does not 
seen to be an important factor in d airy cow health. 
7. Effects of zearalenone in sheep 
Because of its oestrogenic actions zearalenone is likely to influence oestrus, ovulation and 
fertilisation if administered pre-mating in the ewe. The properties of dosed oestrogens 
observed in sheep are a prolongation of oestrous behaviour (Fletcher and Linsay, 1971), 
failure of ewes to ovulate, most probably due to an interference with LH release from the 
pituitary (Scaramuzzi et al., 1971; Smith et al., 1987)) and a reduction in fertilisation rate 
through a possible oestrogenic effect on spenn transport (Croker et al., 1975) 
10 
In a trial by Smith et al., (1986) where ewes were dosed with 25 mg daily for 10 days 
prior to mating, there was a marked reduction in reproductive performance with only 9.1% 
of ewes yielding fertilised eggs compared to 57. 6% of  the control ewes. In the same trial 
46% of the treated ewes were anovular compared to 12% in the controls. The major 
reduction in ewes ovulating and in ovulation rate, coupled with the markedly lower 
fertilisation rates, produced an almost complete failure of the reproductive process. A later 
trial by Smith et al., (1988) showed a decline in ovulation rate in ewes which were dosed 
with 6 mg of zearalenone for 10 days prior to mating . A third trial by Smith et al., ( 1990 ) in 
which ewes were dosed at different rates for 10 days prior to mating showed a linear 
decline in ovulation rate with increasing dose rate, also cycle length decreased and the 
duration of oestrus increased with increasing dose levels. There was no effect of 
zearalenone treatment after mating on pregnancy rate or embryonic loss which is in contrast 
to the findings of Mitton et al., (1975) that zearalenone caused abortions in sheep. In the 
trial of Smith et al. , (1990) the zearalenone dose had significant effects on liver weight, 
ovary weight and the weight of uterus and oviducts but not on the carcass weight. The 
fertilisation rate in this trial was unaffected which is in contrast to the findings of Smith et 
al., (1986). The maximum amount of zearalenone administered to ewes in these trials was 
25 mg per day which represents approximately 1 6  ppm of the dietary dry matter intake. 
This is considerably lower than the zearalenone intakes reported in pigs for a similar degree 
of depression in reproductive performance (Diekman and Long, 1984) and thus the ewe 
would appear to be more sensitive than the sow to zearalenone (Smith et al,. 1986) . 
Given that zearalenone can cause reproductive dysfunction in sheep, it is necessary to 
determine whether sheep grazing pasture would ingest sufficient amounts of zearalenone 
around the time of mating to affect reproduction. This would clearly depend on the 
suitability of the climate and environment for Fusarium species to colonise pasture material 
and to produce zearalenone, and in the grazing behaviour of the sheep. 
1 1  
8. Production of zearalenone 
Levels of zearalenone produced on maize grown in Manawatu and W aikato, New Zealand, 
infected with different Fusarium species, have ranged between 1 and 1 6  mg/kg (Hassan et 
al., 1 987). Zearalenone levels from wheat samples grown in Manawatu, New Zealand have 
ranged from 0.04 to 0.35 mglkg (Agnew et al., 1 986). 
Seven Fusarium species., F. acuminatum, F. avenaceum, F. crookwellense, F. culnwrum, 
F. graminum, F. oxysporum and F. semitectum are the predominant species found in New 
Zealand pastures (di Menna et al., 1 987). Of these the most common species F. culnwrum 
and F. crookwellense produce zearalenone consistently and in greatest amounts (di Menna 
et al., 1987 and 1988). F. culmorum from pasture isolates was also found to produce a­
zearalenol and �-zearalenol (di Menna et al., 1 988). 
The presence of the various fusaria in pasture and their ability to produce significant 
quantities of the oestrogenic metabolite zearalenone indicates that animals grazing these 
pastures might ingest significant quantities of zearalenone to cause reproductive disorders. 
Pasture fusaria populations increase when the combination of grass minimum temperature, 
humidity and day length are favourable for fungal growth. They are present in greatest 
numbers in late summer and autumn (February to April) when counts of 104 to 1 06 
Fusarium macroconidia/g wet weight of leaves have been recorded ( diMenna et al., 1 969. ; 
Smith et al., 1990). There are however more than just macroclimatic conditions to be 
considered in determining the suitability of an environment for Fusarium proliferation in 
pasture. The characteristics of a pasture are influenced by plant species and the grazing 
animal which create separate microclimates within the pasture. These different site types 
within the pasture provide conditions which may or may not be suitable for fusaria 
colonisation. The plant species in the pasture affect the amount of dead material which is 
important as fusaria populations are largely restricted to the dead material in the herbage (di 
Menna et al., 1 99 1  ). Keogh, ( 1973b) found more fusaria macroconidia on dead ryegrass 
blade and sheath than on live ryegrass leaf. di Menna et al., ( 1969) found that fusaria 
numbers were slightly higher on the sheath than on the blade, and much higher on the dead 
litter than on the sheath or blade. Pasture length could be another determining factor of 
within pasture macroconidiwn counts, as the sporulation of Fusarium species. is stimulated 
by light. This may account for the fmdings of di Menna et al., ( 1991 ) which found higher 
1 2  
macroconidia counts in  shorter pasture where light penetration is better. The major effects 
of grazing animals on pasture are herbage removal, treading and the deposition of excreta. 
Most of the soil N is taken up by plants during spring growth; during summer and into 
autumn, further growth in the absence of pasture legumes depends on recycling of N by the 
animal. During the season the ryegrass dominant pasture will become a patchwork of 
excreta and inter-excreta areas each with characteristic growth patterns. In the excreta areas 
such as urine-patch and dung patch sites where there is a high N status the ryegrass grows 
rapidly and becomes densely tillered. Conversely the inter-excreta areas, which are 
generally N-deficient, have sparse tiller populations and are slow growing. The h igh N sites 
also offered favourable conditions for the proliferation of many fungal species which 
include those known to produce zearalenone. A study by Keogh, (1 973a), on the influence 
of the grazing animal on distribution patterns of Fusarium species in ryegrass pastures, 
found that spore loads were higher on herbage from urine patches than on herbage from 
inter -excreta sites. 
It is, however, difficult to predict when zearalenone production will be at its greatest as 
zearalenone is not formed with the spores and, therefore, spore counts cannot be used as 
indicators of zearalenone levels. Zearalenone peaks often appear following peaks in counts 
of Fusarium macroconidia., but after variable time intervals (di Menna et al., 1 99 1  ). Factors 
detennining zearalenone production in pasture seem to be site-related as no correlation 
between zearalenone concentration and Fusaria nwnbers has been observed (di Menna et 
al., 1987). It would appear that the production of zearalenone may have some relationship 
with changing climatic conditions. Mirocha et al., (1969) found that maximum zearalenone 
production in stored maize was obtained when the temperature was reduced to 1 2 °C to 
place a 'stress' on the fungus. Mirocha et al., ( 1977) stated that zearalenone production 
needed alternating low and moderate temperatures which fluctuated over a range of 1 0-
25oC. Findings by Miller et al., (1983), suggested that zearalenone production is associated 
with senescent fusaria populations on field corn inoculated with F. graminearum. A study 
by di Menna et al., ( 1987) found that zeralenone appeared at two sites 2 weeks after the 
peak fusaria counts which is consistent with Miller's findings. However, zearalenone was 
not found at other sites after fusaria numbers had dropped. Wolf and Mirocha., ( 1 973) 
consider that zearalenone is a fungal sex regulating hormone after observing that perithecia 
fonnation was stimulated at low zearalenone concentration and inhibited at high 
concentration. Windels et al., ( 1989) disputed this as there was a lack of correlation 
1 3  
between zearalenone and perithecium fonnation and suggesting instead that factors such as 
nutrition, light quality, and photoperiod are stronger influences on perithecium formation. 
So far the fonnation of zearalenone has proven difficult to characterise as different 
zearalenone producing species may form the metabolite under slightly different conditions. 
No particular weather pattern has yet been associated with high zearalenone levels, nor has 
the aspect of pasture sites had a consistent effect (Smith et al., 1991 ). 
9. Purpose and scope of the study 
The purpose of this present study was to investigate the presence of the oestrogenic 
mycotoxin zearalenone in grazed pasture and determine the effects of ingested zearalenone 
on reproductive perfonnance in sheep. In addition the metabolism of zearalenone and 
related compounds was investigated and the potential use of low zearalenone feeds such as 
chicory to reduce zearalenone intake and the risk of reproductive dysfunction. 
The aims of the experiments in this study were: 
1) To determine the distribution of zearalenone in ryegrass-<lominant pasture and to 
demonstrate that zearalenone can be taken up via the roots of ryegrass plants. 
2) To examine the metabolism of zearalenone in ewes dosed orally and intravenously with 
zearalenone and detennine the subsequent effects on reproductive performance. 
3) To detennine the effects of zearalenone ingested from pasture on the reproductive 
performance of ewes and assess the efficacy of the low zearalenone forage chicory in 
reducing zearalenone levels in the ewe. 
14 
CHAPTER ill 
The distribution of zearalenone in ryegrass pasture and uptake by the ryegrass plant. 
1. Introduction 
Zearalenone-producing Fusarium species are common in New Zealand pasture herbage (di 
Menna et al., 1 99 1  ) . At times the zearalenone concentration in the pasture can be great enough 
to affect the reproductive performance of grazing ewes (Jagusch et al., 1 986). It is not yet 
possible to predict when these high concentrations of zearalenone may occur. Making regional 
or seasonal predictions has been difficult as it is affected by variation in both zearalenone 
concentration and fusaria numbers in a pasture site on any one day. di Menna et al. , ( 199 1 )  
monitored the zearalenone levels and fusaria macroconidia in green and dead herbage fractions 
and on bulk herbage from long and short pasture at a site near Gisbome, in an attempt to 
identify factors which might be responsible for within-site variation. It was found  that 
zearalenone concentrations were higher on dead than on green material and higher in bulk 
samples from short pasture than long pasture. The study concluded that, as zearalenone and 
fusaria populations producing it were largely restricted to the dead material in the herbage, it  is 
this fraction which in the pre-mating period presents a potential hazard to the reproductive 
performance of grazing ewes. 
To better understand the variation in zearalenone concentration between sites and within the 
plant it is necessary to differentiate further between sites in the pasture with different 
characteristics and between components in the plant. The grazing animal has a large influence 
on the pasture characteristics. During the summer months, distinct patchwork areas in the 
pasture are formed by dung and urine from the grazing animal. These areas provide substrate 
and a high N site which suits the development of fusaria populations. Keogh, ( 1973a) found 
that fusaria spore loads were markedly higher on herbage from urine patch than on herbage 
from inter-excreta sites. To date the differences in these sites has been determined for fusaria 
populations, and possible differences in zearalenone levels have not been investigated. di 
Menna et al., ( 1987) found that there was no correlation between zearalenone concentration 
and fusaria numbers. This lack of correlation is not suprising given the different zearalenone-
producing potential of the various Fusarium species and variability in conditions suitable for 
these species to produce zearalenone. 
15  
Another important aspect of  urine patch sites is the rapid growth and tillering of  the grass 
around the patch of dead material. During late summer the rapidly growing areas around the 
urine patch are preferentially grazed by sheep and often provide most of the d iet (Keogh, 
1984). Given the high proportion of the diet contributed by the urine patch areas, it is the 
production and distribution of zearalenone in these ares which are of most importance when 
determining the zearalenone intake of the grazing animal. The characterisation of zearalenone 
levels within these sites is important in understanding the movement of zearalenone into plant 
tissues and determining the likelihood that the grazing animal will ingest sufficient quantities of 
zearalenone to affect reproduction. It is also necessary to investigate the zearalenone 
distribution within the plant in order to characterise movement of the compound to different 
parts of the plant and the eventual transfer to the grazing animal. The aims of this investigation 
were: 
1 .  To characterise zearalenone levels in dung patch, urine patch and inter-excreta sites within a 
rye grass dominant pasture and the distribution of zearalenone within the ryegrass plant 
2.  To determine the uptake of zearalenone by the plant and the subsequent transport to 
different components of the plant 
2. Methods and Materials 
2.1. Distribution of zearalenone in ryegrass pasture 
Herbage samples were collected in late April 1995 from three perennial ryegrass (Lolium 
perenne L.) dominant pastures at AgResearch Aorangi lowland research station consisting of a 
newly sown pasture (Pasture 1 ), and two established pastures, one containing endophytic 
(Acremonium lolii) fungi (Pasture 2), and the other being free of endophytic fungi (Pasture 3). 
The samples consisted of complete ryegrass plants and the soil supporting the roots. Three to 
five dung patch (DP), urine patch (UP), and inter-excreta (lE) sites (as described by Keogh, 
1986) were sampled in each of the three pastures. The samples were kept in sand boxes until 
dissection. The soil was removed from each sample and a small amount kept for zearalenone 
analysis. Six tillers from each sample were divided into tiller tops and roots. A further twenty 
1 6  
ryegrass tillers from each sample were taken and dissected into leaf blade (Bl) and  leaf sheath 
(Sh) which were grouped according to the age of the leaf (Bl l -4 and Sh 2-4). Dead leaf 
material was also divided into blade (DBl) and sheath (DSh). Roots (Rt) were separated into 
young and old tissue. flowering stem (St) and daughter tillers (DT) were also dissected out of 
the twenty tillers. The components were then freeze dried and weighed and ground prior to 
zearalenone analysis. 
2.1 . 1. Zearalenone determination 
Zearalenone levels in the herbage were determined by an indirect competitive ELISA 
immunoassay using partially purified zearalenone binding antibodies (di Menna et al., 1 99 1 ). 
Zearalenone analyses was carried out in the Plant and Fungal Toxins Lab, Agreasearch, 
Ruakura Research Centre (see Appendix 2 for full details of assay). 
Zearalenone yield was determined by multiplying zearalenone concentration by the dry herbage 
weight 
2.2. Uptake of zearalenone by the ryegrass p lant 
Eighty ryegrass tillers were collected from the same site in a pasture which had not been 
recently grazed. The soil was washed from the roots and the entire plant was washed with 
distilled water. The plants were kept overnight with the roots submerged in distilled water. The 
tillers were divided into four treatment groups (n = 20 tillers) and each group of tillers was 
supported in a container with nutrient solution (see appendix 3 for details of nutrient solution). 
In three containers, 50 J.l.g of pure zearalenone was added to 6 litres of nutrient solution (2 
litres per container). The fourth container was filled with 2 litres of nutrient solution without 
zearalenone. 
The ryegrass tillers were grown in the containers for 7 days at which time they were removed 
and dissected into blade (Bl), sheath (Sh), mature blade(MBl), mature sheath (MSh), stem (St), 
daughter tillers (Dn, young roots (YR) and old roots (OR). Each component of the twenty 
tillers was freeze dried, weighed, ground and analysed for zearalenone concentration. 
Samples of the nutrient solutions were taken at the beginning and end of the treatment period 
for zearalenone assay. 
1 7  
'late 3. 1 .  Ryegrass tillers in  nutrient solution 
1 8  
Plate 3.2. Dissection of ryegrass tillers into components 
·--
-
' • ' � '  I • 
19  
2.3. Statistical analyses 
Statistical analysis was carried out using Graph-pad Prism 2.0 software: 10855 Sorrento Valley 
Rd #203, San Diego, CA 92 1 2 1  USA. 
One and two-way analysis of variance was used to compare zearalenone concentrations and 
yields between pastures and between dung patch, urine patch and inter-excreta sites within a 
pasture. The reported probability (P) values were calculated from 'Type Ill ' sums-of-squares, 
after fitting generalised linear model. Sources of variation due to pasture, site type and 
interactions between site type and pasture were determined. Generalised linear model 
procedures were also used to compare zearalenone concentration and yield between plant 
components and the interaction between site type and component. 
Zearalenone levels are given as means ± standard error of the mean (SEM). 
3. Results 
3.1 .  Zearalenone concentration in pasture sites 
The difference in mean zearalenone concentration in the tiller tops between the three pastures 
was found to be statistically very significant (P<0.001 )  with levels in pasture 2 less than half 
those in pastures 1 and 3. There was no significant difference in zearalenone concentration in 
the roots between the three pastures. 
Levels of zearalenone were significantly (P<0.05) higher in the tiller tops in pasture 2 than in 
the roots. There was no significant difference in zearalenone concentration between tiller tops 
and roots in pastures 1 and 3. 
There was no significant interaction between the pasture and the site types within a pasture. 
20 
Figure 3.1. Zearalenone concentrations (mean + SEM) in tiller tops and roots between inter­
excreta, urine patch and dung patch sites in the three pastures . 
0 . 3 
E 
c. 
c. 0 .2 � 
u 
c: 
0 u 0 . 1 
0 .  
0 . 7 
E 
c. 
c. 0 .5 
u 
c: 
0 
u 0 .2 5  
0 . 0 0  
0 .3 
E 
c. 
c. 0 .2 
u 
c: 
0 
u 0 . 1  
0 .0 
P a s t u r e  1 
P a s t u r e  
P a s t u r e  3 
2 
c::J I N T E  R - E  X C  R E TA 
• · ·· · · • U R I N E P A TC H  
lli!IIIIIIIIIII D U N G  P A TC H  
r=::J I N T E R - E X C R E T A 
� U R I N E  P A TC H  
II!IIIIIIIIIIII D U N G  P A TC H 
c::::J IN T E R - E X C R E T A  
I · · ··I U R I N  E P A T  C H 
l!lllllllllll!i D U N G  P A TC H 
In pasture 1 zearalenone concentrations were 0. 137 ± 0.03 1 ppm (mean ± SEM), 0. 1 12  ± 
0.038 ppm and 0. 137 ± 0.01 5  ppm in the tiller tops and 0.028 ± 0.010 ppm, 0. 156 ± 0. 1 06 
ppm, and 0. 170 ± 0.017  ppm in roots for inter-excreta, urine patch and dung patch sites 
respectively. There was no significant difference in zearalenone concentration between the 
dung patch. urine patch and inter-excreta site types in the tiller tops. Zearalenone concentration 
in the dung patch roots were significantly higher (P<0.05) than inter-excreta roots. There was 
no difference in zearalenone concentration between urine patch and dung patch roots and 
between urine patch and inter-excreta roots. 
2 1  
In pasture 2 zearalenone concentrations were 0. 223 ± 0.03 ppm, 0.495 ± 0. 1 09 ppm and 0.33 1 
± 0 .0 84 ppm in the tiller tops and 0.044 ± 0 .027 ppm, 0. 0 47 ± 0. 0 47 ppm, and 0.06 4 ± 0.009 
ppm in the roots for inter-excreta, urine patch and dung patch sites respectively. There was no 
significant difference in zearalenone concentration between the three site types in either the 
tiller tops or roots. 
In pasture 3 zearalenone concentrations were 0.13 1 ± 0 .0 0 3  ppm, 0.168 ± 0 . 0 3 8  ppm and 
0 . 159 ± 0 .0 23 ppm in the tiller tops and 0 .045 ± 0. 0 4 1  ppm, 0. 020 ± 0.020 ppm, and 0.089 ± 
0 . 0 77 ppm in the roots for inter-excreta, urine patch and dung patch sites respectively. The 
was no significant difference in zearalenone concentration between the three site types in either 
the tiller tops or roots. 
Zearalenone concentrations were significantly (P<0.05 ) higher in the tiller tops than in the 
roots. Concentrations were on average 3 .6 ± 1.3 times higher in the tiller tops than in the roots 
of inter-excreta plants, 6.2 ± 2. 8 times higher in urine patch plants and 3.9 ± 1. 3 times higher in 
dung patch plants. 
3.2. Soil samples 
Zearalenone was only detected (0.01 11-g/g) in two soil samples, one from a dung patch site in 
pasture 1 and the other from a dung patch site in pasture 2. 
3.3. Zearalenone yield (Total zearalenone in the plant tissue) 
There was no significant difference in the zearalenone yield between the three pastures. The 
effect of site type on zearalenone yield was significant (P<0.001 ). There was no significant 
interaction between pasture and site type. 
Figure 3.2. Mean ( +SEM) zearalenone yields in tiller tops and roots between inter-excreta, 
urine patch and dung patch sites in each pasture. 
0 .  
C) 0 .  � 
-c 
Cl) 
>- 0 . 1  
0 . 3  
C) 0 .  
'"1lllt 
-c 
>- 0 . 1  
0 .  
tn 0 . 2  
.Q 
::2 
� 
>- 0 . 1  
P a s t u r e 1 
P a s  tu re 2 
ROOTS 
P a s t u r e  3 
I : ! I NTE R- EXC RETA 
!\•"·"'''' UR I N E  P ATC H 
IIIIIIIII D UNG  P ATCH 
c:J I NTE R - EXC RETA 
r;::::::::;J UR I N E  PATC H  
llllmiii DUNG  PATC H 
I = = = = = i i N T E R - E X C  R E TA 
'"",;r.:mi U R I N E  P A TC H 
IIIIIIIII D UNG P A TC H 
In pasture 1 zearalenone yields were 0.038 ± 0.008 J.lg, 0.078 ± 0.032 J.lg and 0. 1 1 1  ± 0.029 
J.lg in the tiller tops and 0.009 ± 0.005 J.lg, 0.017  ± 0.009 J.lg, and 0.009 ± 0.006 jlg in the 
tiller tops and roots for inter-excreta, urine patch and dung patch sites respectively. 
22 
Zearalenone yields were not significantly different between the dung patch, urine patch, and 
inter-excreta sites. 
23 
In pasture 2 zearalenone yields were 0.049 ± 0.002 �g, 0. 102 ± 0.046 �g and 0.200 ± 0.056 
�g in the tiller tops and 0.0 1 8  ± 0.009 �g, 0.004 ± 0.004 �g, and 0.02 1 ± 0.007 �g for inter­
excreta, urine patch and dung patch sites respectively. Zearalenone yield in the tiller tops of the 
dung patch sites were significantly higher than the inter-excreta sites, however, there was no 
d ifference in yield between the urine patch and dung patch sites or the urine patch and inter­
excreta sites. There was no difference in zearalenone yield in the roots between the three site 
types. 
In pasture 3 zearalenone yields were 0.049 ± 0.004 �g, 0.039 ± 0.0 1 0  �g and 0. 2 1 5  ± 0. 1 7 8  
�g in the tiller tops and 0.02 1 ± 0.0 1 5  �g, 0.002 ± 0.002 �g, and 0.029 ± 0.005 �g  for inter­
excreta, urine patch and dung patch sites respectively. 
There was no significant difference in zearalenone yield in the tiller tops between inter-excreta 
and urine patch sites. Zearalenone yield was significantly higher (P<0.05) in dung patch sites 
than urine patch and inter-excreta sites. There was no difference in the zearalenone yield of the 
roots between the three site types. 
There was no significant difference in zearalenone yield between the same site types in the 
different pastures. Yields in the tiller tops were significantly greater than in the tiller roots. 
3.4. Determination of zearalenone concentration and yield in components of ryegrass 
tillers from inter-excreta, urine patch and dung patch sites 
3.4.1. Zearalenone concentration 
The difference in zearalenone concentration between the components was found to be highly 
significant (P<O.OOI )  in pasture 1 and there was a significant difference (P<O.O l )  in 
zearalenone concentration of the components between the dung patch, urine patch and inter­
excreta sites. The interaction between components and site types in pasture 1 was found to be 
24 
highly significant (P<O.OOl). Zearalenone concentrations were generally higher in the leaf blade 
than in the sheath and were highest in the younger parts of the plant 
There was no significant difference in zearalenone concentration between components or 
different site types in pastures 2 and 3 and there was no significant interaction between site 
type and component. 
Figure 3.3. Zearalenone concentrations in dissected components of rye grass tillers. 
3 . 0  
E 
a. 2 . 5  
a. 
2 . 0 
() 
c: 1 . 5 
0 
() 1 . 0  
0 . 5  
0 . 0 
E 3 . 0 
a. 2 . 5 
c. 
2 . 0 
u 
c: 1 . 5 
0 
u 1 . 0  
0 . 5 
0 . 0 
3 . 0 
E 
a. 2 . 5 
a. 
2 . 0 
u 1 . 5  c: 
0 
u 1 . 0  
0 . 5 
0 . 0  
P a s t u r e  1 
C o m p o n e n t  
P a s t u r e  2 
C o m p o n e n t  
P a s t u r e  3 
C o m p o n e n t  
c:::::! I N T E R - E X C R E T A 
c:::::J U R I N E  P A T C H 
mllmlillll D U N  G P A T C  H 
c:::::J I N T E A - E X C  A E T A 
r::::::::J U A I N E  P A T C H  
III!IIIIIII!II! D U N G  P A T C H  
c:::::l i N T E R - E X C  R E T A 
'''""''""'' U R I N E P A T C H 
111111111111 0 U N G  P A TC H  
25 
3.4.2. Zearalenone yield 
Figure 3.4. shows the zearalenone yields of the components between the dung patch, urine 
patch, and inter-excreta sites. Zearalenone yields of the different plant components within a site 
type were also significantly different (P<O.O l)  and there was a significant (P<0.05) interaction 
between site type and component. 
In pasture 1 zearalenone yields were generally higher in the leaf blade than in the sheath. Yields 
were on average higher in components from dung patch plants than urine patch which were 
higher than inter-excreta. Relatively high yields were obtained from the roots of urine and dung 
patch plants however. 
In pasture 2 there was a significant difference (P<0.05) in zearalcnone yield between 
components from urine patch, dung patch and inter-excreta. There was no significant 
difference in zearalenone yield between components within a site type and no significant 
interaction between site type and component. 
In pasture 3 there was no significant difference in zearalenone yield between components of 
each site type and between components within a site type and there was no significant 
interaction between site type and component. 
26 
Figure 3.4. Zearalenone yields in dissected components of ryegrass tillers. 
P a s t u r e  3 
I o ! I N T E R - E X C R E T A 
0 .6 � U R I N E  P A TC H  
0 .5 111111111111111 D U N G P A T C H C) 
� 0 .4 
"C 
., 0 .3 
> 
0 .2 
J 0 . 1  
0 .0 
C o m p o n e n t  
27 
3.5. Zearalenone uptake 
The ryegrass components grown in the solution containing zearalenone all had higher 
zearalenone concentrations than the control. The highest zearalenone concentration in the 
zearalenone treatment was found in the dead leaf material at 0.25 ± 0.05 ppm compared to 
0.06 ppm in the control dead leaf. The highest concentration in the control plants was found in  
the blade (0. 1 ppm) which was as  high as the blade in the zearalenone treatment (0. 103 ± 
0.003 ppm).  
Figure 3.5. Zearalenone concentration in the blade (Bl), sheath (Sh) , mature blade (MBl),  
mature sheath (MSh),  dead leaf (DL), daughter tillers (DT) ,  stem (St), old root (OR) and 
young root (YR), components in ryegrass tillers grown i n  a solution which contained 
zearalenone. 
-
E 
a. 
a. -
0 
c: 
0 
0 
0.4 
0.3 
0.2 
0.1 
0.0 Bl  Sh MBI MSh DL DT St OR YR 
component 
3.5.1. Zearalenone yield 
c::::J zearalenone trt. 
1, ••• ,. ,  ... _.1 control 
The highest zearalenone yields in the zearalenone treaunent (highest to lowest) were in the old 
root, mature blade, dead leaf and daughter tillers. These components were also highest yielding 
in the control plants with the exception of the old roots. 
Figure 3.6. Mean zearalenone yields in the blade (Bl), sheath (Sh), mature blade (MBl), 
m ature sheath (MSh), dead leaf (DL), daughter tillers (DT), stem (St), old root (OR) and 
young root (YR), components in ryegrass tillers grown in a solution which contained 
zearalenone. 
Cl 
� 
0 . 1 00  
0 .075  
3:! 0 .050  � 
0 .0 2 5  
e o  m p o n e n t  
3.5.2. Zearalenone concentration i n  nutrient solutions 
c::::::J zea ra le none  trt .  
"'·''"''·"'· ·' c on tro l 
28 
The pre-treatment zearalenone concentration in the nutrient solutions was 0.46 ng/ml and 0 
ng/ml for the zearalenone and control treatments respectively. After the tillers were removed 
from the solutions the zearalenone concentrations were 0.04 ng/ml and 0 ng/m l for the 
zearalenone and control treatments respectively. These results mean that there was 
approximately 1 .8 4  11g present in solution in the 4 litres of nutrient solution which was reduced 
to 0.0 1 6  11g after the plants were removed. Therefore 1 .82 11g of zearalenone was removed 
during the treatment period. The total zearalenone yield in the tillers grown in this solution was 
1 . 8 1 �-Lg. 
4. Discussion 
4.1. Zearalenone distribution in pasture 
The results obtained in the investigation into zearalenone distribution within ryegrass pasture 
varied between areas and site types within an area. Zearalenone concentrations were generally 
lower in pasture 1 which was the youngest and hence had less dead material in the sward and 
soil near the surface for fusaria to colonise. Zearalenone concentrations in the tiller tops were 
significantly higher in Pasture 2 (endophytic) which may suggest that the presence of 
29 
endophyte fungi affects zearalenone levels. In all three areas sampled, there was no significant 
difference in zearalenone concentration between inter-excreta, urine patch and dung patch 
sites. This lack of significant difference is indicative of the large variability in zearalenone 
concentration within each site type. 
High N sites such as urine and dung patch sites offer favourable conditions for the proliferation 
of many fungal species which include various Fusarium species known to produce zearalenone 
(Keogh, 1 973b). Zearalenone production has been linked to changing climatic conditions 
(Mirocha et al. , 1 978) such as temperature and light, although no particular weather pattern 
has been associated with high zearalenone concentrations. Given the large number of variables 
present in zearalenone production, it is difficult to determine the conditions which result in 
production of zearalenone by the fusaria present in the pasture. These variables explain the lack 
of correlation between Fusarium numbers and zearalenone concentration observed by di 
Menna et al., ( 1 987). The samples in this investigation were taken on one occasion and 
therefore offer no information on zearalenone production resulting from changes in climatic 
conditions and substrate available for zearalenone-producing Fusarium colonies. The samples 
were taken in late April which is later than the months in which zearalenone production is 
considered to be at its peak. The pastures had not been recently grazed and there was new 
regrowth and very little dead material present. These factors and the fact that zearalenone 
production was found to be lower in 1 995 than previous years (Keogh, pers. comm) reduces 
the possibility of detecting differences between the sites. 
The zearalenone concentrations found in this investigation were all below concentrations found 
between January and April in other New Zealand pastures from Wanganui and Gisborne in an 
earlier study by di Menna et al. , ( 1 987), which reported zearalenone levels of between 0.4 and 
4.0 mglkg dry weight of herbage. 
In most cases zearalenone was not detected in the soil samples which indicates that most of the 
zearalenone at these sites was associated with the plant material above and below ground. It is 
possible that zearalenone was leached from the soil by watering during the period the plants 
were kept in the sand boxes prior to dissection as the soil samples containing zearalenone were 
among those dissected first 
In all areas sampled the zearalenone yields were highest in the tillers from dung patch sites 
followed by urine patch and inter-excreta sites. This result reflects the larger herbage mass of 
the sites with higher N levels and the lack of grazing pressure on the dung patch sites. At the 
time of sampling the areas were not being grazed and some regrowth had occurred which 
resulted in the higher herbage masses in the sites with higher N levels. The herbage around 
urine patch sites is preferentially grazed (Keogh, 1984) and it is from this portion of the 
pasture that the grazing animal is likely to ingest zearalenone. 
30 
The ryegrass dissection results showed that zearalenone concentrations were generally h igher 
in the leaf blade than the sheath which was most evident in the youngest leaves. The daughter 
tillers also had relatively high levels of zearalenone present As was seen in the whole tiller 
samples there was no significant difference in zearalenone concentration between inter-excreta, 
urine patch and dung patch sites within a pasture and there was a large variability in 
zearalenone concentration within a site type. The higher zearalenone concentrations in the 
young parts of the plants would suggest that zearalenone taken up by the roots is translocated 
p redominantly to these areas along with most of the nutrients required for growth. This has 
implications for the grazing animal as it is these parts of the plants which are selectively grazed 
and become the primary source of zearalenone for the animal. 
Zearalenone yields were greater in the leaf blade than in the sheath which is due to the higher 
zearalenone concentration and dry weight associated with the blade. Yields of zearalenone 
were highest in the dung patch and urine patch plant components which is due to a larger 
herbage mass than any difference in zearalenone concentration. The measurement of 
zearalenone yield in the different plant components enables the quantification of zearalenone 
intakes by the grazing animal as it allows comparison between parts of the plant which are 
eaten by the animal and those which are not normally eaten and the amount of plant material in 
each component. 
4.2. Assessment of method of estimation of zearalenone distribution 
The samples taken in this investigation gave an instantaneous representation of the zearalenone 
distribution at the various site types and these allowed comparison between the site types and 
pasture types. However, the method could not show the possible changes in zearalenone 
concentration and distribution over time within a site type which would illustrate some of the 
seasonal and grazing management effects on zearalenone production. Samples were kept 
outside in sand boxes until dissection which was carried out over a two week period. The 
3 1  
length of time from sampling to dissection may have influenced the zearalenone present in the 
plant causing difficulty in comparing samples which were dissected days apart. Despite these 
limitations, this investigation provided information on zearalenone distribution within different 
pasture site types and within the ryegrass planl 
4.3. Zearalenone uptake by the ryegrass plant 
The zearalenone uptake trial was aimed at determining whether zearalenone could be taken up 
by the roots and translocated in the ryegrass plant. The highest concentration was found in the 
dead leaf material (0.25 ± 0.05 ppm) which was not expected considering there would be no 
active transport to this tissue. The next highest concentration was found in the daughter tillers 
(0. 1 47 ± 0.045 ppm). The daughter tillers, and other new growth areas on the plant, are the 
sites of most of the nutrient transport and therefore nutrients taken up by the root would be 
transported and deposited in these tissues along with other compounds such as zearalenone 
which may have been taken up by the roots. The relatively high concentration of zearalenone in 
the old and young roots of the tillers grown in the solution containing zearalenone may be as a 
result of zearalenone adhering to the root surface rather then deposition of zearalenone in the 
root tissue. Analysis of the zearalenone concentration in the nutrient solution showed that only 
approximately 1 .84 j.ig of the 50 jlg added to the 6 litres of solution stayed in solution. 
Zearalenone's low solubility in water and possible adherence to the container walls are the 
most likely reasons for the loss of zearalenone. However, it appeared that most of the 
zearalenone remaining in solution was taken up by the plant as the difference in total yield 
between the zearalenone treatment and the control plant was approximately 1 .6 jlg and the loss 
of zearalenone from the nutrient solution was 1 .82 jlg which leaves 0.22 J.Lg of zearalenone 
unaccounted for. 
4.4. Assessment of the method 
The zearalenone uptake trial was a preliminary investigation into measuring zearalenone uptake 
via the roots and subsequent transport within the plant. The trial was a first attempt at 
. determining zearalenone uptake in the ryegrass plant and was important in the further 
development of a suitable method for achieving this. Major concerns were in keeping 
zearalenone in an aqueous solution in significant amounts so that uptake by the roots can be 
determined. Once developed fully, this method will be valuable in characterising zearalenone 
uptake and distribution by many species of pasture plants. 
4.5. Conclusions 
32 
The main factors which affect the  ingestion of  zearalenone by the grazing animal are grazing 
behaviour and the production and distribution of zearalenone in the forage. Grazing behaviour 
can be easily determined and is well understood, however, the difficulty in characterising 
zearalenone production and distribution within the pasture and plant does not allow accurate 
quantification of zearalenone intake. It is necessary to further investigate the distribution of 
zearalenone in different pasture sites and within the plant so that the relationship between 
zearalenone production in the pasture and subsequent ingestion by the animal can be 
determined. 
The characterisation of zearalenone transport in the ryegrass plant is an important part in 
understanding how zearalenone, once produced, is ingested by the grazing animal. If we 
assume that zearalenone is more likely to accumulate in the youngest parts of the plant once 
taken up by the roots it is likely that a large proportion of this will be ingested by grazing 
animals. Further work needs to be done to better understand the transport of zearalenone in 
the plant and the implications to the grazing animal. 
33 
CHAPTER IV 
Zearalenone and related compounds in the blood and urine of ewes intravenously and 
orally dosed with zearalenone and the effects on reproductive performance. 
1. Introduction 
Zearalenone is a naturally occurring mycotoxin produced by Fusarium species which are 
present in New Zealand pasture (di Menna et al ., 1987; di Menna et al., 199 1 and Lauren et 
al. , 1 988). Zearalenone and its metabolites a- and �-zearalenol have been shown to have 
oestrogenic properties (Miksicek, 1 994). The adverse effects of zearalenone on reproduction 
have been extensively reported in pigs (Aucock et al., 1982; Long et al., 1982 and Ruhr et al. , 
1983),  poultry (Alien et al., 198 1 and Maryamma et al., 1 992) and cattle Mirocha et al., 1 968; 
Danko and Aldsay. , 1969, and Miller et al., 1973). To date only a few studies have examined 
effects of zearalenone on ewes. Reproductive performance was markedly reduced in ewes 
dosed daily with Fusarium cultures containing 25 mg zearalenone for 10 days prior to mating 
(Smith et al., 1986). Later Smith et al., ( 1 988) observed a significant reduction in ovulation 
rate in ewes dosed daily for 10 days with Fusarium cultures containing 6 mg zearalenone. 
Intakes of zearalenone of 3 mg/ewe/day or more during a ten day period prior to mating have 
been reflected as depressed ovulation rate and lower lambing percentages (Smith et al. ,  1990). 
In these trials investigating the effects of zearalenone on sheep reproduction, the zearalenone 
was dosed as part of a Fusarium culture which is likely to contain other compounds, some of 
which will also have bioactive properties, which may add to the effect on reproductive 
performance. In the studies where pure zearalenone was dosed orally, only the reproductive 
parameters were examined and no account was taken of how much of the dosed zearalenone 
entered the blood stream and in particular the amount of unconjugated or free zearalenone 
which is the oestrogenically active portion. The conversion of zearalenone into other 
metabolites was also not taken into account. Kiessling et al.,( 1984) showed that zearalenone 
was reduced to a-zearalenol and to a lesser extent �-zearalenol in rumen fluid. It is likely that a 
large proportion of ingested zearalenone is converted in the rumen to a-zearalenol which is of 
higher oestrogenic potency than zearalenone (Miksicek, 1994). Fusarium species present in 
New Zealand pasture are capable of producing a- and �-zearalenol which may result in sheep 
34 
ingesting these metabolites with the forage (di Menna et al . ,  1987). There is also evidence that 
zearalenone is converted to zearalenols and zearalanols in the animal (Miles et al., 1996) 
Therefore, it is necessary to consider the other metabolites of zearalenone in addition to 
zearalenone when determining effects on reproductive performance. The aims of this study 
were: 
l .  To determine free and conjugated zearalenone and other zearalenone metabolites in the 
blood and urine of ewes dosed orally or intravenously with pure zearalenone for ten days prior 
to mating. 
2. To measure the effects of zearalenone on the reproductive performance in sheep. 
2. Methods and Materials 
2. 1 .  Animals and treatments 
Sixty mixed-age Romney ewes were divided into two groups (n=30) which were either grazed 
on perennial rye grass (Lolium perenne. L.) pasture or on chicory ( Chicorium intybus L. cv 
Grasslands Puna), which has low levels of zearalenone ( <0. 1 �g/g). Within each grazing 
treatment the ewes were divided into three sub-groups (n = 10) which were allocated to either 
oral (0) or intravenous (IV) dosing with zearalenone, and a control (C). The oral dosed group 
received a dose of 5 mg of pure zearalenone in 10% ethanol solution daily for 10 days prior to 
mating. Half of the ewes in the intravenous dosed group received 2 mg of zearalenone (NH) 
in 10% ethanol injected intravenously daily for 10 days prior to mating and the remaining half 
received 0.5 mg of zearalenone (IVL) in the same manner. 
Ewes were treated with synchronisation devices (CIDR, type G, containing 0.3 g 
progesterone) for 14 days before mating (see Fig. 4. 1 .). 
All ewes were blood sampled pre-treatment by jugular venipuncture with hypodermic needle 
and evacuated collection tube (vaccutainer 10 ml draw). Subsequent blood samples were taken 
daily before dosing. On day 5 of dosing blood and urine samples were collected at 2, 4 and 24 
hours after dosing in the pasture group and 1, 4 and 24 hours after dosing in the chicory group. 
Samples of chicory and the ryegrass pasture were taken for zearalenone determination. 
Mter the 10 days of treatment, the CIDRs were removed and the ewes were run on their 
respective forages with an entire ram fitted with harness and crayon and inspected daily for 
mating marks. After all ewes had shown oestrus, the crayon colour was changed to identify 
returns. Ovulation rates were determined 7 days after mating by laparoscopic examination. 
Figure 4.1. Diagram showing treatment groups 
10 DAYS 
ORAL DOSED ( 10  EWES) 
2 DAYS 
INTRA VENOUS IDG H DOSE (5 EWES) 
PASTURE (30 EWES) 
INTRA VENOUS LOW DOSE (5 EWES) 
CONTROL (IO  EWES) 
ORAL DOSE (10 EWES) 
INTAVENOUS IDGH DOSE (5 EWES) 
CHICORY (30 EWES) 
Il\'TRA VENOUS LOW DOSE (5 EWES) 
CIDR INSERTED CONTROL (10 EWES) 
35 
START OF DOSING RAM INTRODUCED 
2.2. Determination of zearalenone and its metabolites in blood and urine 
Whole blood and urine samples were analysed for conjugated and free "zearalenone" 
equivalents by enzyme-linked immunoassay (ELISA) (di Menna et al., 199 1 (appendix ll)).The 
ELISA recognises zearalenone ( 100% ), but also has appreciable cross-reactivities with a­
zearalenol (220%), �-zearalenol (60%), a-zearalanol ( 1 10%),Taleranol (35%) and zearalanone 
(46%). 
36 
Blood and urine taken at 24 hour intervals was subsampled and combined to make composite 
samples representing each treatment group at each sampling and were analysed for total 
zearalenone, zearalanone, a-zearalenol, P-zearalenol, zeranol and taleranol by a multiresidue 
assay for a number of anabolic and oestrogenic compounds and fungal metabolites (Erasmuson 
et al,. 1994). Aliquots (5ml) were analysed by glucuronidase re-formation of the alcohols, 
extraction with hexaneltBME (70:30), and then HPLC cleanup of the extracts with 
programmed fraction collection . A fraction was evaporated under nitrogen, then trimethylsilyl 
derivatized with MSlFA, and subject to GC-MS analysis (see appendix I for full details of 
assay). 
2.3. Statistical analyses. 
Statistical analyses were done using Graph-pad Prism 2.0 software: 10855 Sorrento Valley Rd 
#203, San Diego, CA 92 1 2 1  USA. 
Differences in ovulation rate between oral, intravenous and control groups were compared by 
the Kruskal-Wallis test which is a non-parametric test to compare three or more unpaired 
groups. Difference in ovulation and conception rate between groups grazed on chicory and 
groups grazed on pasture were compared by Wilcoxon signed rank test which is a 
non parametric test that compares two paired groups. 
Levels of zearalenone and related metabolites in the blood and urine were compared by one­
way analysis of variance (ANOV A) techniques. 
3. Results 
3.1 .  Liveweight 
Ewes grazed on chicory lost an average of 0.5 ± 0.72 kg and ewes grazed on pasture lost an 
average of 1 .0 ± 0.43 kg. There was no significant difference in the weight loss between the 
grazing treatments. 
3.2. Ovulation rate 
There were no significant differences in ovulation rate between oral dosed (0), intravenous 
high (IVH) and low (IVL) dosed and the control (C) groups within either the pasture or the 
chicory treatments (Fig 4.2.). 
However, a significant difference (P<0.05) was found in mean ovulation rate between the 
group grazed on chicory ( 1 .57 ± 0. 1 1 ) and the group grazed on pasture ( 1 .23 ± 0.08). 
Figure 4.2. Mean ovulation rate ( + SEM) for each treatment group. 
2 
CD -
C'CI 
... 
c 
.2 
-
..!!! 1 
::::7 
> 
0 
pc po 
3.3. Conception 
CC CO 
Treatment 
37 
There was no significant difference in the number of returns to oestrus between the groups of 
ewes grazed on chicory and those grazed on pasture (Fig. 4.3.). There were no returns in the 
pasture control or chicory oral groups. The number of returns were 33% and 20% for the 
groups grazed on pasture and chicory respectively however, they were not significantly 
different 
Figure 4.3. Number of returns in each treatment group. 
1 00 
ctl 
c 7 5  
.. 
::::7 -CD 5 0  .. 
� 0 
25  
0 
. . . . .. . .  
. . . . . . .  · · · · · · · · 
pc po pM pivh CC CO 
T r e at m e n t  
3.4. Zearalenone in the forages 
The zearalenone concentrations in the forages were 6.0 Jlg/g and 0. 1 8  Jlg/g for the ryegrass 
pasture and chicory respectively 
3.5. Zearalenone and its metabolites in blood 
38 
Free zearalenone concentrations (Fig 4.4.) in the intravenously dosed pasture groups 2 hours 
after dosing in the IV groups were 0.20 ± 0. 1 1  ng/ml and 0.858 ± 0.47 ng/ml for the low and 
high dose respectively. The highest concentration recorded (0.69 ± 0.20 ng/ml) in the low IV 
group was at 4 hours after dosing. The free zearalenone concentration in the oral dosed 
pasture group was greater than the IV groups with a concentration of 1 .56 ± 0. 14 ng/ml at 4 
hours after dosing. By 24 hours the levels of free zearalenone in the blood of all the dosed 
pasture groups had fallen to 0.09 ± 0.05 ng/ml, 0.046 ± 0.05 ng/ml for the oral and high IV 
dose respectively and were not detected in the low IV dose group. The free zearalenone 
concentration in the groups grazing chicory were on average higher that the levels in the 
pasture groups. The highest free zearalenone concentrations recorded were 0.67 ± 0.29 ng/ml, 
1 .35 ± 0. 1 6  ng/ml and 0.75 ± 0. 19  ng/ml for the oral, low IV and high IV dose groups 
respectively,  1 hour after dosing. By hour 4 the free zearalenone concentrations had decreased 
to 0.43 ± 0.05 ng/ml, 0.28 ± 0.08 ng/ml, 0.28 ± 0. 1 2  ng/m1 for the oral, low IV and high IV 
dosed groups respectively. 
The conjugated zearalenone concentration in the pasture groups follows a similar pattern after 
dosing and was at similar levels to the free zearalenone. Conjugated zearalenone 
concentrations were 1 .50 ± 0.29 ng/ml, 0.8 1  ± 0.33 ng/ml and 0.82 ± 0.30 ng/ml for the oral, 
low IV and high IV dosed groups respectively 4 hours after dosing. The oral and low IV dosed 
groups had highest conjugated zearalenone levels at 4 hours after dosing whereas the high IV 
group had similar concentration for all samples. 
The levels of conjugated zearalenone (Fig 4.5.) in the groups grazing chicory follow a similar 
pattern to the concentrations of free zearalenone. The conjugated zearalenone concentrations 
were highest 1 hour after dosing being 1 . 1 8  ± 0.44 ng/ml, 0.98 ± 0.56 ng/ml and 1 .6 1  ± 0.46 
ng/ml for the oral, low N and high IV groups receptively. Levels of free and conjugated 
zearalenone appeared to be very high in the chicory control group 1 hour after dosing ( 1 .65 ± 
0.23 ng/ml and 2.62 ± 0.3 1 ng/ml for free and conjugated respectively) which cannot be 
explained. 
Figure 4.4. Mean levels of unconjugated (free) zearalenone in the blood of ewes in the OD, 
IVH, IVL and CON groups grazed on either pasture or chicory, at four times during a 24 h 
period after dosing. 
3 
- 2 
E 
a, 
c: 
1 
a ......... �..__ 
� 
c 
3 
2 
1 
0.0 2.0 4.0 24.0 
Time after dosing (hours) 
0 .. . . . . 0.0 1 .0 4.0 24.0 
Tirre after dosing (hours) 
EII2J pc 
,�,::,�,���'''  p o  
fill1i'llll p i vi 
- p i vh  
LIITITJ] cc 
� CO 
1111111111 ci � 
-ci\kl 
39 
40 
Figure 4.5. Mean levels of conjugated zearalenone in the blood of ewes in the OD, IVH, IVL 
and CON groups grazing either rye grass pasture or chicory, at various times during a 24 h 
period after dosing. 
3 
2 
1 
o � ............ 
0 .0  2 .0  4.0 24.0 
3 
1 
0 
Time after dosing (hours) 
0.0 1 .0 4.0 24.0 
lirre after dosing (hcus) 
EIJJ pc 
ll!!!ll!lli!il p i \A  
- p i \tl  
l = : : l cc  
l�:i,��'!!.q CO 
111'1 ci� 
-ci\h 
Levels of alkane metabolites were very small (<1 %) compared to alkene metabolites. 
Zearalenone was the major metabolite present ( 16.7 ng/ml) in the oral dose ewes. Alpha and 
beta zearalanol and zearalenol were found in both control and oral dosed groups. 
Figure 4.6. Levels of alkene zearalenone metabolites in the blood of oral dosed ewes and 
control ewes grazed either pasture or chicory. 
20 
E 
c, 1 0  c:: 
a-zearalenol 
metabolite 
b-zearalenol 
c:J pasture control 
,, ,,,, , pasture oral 
llll!l!lllllill chicory control 
- chicory oral 
Figure 4.7. Levels of alkane zearalenone metabolites in oral dosed and control ewes grazing 
either pasture or chicory. 
0.3 
0.2 
E 
a, c 
0 . 1  
0.0 
" '  
. ,  
" '  
" ' 
, . ,  
metabolite 
l 'I pasture control 
'''"''""ij pasture oral 
1!1111111111111 chicory control 
-chicory oral 
4 1  
Zearalenone was also the predominant metabolite present (2. 1 ng/ml) in the oral dosed group 
grazing chicory. Levels of zearalenone in the oral dosed group grazing pasture were eight 
times higher than levels in the oral dosed group grazing chicory. Zearalenone, a-zearalenol, 13-
zearalenol and a-zearalanol were not detected in the chicory control group. Levels of alkane 
metabolites were <1% of the alkene metabolites where both alkene and alkane metabolites 
were present. 
Levels of both alkene and alkane metabolites were higher in the groups grazing pasture than 
those grazing chicory (Fig 4.7.). 
3.6. Zearalenone and its metabolites in the urine 
After two days of grazing the different forages the levels of free and total zearalenone were 
significantly higher (P<0.05) in the urine of ewes grazed on the pasture treatment (Fig 4.8.) . 
The free zearalenone:creatinine ratios were 0.055 ± 0.009 mmollmol and 0.027 ± 0.005 
mmol/mol for the pasture and chicory groups respectively. The conjugated 
zearalenone:creatinine ratios were 0.389 ± 0.037 mmollmol and 0. 1 34 ± 0.020 mmollmol for 
pasture and chicory treatments respectively. Levels of free zearalenone in the urine of ewes 
grazing chicory were approximately half those in the urine of ewes grazing ryegrass pasture 
after two days of grazing. 
42 
Figure 4.8. Free and conjugated zearalenone/creatinine ratios in the urine of ewes which have 
been grazed on either ryegrass pasture or chicory for two days before dosing treatments 
started. 
.2 -
ea 
... 
... 
0 -N 
0 . 4 
0 .  
0 .  
0 .  Q....L.---!;.;.;;;.;.;.;.;;.;;.;L._ pasture 
g r a z i n g  t r e a t m e n t  
lo o : o : o : o o) f re e  
� c o n j u g a t e d  
43 
Figure 4.9. Free and total zearalenone:creatinine ratios on day 6 of dosing in the urine of the 
OD, IVL, IVH and control (C) ewes grazed on either ryegrass pasture or chicory. 
0 
-
� ... 
... 
() -
N 
3 
0 
-; 2 
a.. 
a.. 
() 
r::J 1 
CO 
t r e a t m e n t  
civh 
t r e a t m e n t  
cc 
!: : : : : ; ; : : : :! f r e e  
J;;" "'"'""' c o n  ju g a te d 
,, , , , , , , , , , ,, free  
'"""'�'ti�!''  conjugated 
Conjugated zearalenone levels in the pasture control group were significantly higher (P<0.05) 
than in the chicory control group, however there was no significant difference in free 
zearalenone. The oral dosed groups were significantly higher (P<0.05) in both free and total 
zearalenone than their respective control groups. There was no significant difference in either 
free or conjugated zearalenone concentration between oral and intravenously dosed groups 
within or between the chicory and pasture groups (Fig 4.9.). 
There was no significant difference in the levels of any of the metabolites in the urine (Fig 
4. 10.)between the dosed and non-dosed groups grazed on pasture, however, there was 
significantly (P<0.05) more of the metabolites in the groups grazed on pasture than those on 
chicory. Zearalenone and �-zearalenol were the most abundant metabolites in both dosed and 
non-dosed ewes. The zearalenone related metabolites were predominantly present in alkene 
form. 
Figure 4. 10. Levels of alkene zearalenone metabolites in the urine of zearalenone dosed and 
non-dosed ewes grazed on either ryegrass pasture or chicory. 
1 5 
E 1 0 -
Cl 
5 
zearaleoone a-zearalenol 
m e t a  b o  l i t e 
c:::::J p a s tu r e  c o n t r o  I 
P''""'''"'''l p a s tu r e o r a I 
l!lllllllllllll c h i c o r y c o n t r o l  
- c h ic o r y o ra l  
44 
Concentrations of a-zearalanol and �-zearalanol (Fig 4. 1 1 .)were significantly greater (P<0.05) 
in the urine of the group dosed with zearalenone. No zearalenone was detected in the non­
dosed group. 
Concentrations of all metabolites in the dosed and non-dosed groups grazed on chicory were 
significantly lower (P<0.05) than the groups grazed on pasture. 
Figure 4.11. Levels of alkane zearalenone metabolites in the urine of ewes which are either 
dosed or non-dosed with zearalenone and grazed on either ryegrass pasture or chicory. 
E -
C) 
c 
a-zearalanol 
m e t a b o l i t e  
b-zearalanol 
!:: : : : : : : : : : :! p a s tu r e  c o n tr o l  
� p a s tu r e  o r a l  
IIIIIIIIIB c h i c o r y c o n tr o l  
- c h i c o r y  o r a l  
4. Discussion 
4.1. Animal measurements 
The weights of the ewes fell in both the chicory and pasture treatments but there was no 
significant difference in the mean liveweight loss between the two grazing treatments. 
45 
The results obtained in the zearalenone dosing trial showed no significant difference in 
ovulation rate between the type of dosing or the amount dosed within the pasture or chicory 
groups. However, there was a significant difference (P<0.05) observed in ovulation rate 
between the groups grazed on pasture and the groups grazed on chicory. Because of  the 
relatively small number of animals used in each dosing treatment it is likely that the differences 
in ovulation rate between oral dosed, intravenous dosed and the control ewes within each of 
the grazing treatments, and in particular the chicory treatment, would not be significant. The 
difference in mean ovulation rate between the ewes grazed on chicory and the ewes grazed on 
rye grass pasture could be an indication of the difference in zearalenone intake between the 
ewes grazing the ryegrass pasture and those on chicory or could be a reflection of the different 
nutritional properties of the two feed types. Although chicory has been shown to have 
nutritional qualities which are superior to ryegrass pasture (Fraser et al. ,  1988), the change in 
liveweight between ewes grazed on chicory and ewes grazed on pasture was not significantly 
different which reduces the possibility that the difference in ovulation rate was due to a 
difference in nutrition. 
Smith et al., ( 1988) found that conception rate was only reduced in ewes which were ingesting 
more than 12 mg of zearalenone per day. There were no significant differences in returns to 
service in the groups grazed on chicory or pasture which may be due to the small number of 
animals in each treatment and/or insufficient free zearalenone to affect conception rate. 
In addition to better defining the relationship between zearalenone ingestion and reproductive 
performance in ewes, the other important aspect of this trial was to characterise the 
metabolism of free and conjugated zearalenone and its various metabolites in the sheep and 
attempt to relate this to the reproductive data obtained. The intravenous groups were dosed at 
rates of 0.5 mg and 2.0 mg for the low and high dose rates respectively, and the oral dosed 
groups received 5 mg at each dosing. Although the zearalenone concentrations of the chicory 
and the ryegrass pasture were determined, it would be difficult to know how much zearalenone 
46 
was ingested by the ewes as the daily intakes were not determined. However, assuming that the 
intakes were similar for the pasture and chicory treatments and given the relative zearalenone 
concentrations at approximately 6.0 !lg/g and 1 .3 llg/g of plant tissue for pasture and chicory 
respectively, it is likely that the zearalenone intake in the ewes grazing the ryegrass pasture was 
considerably higher than in ewes grazing chicory. Smith et al. , ( 1990) concluded that intakes of 
3 mg/day and above were sufficient to reduce ovulation rates which would suggest that the 
levels of zearalenone dosed in this trial, and in particular the pasture groups, were more than 
sufficient to evoke a response in ovulation rate 
4.2. Zearalenone in the blood 
Blood samples taken at 24 hour intervals, directly before the daily doses of zearalenone were 
administered, showed the relative free and total zearalenone concentration in each of the 
dosing groups and grazing treatments. It was only possible to compare the groups grazed on 
pasture with the groups grazed on chicory at 0, 4 and 24 hours after dosing. The levels of free 
and conjugated zearalenone in the blood are representative of the net effect of dosing free 
zearalenone and the influences of ingestion, conjugation, excretion and recycling of 
zearalenone. Conjugation and deconjugation of zearalenone by the liver and recycling via the 
bile are important factors that may have a large contribution to changes in free and conjugated 
zearalenone concentrations in the blood in addition to the effect due to the amount ingested or 
dosed. No blood samples were taken between 4 and 24 hours after dosing so determination of 
zearalenone concentrations between these times was not possible. 
4.3. Zearalenone in the urine 
Analysis of free and conjugated zearalenone in the urine provided a better indication of the 
relationship between zearalenone intake and passage through the animal because once in the 
urine, zearalenone was not recycled back into the blood. Urine samples taken before dosing 
began and when the ewes had been on the grazing treatments for two days showed clear 
differences in zearalenone concentration in the animals grazing chicory from those grazing 
pasture. The results showed that both free and conjugated zearalenone levels were significantly 
higher in ewes grazed on pasture than on chicory. 
47 
The urine samples taken on the sixth day of dosing also had concentrations which were 
indicative of the zearalenone intake. There were significantly (P<0.05) higher levels of free and 
conjugated zearalenone in the dosed groups than in the control groups on both chicory and 
pasture treatments. These results reflect the larger amounts of zearalenone present in the dosed 
animals. The difference in zearalenone intakes between ewes grazed on pasture and those 
grazed on chicory was also illustrated by the zearalenone concentration in the urine samples. 
Levels of free zearalenone were not significantly different in the pasture and chicory control 
groups, however, the amounts of conjugated zearalenone were higher in the pasture control. 
The lack of difference in the free zearalenone concentration is largely due to conjugation of 
most of the zearalenone before passing into the urine. Unlike levels of free zearalenone in the 
blood, which often represented more than 50% of the total zearalenone present, the proportion 
of free zearalenone in the urine was generally lower at around 20% of the total zearalenone 
present. However, the reduction in the proportion of free zearalenone from the blood into the 
urine observed in this trial indicates that the process of conjugation may not be very efficient 
Many other toxic compounds exist solely in the conjugated form once they reach the urine and 
the proportion of free compound in the blood is much lower than that seen with zearalenone. 
The inefficient conjugation of zearalenone by the ewes means higher proportions of the 
oestrogenically active or free zearalenone may be present in the system. However, the levels of 
free and conjugated zearalenone in the urine and blood are not conclusive as they often don' t  
reflect the level of  zearalenone intake by the animal. 
A urinary total zearalenone/creatinine (as measured by ELISA) marker for zearalenone 
intoxication in sheep has been established by Sprosen et al. ( 1995) which indicates that levels 
in excess of 12.5 mmol zearalenone per mol creatinine may be associated with significant 
reductions in lambing percentages. The urine results obtained in this dosing trial were 
significantly lower than this marker level. However, urine samples were taken later in the 
morning when most of the ewes had already urinated thereby removing the zearalenone 
excreted during the previous night and possibly explaining the lower levels measured in the 
urine. 
48 
4.4. Metabolism of zearalenone 
Previous studies have regarded zearalenone alone when considering reproductive dysfunction 
which takes no account of the likely oestrogenic effects of other zearalenone metabolites. 
Kallela and V asenius, ( 1982) made the assumption that ruminants would be less affected by the 
oestrogenic effects of zearalenone because it was degraded in the rumen and, therefore, 
rendered inactive. The fact that it may be degraded to metabolites, of which some are more 
potent in oestrogenic activity than zearalenone (Fitzpatrick, 1989; Miksicek, 1 994) was 
unknown at that time. The reduction of zearalenone by rumen protozoa and bacteria to a­
zearalenol and to a lesser degree to �-zearalenol was demonstrated by Kiessling et al. , ( 1 984). 
In addition recent fmdings by Erasmuson et al., ( 1 994) and Kennedy et al., ( 1 995) have found 
zeranol, taleranol a-zearalenol and �-zearalenol in the bile and urine of grazing animals and 
that the source of these compounds could be intrinsic i.e. resulting from transformation of 
zearalenone in the animal, or extrinsic i.e. being produced by Fusarium species prior to 
ingestion by the animal. Fitzpatrick et al., ( 1 986) found that the relative order of oestrogenic 
potency for zearalenone and its major metabolites in order of strongest to weakest was a­
zearalenol, zearalenone and �-zearalenol. Zearalenone metabolised into a-zearalenol in the 
pasture or in the animal, will result in a higher risk of reproductive dysfunction than if 
zearalenone intake alone was considered. The presence of zearalenone metabolites in the urine 
of zearalenone dosed sheep has been examined subsequent to this investigation, by Miles et al. , 
( 1996). The relative proportions of the alkane and alkene metabolites were similar to those 
. 
found in the urine of orally dosed ewes in this trial with the major metabolites present being 
zearalenone, a- and �-zearalenol and relatively small levels of the alkane metabolites i.e. 
zearalanone, a-and �-zearalanol. The study by Miles et al. , ( 1996) also determined free and 
conjugated zearalenone concentrations relative to time after dosing. Urine samples in this 
investigation were not taken in a sequence after dosing, however, the decline of free 
zearalenone in the blood samples after intravenous dosing and the increase after oral dosing are 
similar to urine samples taken after dosing by Miles et al., ( 1996) The similarities found in 
relative zearalenone metabolite levels indicate that there are specific metabolic pathways 
involving zearalenone within the sheep which must be examined further. Therefore further 
investigations are required into the presence of these zearalenone metabolites in sheep and their 
role in reproductive dysfunction. 
4.5. Zearalenone related metabolites in the urine 
In all the metabolite concentrations determined, the levels in the pasture groups were 
significantly (P<0.05) higher than those in the chicory groups. 
49 
This finding provides some interest in light of results of Kiessling et al., ( 1984) where a.­
zearalenol was the predominant metabolite remaining after the reduction of zearalenone in the 
rumen suggesting that a.-zearalenol would be the predominant metabolite absorbed into the 
blood stream of the sheep after zearalenone was ingested. This, however, does not account for 
any further transformations which may take place once in the blood stream. However, the 
relative proportions of the alkane metabolites determined in the urine differ from the alkenes in 
that the proportion of a.-zearalanol is twice that of �-zearalanol and up to ten times that of 
zearalanone. This result follows more closely the fmdings of Kiessling et al., ( 1 984) which may 
be due to the fact that the alkane metabolites obtained from the forage were already in the a.­
and �-zearalanol forms and that all ingested alkane fonns would be further metabolised in the 
rumen. 
4.6. Conclusions 
Blood results were not correlated to the level of zearalenone intake, however the urine results 
provided a good indication of differences in zearalenone levels and metabolism in sheep in 
relation to zearalenone intake. 
The results obtained in this trial showed no difference in ovulation rate between dosed and 
non-dosed animals despite differences in zearalenone intake. The lack of difference may have 
been due to the small group sizes. There was, however, a difference in ovulation rate between 
groups grazed on chicory and those on pasture which could be due to the higher zearalenone 
intake of ewes grazed on pasture given that there appeared to be no nutritional differences 
between the groups. Further work is required dosing zearalenone to a larger number of animals 
to identify effects on reproductive performance. 
CHAPTER V 
Zearalenone in ewes grazed on pasture or chicory and subsequent effects on 
reproductive performance. 
1. Introduction 
Lambing performance on many New Zealand fanns is often below the expected level despite 
adequate ewe condition, management and absence of disease. 
50 
Zearalenone-producing Fusarium species are common in New Zealand pasture herbage (di 
Menna et al. , 1 99 1 ). The oestrogenic effects of zearalenone have been reported in swine (Long 
et al., 1982; Diekman & Long, 1989) and cattle (Mirocha et al., 1968; Dank:o & Aldsay, 1 962; 
Miller et al., 1973). Recent fmdings have indicated that zearalenone present in pastures may be 
sufficient to reduce reproductive performance in ewes (Smith et al. , 1986; Smith et al., 1 987 ;  
Smith et al., 1 988 ;  Smith e t  al., 1990; Jagusch e t  al. , 1986). These studies found the exposure 
of  ewes to zearalenone around the time of mating reduced ovulation rate and the subsequent 
lambing percentage. Numbers of fusaria are greatest in late summer and autumn when suitable 
environmental conditions and substrate exist for proliferation of the fungi. This seasonal 
increase in fusaria activity coincides with mating on many New Zealand sheep farms and it 
seems possible that zearalenone produced by the Fusarium fungi at this critical time of the year 
may be sufficient to reduce reproductive performance in grazing sheep. Smith et al., ( 1990) 
concluded that intakes of 3 mg/ewelday or more during the period around mating would be 
reflected as depressed ovulation rates and lower lambing percentages. Levels of zearalenone 
between 0.4 and 4.0 mglkg dry weight have been reported in some New Zealand pastures (di 
Menna et al., 1 987) which could result in sufficient zearalenone intakes by the ewes to reduce 
reproductive performance. However, these zearalenone levels were determined from pasture 
samples which took no account of the various site types present in pasture during the late 
summer and autumn. High N sites such as urine patch areas, which can represent as much as 
40 % of the pasture, are often associated with the highest numbers of fusaria (Keogh, 1 986) 
and, therefore, could be expected to contain higher levels of zearalenone. Urine patches are 
grazed more frequently and intensively than other sites in the pasture and can, therefore, 
contribute disproportionately to the acquisition of fungal toxins by livestock (Keogh, 1 986). 
The grazing behaviour of the ewe increases the likelihood that sufficient zearalenone will be 
ingested around mating to reduce reproductive perfonnance. 
The aims  of this trial were: 
5 1  
1 .  To measure free and conjugated zearalenone in the blood and urine of ewes grazing grass­
dominant pastures or chicory. 
2. Determine any subsequent effects on reproductive perfonnance. 
2. Methods and materials 
2.1 .  Animals and treatments 
Finish Landrace x Romney 2-tooth ewes were ear tagged and allocated to four groups 
(n= l l O) .  Thirty ewes in each group were treated with synchronisation devices (CIDR, type G, 
containing 0.3g progesterone) inserted into the vagina for 14 days prior to mating and the 
remaining 80 ewes were not synchronised. Two groups were grazed on chicory (Chi corium 
intybus L. cv Grasslands Puna), which is a forage herb with very low zearalenone 
concentrations ( < 0. 1 �g/g), and the remaining two groups were grazed on grass-dominant 
pasture. The groups remained on these treatments for two weeks at which time one group on 
each forage type was interchanged (Fig. 5. 1 .). Entire rams, with harnesses and crayons, were 
introduced to all groups after the first two weeks and checked daily for mating marks. 
Figure 5.1. Grazing treatments for each group of 30 synchronised + 80 non-synchronised 
ewes. 
g r o u p  w e e k  1 & 2 w e e k 3 & 4 
1 
� _________ 
p
_
a
_
s
_
t
_
u 
__ 
r
_
e
----------�----------
p
--
a
_
s
_
t
_
u
_
r
_
e
----------� 
2 p a s t u r e c h i c o ry 
3 c h i c o ry p a s t u re  
4 c h i c o ry c h i c o r y 
R a m i n tro d u c e d  
52 
Seven days after the beginning of mating all synchronised ewes were examined by laparoscopy 
to determine ovulation rate. After all ewes were mated for the first time the crayon colour was 
changed and the ewes were checked twice weekly for returns to service. The number of lambs 
carried per ewe was determined by ultrasound scanning 90 days after the end of mating. 
2.2. Sampling 
All the ewes were weighed pre-treatment, at the beginning of mating and after the first cycle 
with the ram. Blood samples were taken from synchronised ewes by jugular venipuncture with 
evacuated collection tubes ( 1 0  ml draw vaccutainerni ) and hypodermic needle on the days the 
ewes were weighed. Urine samples were taken from synchronised ewes at the beginning of 
mating. 
Samples of chicory and the various pasture species were taken weekly during the trial period 
for zearalenone analysis. 
2.3. Zearalenone determination in herbage, blood, and urine 
Zearalenone concentrations determined by an indirect competitive ELISA immunoassay using 
partially purified zearalenone binding antibodies (di Menna et al., 1 99 1 )  (see appendix ll for 
full details of the assay). 
2.4. Statistical analyses 
All statistical analyses were done using Graph-pad Prism 2.0 software: 10855 Sorrento Valley 
Rd #203, San Diego, CA 9212 1  USA. 
Ovulation rate and pregnancy scanning data for each treatment group were compared using 
Friedman test and Dunn's multiple comparison test Differences in weight change were 
compared by analysis of variance (ANOV A) and Tukey' s multiple comparison test. Free and 
conjugated zearalenone concentration in the blood and urine were compared by t-test Results 
are displayed as mean values ± standard error of the mean (SEM). 
53 
Plate 5. 1 .  Laparoscopic examination of ewes 
54 
Plate 5.2. Ewes grazing grass-dominant pasture 
-llo � -.... 
. ...  ,......_ 
, .  
·� 
55 
Plate 5.3. Ewes grazing chicory 
3. Results 
3.1. Weight change and reproductive performance 
Tables 5. 1 .  and 5.2. shows reproductive data and weight change for synchronised and non­
synchronised ewes. 
Table 5.1.  Weight change (over 44 day trial period),  ovulation rate, returns to service and 
number of lambs carried in synchronised ewes in each treatment group. 
Weight Change 
(kg) 
Ovulation rate 
(corpora 
lutea/ewe) 
Returns to 
service (%) 
Pregnancy 
scanning W 
lambs carried) 
1 
1 .25 ± 0.34" 
2.40 ± 0. 14  
1 7  
1 .43 ± 0. 1 5  
Treatment 
groups 
2 
1 . 1 5  ± 0.37" 
2 .3 1 ± 0. 1 5 
30 
1 .63 ± 0. 12 
3 4 
-0.35 ± 0.49b -0.87 ± 0.34b 
2.23 ± 0. 1 2.29 ± 0. 14  
30 40 
1 .63 ± 0. 1 0  1 . 87 ± 0. 1 0  
Means (+ SEM)in each row with different superscript letters differ significantly (P<O.Ol)  
Table 5.2. Weight change (over 44 day trial period), returns to service, and number of lambs 
carried in non-synchronised ewes in each treatment group. 
Weight Change 
(kg) 
Returns to 
service (%) 
Pregnancy 
scanning W 
lambs carried) 
Treatment 
groups 
1 2 3 4 
7.06 ± 0.21 8  5.80 ± 0.27b 5 .48 ± 0.23b 5.69 ± 0.3 lb 
7 6 1 0  13  
1 .7 1  ± 0.08 2.22 ± 0.28 1 .76 ± 0.08 1 .91  ± 0.07 
Means (+ SEM)in each row with different superscript letters differ significantly (P<O. O l )  
56 
57 
3.2. Zearalenone levels in the herbage 
Forage samples taken from the pasture including the various site types and species. 
Zearalenone concentrations per dry weight of herbage, were 3.01 ± 2.50 jlg/g in grass pasture 
and 0.25 ± 0.08 jlg/g. in the chicory. 
3.3. Zearalenone in the blood 
The levels of conjugated and free zearalenone in the blood of the ewes pre-treatment were 
0. 1 63 ± 0.035 ng/ml and 0.219  ± 0.034 ng/ml respectively. 
There was no significant difference in the levels of conjugated zearalenone after two weeks 
between the ewes grazed on pasture (0. 170 ± 0.023 ng/ml) and those grazed on chicory (0.238 
± 0.030 ng/ml). The level of free zearalenone was significantly greater in the blood of ewes 
grazing pasture (0.295 ± 0.027 ng/ml) than in the ewes grazing chicory (0. 1 22 ± 0.0 14 ng/ml). 
Free zearalenone represented 67% of the total zearalenone present in the ewes grazed on 
pasture and 40% of the total zearalenone present in the ewes grazed on chicory. 
There was no significant difference in the levels of conjugated zearalenone in the blood after 
six weeks of grazing between any of the treatments, however the levels of conjugated 
zearalenone in the blood had increased significantly (P<0.05) in all the grazing treatments since 
the pre-treatment sampling and two week sampling, and were often up to ten times the 
concentration (see Figure 5.2.). 
There was no significant difference in the levels of free zearalenone after six weeks in the blood 
of ewes on the pasture or chicory treatments, although levels of free zearalenone were 
significantly lower (P<0.05) than at the pre treatment and week 2 samplings. Free zearalenone 
represented between 2% and 3% of the total zearalenone present (see Figure 5.3.). 
58 
Figure 5.2. Conjugated zearalenone in the blood of ewes grazed either on chicory or pasture. 
3 
-
E 2 a, 
c 
. 
(.) 
c 
0 
(.) 1 
pasture c h i co ry 
T reat me n t  
t 1 pre-treatment 
�h4c<t.H'iff�jJ 1 0/3/95 
lllililillll 1 2/4/9 5 
Figure 5.3. Free zearalenone in the blood of ewes grazing either on chicory or pasture. 
0 . 4  
e o . 3  
a, 
c: 
0 0 . 2 
c: 
0 
0 
0 . 1  
o . o...I.-...J=� pasture ch icory 
Treat ment 
l '  , , , pre-treatment 
li!i1l 1 0/3/95 
llllll!ll 1 2/4/95 
59 
3.4. Zearalenone in the urine 
There was no significant difference in the levels of conjugated zearalenone in the urine between 
ewes grazing chicory (0.099 ± 0.027 ng/ml) or pasture (0.086 ± 0.024 ng/ml). There was a 
significantly higher concentration of free zearalenone in the urine of ewes grazed on pasture 
(0. 1 34 ± 0.0 1 5  ng/ml) than in ewes grazed on chicory (0.0 19  ± 0.004 ng/ml). (See Figure 
5.4.) 
The proportion of the total zearalenone in the urine present in free form was 6 1%  for the 
pasture group and 1 6.7% for the chicory group. 
Figure 5.4. Free and conjugated zearalenone in the urine of ewes either grazing pasture or 
chicory for two weeks prior to mating. 
E 
-e, 
c: 
u 
c: 
0 
u 
0.4 
0 
0 
0. 0-L---L:.;==....__....t.===""--
Pasture Chicory Pasture Chicory 
Treatment 
1 I Conjugated 
''"""'"'"'' Free 
60 
4. Discussion 
4.1. Animal performance 
Results obtained in this trial showed no significant difference in  ovulation rate, conception rate 
or the number of lambs carried per ewe between any of the grazing treatments. However, 
several factors may have contributed to a lack of difference being observed. The live-weight 
data showed that ewes grazing the chicory prior to mating lost weight on average whereas 
those grazing the pasture gained weight. It is possible that the loss of weight in the groups 
grazing the chicory treatment prior to mating would have reduced ovulation rates. Therefore, 
any positive effects on ovulation rate which may have resulted from grazing the lower 
zearalenone feed would have been diminished by the poorer feed availability in the chicory 
groups. The liveweight data showed that, although the synchronised and non-synchronised 
ewes in each treatment group were run together, the synchronised ewes were on average 4-5 
kg lighter than the non-synchronised ewes despite having similar average liveweights at the 
start of the trial. The reasons for the l ighter live-weights of the synchronised ewes are not clear 
although, possibilities include the synchronisation treatment and/or the laparoscopy procedure 
and generally the animals were handled more often than the non-synchronised ewes. 
4.2. Zearalenone in the herbage 
Levels of zearalenone in the herbage were highly variable depending on the species sampled 
and from where the sample was taken. Pasture samples taken include species such as ryegrass, 
brown top, and Yorkshire fog. Levels were generally lower than recorded in previous years 
(Keogh, pers. comm.). Despite the lower levels of zearalenone recorded this year, the levels of 
zearalenone in the chicory were still well below those in the grass species. 
The relatively low levels of zearalenone recorded in the pasture during this trial could be 
another reason why no difference was observed in reproductive performance between ewes 
grazing pasture and those grazing chicory. Zearalenone intakes above 3 mg/ewelday are 
61 
sufficient to depress ovulation rate and lower lambing percentages (Smith et al., 1990). The 
average levels of zearalenone in the pasture are often below 3 mglkg of dry plant tissue which 
would mean that ewes harvesting an average of 1 kg dry matter per ewe per day would be 
ingesting levels of zearalenone below 3 mg/day. The low pasture zearalenone levels were 
reflected in the urine and blood samples taken pre-treannent and on the l Oth March which 
were more critical times when considering likely effects on reproductive performance. 
4.3. Zearalenone in the blood 
Analysis of the blood for free and conjugated zearalenone showed that there were clear 
differences between ewes grazed on chicory and those grazed on pasture. After introduction to 
the grazing treatments the levels of conjugated zearalenone remained relatively similar and 
after two weeks of grazing there was no significant difference in the levels of conjugated 
zearalenone between the groups grazed on pasture or on chicory. A possible reason for the 
lack of change in the conjugated zearalenone levels is that much of the conjugated zearalenone 
may be recycled by the liver via the bile and therefore remains in the body much longer (Smith, 
J.F., pers. comm.) .  The possible recycling of conjugated zearalenone has made the 
interpretation of blood results difficult particularly when results have been in terms of total 
zearalenone and have not discerned between the conjugated portion and the oestrogenically 
active free zearalenone. The blood analysis showed clear differences in the levels of free 
zearalenone by the second week of grazing with levels in the pasture group increasing 
significantly (P<0.05) whereas the free zearalenone levels in the ewes grazing chicory had 
decreased. The differences in free zearalenone levels in the blood and the lack of significant 
change in the levels of conjugated zearalenone in both the chicory and pasture groups resulted 
in a higher proportion of the total zearalenone present in the group grazed on pasture existing 
in the free 'active' form, compared to the group grazed on chicory. 
The fmal set of blood samples taken four weeks after the start of mating showed up to ten-fold 
increases in the amount of conjugated zearalenone present since the two week and pre­
treatment sampling and a reduction in the amount of free zearalenone present in all treatment 
groups. This change in the conjugated and free zearalenone levels cannot be easily explained as 
the levels of zearalenone determined in the blood pre-treatment and at the start of mating were 
normally associated with background levels of zearalenone which are much lower than levels 
associated with increasing zearalenone levels in the pasture. 
4.4 Zearalenone in the urine 
62 
The urine samples taken at two weeks showed a similar pattern to the blood samples. As with 
the blood levels, there was no significant difference in the levels of conjugated zearalenone 
between the groups grazed on pasture and those on chicory. The difference between the 
chicory and pasture treatments is clearly expressed in the levels of free zearalenone present in 
the u rine and the proportion of the total zearalenone present in free form. The difference in the 
levels of  free zearalenone in the urine between the chicory and pasture groups is greater than in 
the blood which indicates that a larger proportion of the free zearalenone present in the blood 
e>f the chicory group is in the conjugated form by the time it has entered the urine. The high 
proportion of free zearalenone in the urine of the pasture group could result from an inability 
of the ewe to conjugate the greater amounts of zearalenone in the blood or could indicate a 
large amount of conjugated zearalenone being recycled and remaining in the body. 
�.5. Conclusions 
The lack of a significant difference in reproductive performance between ewes grazing chicory 
md those grazing pasture may be due to lower feed availability in the chicory treatment and 
:he comparatively low levels of zearalenone in the pasture during the trial period. 
learalenone levels in the blood were difficult to interpret because of recycling which occurs in 
:he body, however levels of free zearalenone were significantly lower in the ewes grazing 
:::hicory at the start of mating. Urine analysis gave a better indication of zearalenone intake 
:::ompared to blood as the urine is the major route of excretion of zearalenone from the body. 
:hicory was effective in reducing zearalenone intake and it is likely that this would be the case 
n years where conditions for zearalenone production are better. More work, especially in years 
Nhen higher zearalenone levels prevail, will be necessary to establish if an important difference 
!xists. 
63 
CHAPTER VI 
General discussion and conclusions 
The main objective of this study was to characterise zearalenone distribution in pasture and its 
possible role in reducing reproductive performance in sheep grazed on pasture. The 
distribution of zearalenone in different pasture sites was examined and a preliminary 
investigation into zearalenone uptake by the ryegrass plant was conducted. The distribution of 
zearalenone in pasture is a major piece of information in understanding the relationship 
between zearalenone and the grazing animal. The dosing trial was conducted to add evidence 
to previous studies which found that zearalenone causes reductions in reproductive 
performance in sheep. The grazing trial was aimed at linking zearalenone produced in pasture 
to reductions in reproductive performance in the grazing animal. 
The presence of zearalenone-producing Fusarium species in New Zealand pastures has been 
demonstrated (di Menna et al. , 1987) and under certain favourable conditions zearalenone is 
produced in appreciable amounts. The distribution of zearalenone within the pasture is of great 
importance in determining the amount ingested by the grazing animal. Certain areas in the 
pasture, such as high N areas caused by animal excreta, have significantly higher populations of 
fungal saprophytes, which include fusaria (Keogh, 1 986). It follows that zearalenone levels will 
generally be greatest in these areas. This characteristic of zearalenone distribution is of greatest 
�onsequence to the grazing animal as pastures are not uniformly grazed and defoliated by 
livestock during the summer and autumn. Herbage in urine patch sites, which can cover up to 
40% of the pasture, is grazed in preference to other areas of the pasture (Keogh, 1984; Keogh, 
1986). Acquisition of fungal toxins is, therefore, dependant on suitable conditions for fungal 
�rowth and toxin production, and grazing behaviour of the livestock. 
Ibe results (Chapter 3) showed that zearalenone concentrations varied greatly within sites and 
:>etween site types indicating possible differences in conditions suitable for zearalenone 
>reduction. However, the samples were taken in late autumn when zearalenone levels would 
tave been falling and in addition the zearalenone levels in pasture during 1995 were lower on 
tverage than in previous years. Regular sampling during the summer and autumn from the 
tifferent pastures and site types is required to better characterise zearalenone distribution in the 
>asture. The analysis of the components from the pasture samples and in the uptake trial 
showed high concentrations in the youngest parts of the plant and in particular the daughter 
tillers. It appeared that zearalenone in solution was taken up by the plant and translocated to 
the youngest parts of the plant (Fig 3.5. ) with nutrients required for growth. 
64 
Further investigations into the uptake of zearalenone using improvements to the method 
developed in this investigation, will enable characterisation of zearalenone uptake, not only in 
the ryegrass plant. but in other common pasture species. This will give further insight into the 
relationship between zearalenone in the pasture plant and the grazing animal. 
In the animal the importance of levels of free and conjugated zearalenone were difficult to 
interpret (Chapter 4, section 4.2.) because in many cases there appeared to be no correlation 
between the amount of zearalenone ingested or dosed and the levels in the blood. Dosed 
zearalenone and its metabolites have been detected in the bile (Kennedy et al., 1 995; Hewitt et 
al. , 1 996) which offers a route for excretion and subsequent reabsorption of zearalenone into 
the blood. In addition to this recycling, it is likely that zearalenone is constantly being 
conjugated and deconjugated in the liver and metabolised to other related compounds 
(Erasmuson et al., 1 994). All these factors affect levels of free and conjugated zearalenone in 
the blood and are responsible for much of the variability. Urinary levels of free and conjugated 
zearalenone could be correlated with zearalenone intake (Chapter 4 section 4.3.) and were the 
best indication of zearalenone levels in the animal. Urine is the major excretory pathway for 
zearalenone and once in the urine zearalenone cannot re-enter the blood stream which is the 
reason why urinary zearalenone levels are a good indication of zearalenone present in the 
animal. Levels of free zearalenone were of most interest as it is this portion of the zearalenone 
present in the animal which has the oestrogenic effects. 
Although zearalenone was the major compound considered in this investigation, other related 
compounds with significant effects on the grazing animal, were also examined. The metabolism 
of zearalenone to several related compounds by fusaria in the pasture (di Menna et al. , 1987), 
by micro organisms in the rumen (El-Sharkawy and Abdul-Hajj, 1988) and once in circulation 
in the animal (Miles et al., 1 996) has been shown and therefore the effects of these compounds 
cannot be ignored. Aside from oestrogenic properties common to many of the zearalenone 
related metabolites, some metabolites, namely zeranol, have been shown to have anabolic 
properties and have been used widely as a means of improving animal growth rates. Now 
banned as an anabolic drug for fann animals in European countries, the issue of zeranol 
66 
In conclusion, the results obtained in the three trials were unable to show a direct link between 
zearalenone in pasture and reproductive performance in ewes, however, there were clear 
indications that these links may exist and given appropriate conditions for zearalenone 
production in pasture, it is likely that the zearalenone concentration in the plant and therefore 
the amount of zearalenone ingested by the animal will be sufficient to affect reproductive 
performance. It was also found that several other zearalenone-related metabolites were present 
in sheep grazed on pasture which were produced both within the animal by metabolism of 
zearalenone and within the pasture to be subsequently ingested by the animal. Further studies 
into the effects of zearalenone on ewe reproduction will also have to consider more closely the 
other compounds present which may also have oestrogenic effects. 
Finally, this study was successful in evaluating the forage herb chicory as a feed which 
significantly reduces the risk of free zearalenone in ewes when fed during mating. 
There is a need for further research to identify and clarify links between pasture zearalenone 
and the reproductive performance of New Zealand ewe flocks. 
Appendix 1 
GC-MS analysis of zearalenone carried out by Dr Anton Erasmuson at National Chemical 
Residues Analytical Laboratory, MAF, Wallaceville Animal Research Centre, Upper Hutt, 
New Zealand. 
67 
Sample treatment . Blood and urine samples were stored frozen. Aliquots (5ml) were analysed 
by glucuronidase re-formation of the alcohols, extraction with hexane/tBME (70:30), and then 
HPLC cleanup of extracts with programmed fraction collection. A fraction was evapoprated 
under nitrogen, then trimethylsilyl derivatized with MSTFA, and subjected to GC-MS analysis. 
Chromatography. The normal phase HPLC separation used cyanopropyl Whatman PAC 
column (4.6 mm x 100 mm) in a Varian Vista 5500 HPLC, isocratically pumping 
hexane/tBMEJmethanollacetic acid (695:250:50:5) at 0.9 mUmin. An azo dye 
(phenylazohomovanillyl alcohol) was used to monitor for drift in retention times. Samples were 
automatically injected by a Shimadzu SCL-6B and fractions collected by a Pharmacia FR.AC-
300. 
Residue quantitation used a Hewlett-Packard 5890 gas chromatogragh interfaced to a 
low- resolution mass selective detector (MSD 9570). 
• 
Appendix 3 
Nutrient stock solution used for zearalenone uptake trial (Chapter 3.). 
Stock solutions used to make nutrient solution. 
Stock A CaN03 ( 1 59.3 g/1), NI4N03 (80. 1g!l), FeCh ( 10.82g!l) and DTPA ( 15.74g!l); 
Stock B KH2P04 (3. 1 2g!l) and K2HP04 ( 1 .33g!l) ;  
Stock C KN03 (7.99g!l), MgS04.?H20 (3.36g!l) and Na2S04. 1 0H20 ( 10.07gll); 
Stock D CuS04.SH20 (O.O l Og/1), hydrated HnCl (0.409gll), H3B03 (0.350gll), 
Na2Mo04.2H20 (0.002gll) and ZnS04.?H20 (0.055g!l) 
69 
The relative concentrations of the stock solutions in the nutrient solution were 1 ml/1, 4 ml/1, 8 
mlll and 1 ml/1 for stock solutions A, B , C, and D respectively. 
70 
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