effects of ergot on health and performance of ruminants ... · lysergic acid lysergic acid amide...
TRANSCRIPT
Aus dem Physiologischen Institut
der Tierärztlichen Hochschule Hannover
und dem Institut für Tierernährung
der Bundesforschungsanstalt für Landwirtschaft in Braunschweig
Effects of ergot on health and performance of ruminants and carry over of
the ergot alkaloids into edible tissue
INAUGURAL-DISSERTATION
Zur Erlangung des Grades einer
DOKTORIN DER VETERINÄRMEDIZIN
(Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von
Barbara Schumann
aus Minden
Hannover 2007
Wissenschaftliche Betreunung: Prof. Dr. Gerhard Breves (TiHo) PD Dr. habil. Sven Dänicke (FAL) Prof. Dr. Gerhard Flachowsky (FAL) 1. Gutachter: Prof. Dr. med. vet. Gerhard Breves 2. Gutachter: Prof. Dr. med. vet. Jürgen Rehage Tag der mündlichen Prüfung: 24.05.2007
__________________________________________________________________________
Contents Page Introduction 1 Scope of the thesis 13 Paper I
Effects of different levels of ergot in concentrates 15 on the growing and slaughtering performance of bulls and on carry-over into edible tissue
Archives of Animal Nutrition, submitted Paper II
Effects of different levels of ergot in concentrate on 37 the health and performance of male calves Mycotoxin Research, in press
Paper III
Effects of the level of feed intake and ergot 59 contaminated concentrate on ruminal fermentation, ergot alkaloid metabolism and carry over into milk, and on physiological parameters in cows Food Additives and Contaminants, submitted
General discussion 95 Conclusions 108 Summary 110 Zusammenfassung 113 References (cited in the introduction and general discussion) 116
Abbreviations
AP alkaline phosphatase
AST aspartate aminotransferase
BW body weight
bzw beziehungsweise
ca circa
CK creatine kinase
d day
DM dry matter
DMI dry matter intake
EFSA European food safety authority
et al. et alii
GLDH glutamate dehydrogenase
γ-GT γ-Glutamyltransferase
i.v. intravenous
l Liter
LG Lebendmasse
LMZ Lebendmassezunahme
LW live weight
LWG live weight gain
N nitrogen
NDF neutral detergence fibre
NH3-N Ammoniak-Stickstoff
NOEL no-effect level
OMI organic matter intake
TSA Trockensubstanzaufnahme
U units
UK United Kingdom
US United States
USA United States of America
Introduction
Introduction
The surviving forms of several parasitic fungi of the genus Claviceps are referred to as ergot
(Figure 1). Claviceps purpurea is the most important in terms of frequency of occurrence
(EFSA, 2005).
These banana-shaped solidified mycelia may differ in colour
from purple to black (Lutrell, 1980). Also their weight and bulk
may vary in wide ranges. The size is mainly host dependent,
and may reach a length of up to 4 cm (Jungehülsing, 1995).
After rainy springtime seasons, they form perithecia and these
again produce ascospores (Engelke, 2002). The ascospores are
carried by the wind on blossoms of grasses and grains which is
called the primary infection. The initially pale fungus tissue
develops at the site of grain development instead of the host
grains and exudes the so-called honey dew which is a sweet and
sticky fluid. It contains infectious conidia and serves to attract
insects, which in turn carry the adhering conidia to the next ear
in an ambit up to 1000 m (Wolff, 1998). It may also be spread
from plant to plant or through the washing action of rain. This is
called the secondary infection. The mycelium develops to the
typical sclerotium by becoming harder and bigger whereas the
outside mostly changes its colour to dark.
Figure 1: Ergotsklerotia on a grainear (Bös, 2000)
Figure 1: Ergotsklerotia on a grainear (Bös, 2000)
Invasion is possible in more than 400 gramineae (Klug, 1986; Teuteberg, 1987).
Theoretically, rye, wheat and triticale are equally susceptible to ergot, but especially rye is
more frequently affected. This is due to the fact that rye is a cross-fertilising grain with
differing flower characteristics (Engelke, 2002), and especially during wet summers, the
flower remains open for a long time, which facilitates the invasion by the fungus.
Additionally, an increased cultivation of hybrid rye within the past years, which shows a
worse polling pouring ability than normal rye, resulted in an increasing ergot incidence
(Mielke and Betz, 1995). Moreover, grasses growing at the site of grain fields may also be
infected with Claviceptaceae and boost the infection pressure for the grain. The trend of
ploughless tillage and mulching and of leaving grasses fade also aids an increasing incidence
of ergot (Teuteberg, 1987; Jungehülsing, 1995).
1
Introduction
Furthermore, rye has been enjoying increasing popularity as a feedstuff since the abolition of
the rye intervention in 2002 (Pottgütter and Schaar, 2000).
Occurrence of ergot
From 1995 till 2004 a mean ergot incidence of 0.11 weight percent was found in German rye
samples (Bundesministerium für Verbraucherschutz, 2005). Wolff (1998) tested 606 rye
samples in the years 1992 – 1995 and found a fraction containing more than 0.005 % ergot
which increased from 4% in 1992 up to 32% in 1994. In 1995 it reached 29%. Coenen et al.
(1995) tested 106 unsorted grain samples in 1993 and 30% of them were macroscopically
ergot-contaminated, of which, in turn, 25% contained more than 1000 mg visible ergot
particles/kg. 68% of these contaminated samples were rye samples. In 2004 Lindhauer et al.
(2004) found a mean ergot weight percentage of 0.1 in German rye samples, which was less
than the year before, when it amounted to 0.17%. The EFSA (2005) recently reviewed the
literature and reported a range in European grain contamination with ergot between 0.1 and
0.3%.
Up to now in Germany for farm animals only a weight based upper limit of 1000 mg ergot per
kg unground cereal grains applies (Council Directive 2002/32/EC of 7 May 2002). The UK
standards for ergot are 0.001% ergot for feed grain, and a zero tolerance for all other grains.
For Australia a limit of 0.05% ergot in food cereal grains applies (EFSA, 2005). In the US and
Canada ergoty rye or ergoty wheat with a content of 300 mg ergot/kg or more may become
discharged or extended with non-contaminated batches (Weipert, 1996; cited by (Engelke,
2002)).
Ergot alkaloids
Figure 2: Ergoline ringFigure 2: Ergoline ring
The toxicity of ergot is mainly due to its alkaloid content (Mühle
and Breuel, 1977; Klug, 1986; Gloxhuber et al., 1994; Keller,
1999). More than 40 different alkaloids have been isolated from
Claviceptaceae so far.
These indole alkaloids are synthesized from the amino acid L-
tryptophan (Lorenz, 1979; Klug, 1986; Buchta and Cvak, 1999)
with the tetracyclic ergoline ring system (Figure 2) as their basic
structure (Keller, 1999). The alkaloid synthesis is dependent on
the nitrogen supply since waste amino acids are used for
alkaloid production (Karlson, 1977).
2
Introduction
Alk
aloi
dEm
piric
al
form
ula
Rab
bit (
mg/
kg i.
v.)
MPh
enyl
alan
ine
Ergo
tam
ine
C33
H35
N5O
53
α-hy
drox
y-al
anin
Leuc
ine
Ergo
sine
C30
H37
N5O
5
Val
ine
Ergo
valin
eC
29H
35N
5O5
Phen
ylal
anin
eEr
gocr
istin
eC
35H
39N
5O5
1.9
Prol
inα-
hydr
oxy-
valin
Leuc
ine
Ergo
cryp
tine
CH
NO
0.8
Com
pone
nts o
f the
pep
tide
LD50
(Grif
fith
Tab
le 1
: “Pe
riodi
cal s
yste
m”
of th
e er
got a
lkal
oids
acc
ordi
ng to
Hof
fman
n (1
964)
mod
ified
by
Müh
le a
nd B
reue
l (19
7
Gro
upFu
ngal
sour
ce (C
ole
and
Schw
eike
rt, 2
003)
ouse
(mg/
kg o
ral)
3200
CP
Ergo
tam
ine-
CP
CP,
ET,
AC
CP
3241
55
Ergo
toxi
ne-
CP
Val
ine
Ergo
corn
ine
C31
H39
N5O
50.
920
00C
PPh
enyl
alan
ine
Ergo
stin
eC
34H
37N
5O5
CP
α-hy
drox
y-α-
amin
o-bu
tyric
aci
dLe
ucin
eEr
gopt
ine
C31
H39
N5O
5Er
goxi
ne-
CP
Val
ine
Ergo
nine
C30
H37
N5O
5C
PEr
gom
etrin
eC
19H
23N
3O2
3.2
460
CP,
BE,
BH
, BC
et a
l., 1
978)
7) a
nd e
xten
ded
by e
rgom
etrin
e
CP
= C
lavi
ceps
pur
pure
a, E
T =
Epic
hloe
typh
ina
, AC
= A
crem
oniu
m c
oeno
phia
lum
, BE
= Ba
lans
ia e
pich
loe,
BH
= B
alan
sia
henn
ings
iana
, BC
= B
alan
sia
clav
icep
s
3
Introduction
Natural ergot alkaloids can be divided into three groups according to their structure: Alkaloids
of the clavine type, simple amides of lysergic and paspalic acids, and alkaloids of the peptidic
type (partly shown on Figure 3). The latter group (Table 1) consists of ergotamines
(ergotamine, ergosine, ergosecaline, etc.), ergotoxines (ergocryptine, ergocornine,
ergocristine, etc.) and ergoxines (for example ergostine). Additionally the relatively new
group of ergopeptams belongs to the peptidic alkaloids (Buchta and Cvak, 1999). Some other
authors prefer to combine the peptidic alkaloids and the amides (ergine, ergometrine, etc.)
under the term of lysergic acid derivates (Rutschmann and Stadler, 1978; Mainka, 2006) since
biosynthetically the peptidic alkaloids can be understood as tetrapeptides containing lysergic
acid as the first member of the peptidic chain (Buchta and Cvak, 1999).
Ergosine
Ergotamine
Ergostine
Ergocornine
α-Ergocryptine
β-Ergocryptine
Ergocristine
Lysergic acid
Lysergic acid amide
Lysergic acid diethylamide (LSD)
Ergometrine
Alkaloids of the peptidic type
Simple amides of lysergic acid
Ergosine
Ergotamine
Ergostine
Ergocornine
α-Ergocryptine
β-Ergocryptine
Ergocristine
Lysergic acid
Lysergic acid amide
Lysergic acid diethylamide (LSD)
Ergometrine
Alkaloids of the peptidic type
Simple amides of lysergic acid
Figure 3: Structure of simple lysergic acid amides and peptide alkaloids (Bös, 2000)
The alkaloid concentration in ergot from Germany was reported to vary between 863 and
1620 mg/kg ergot DM sampled from the harvest 2004 (Mainka, 2006), in 2003 from 42 to
343 mg/kg (Mainka, 2006) and in previous years between 900 and 2100 mg/kg (Wolff, 1989).
Ergot alkaloids from Canadian rye ranged between 100 and 4500 mg/kg (Young, 1981).
Further components of ergot
Ergot mostly consists of fat (30-35%), crude fibre (about 30%) and crude protein (18-26%)
whereas starch and other carbohydrates are just found in very small concentrations (Coenen et
al., 1995; Mainka, 2006).
4
Introduction
Also mentionable as toxic components in ergot are the pigments, the yellow ergochromes and
the red derivates of anthrachinon, which are found in concentrations between 1 and 2 %
(Wolff and Richter, 1989; Wolff, 1992). The ergochromes are synthesized from acetic acid
and known for their antimicrobial effects (Guggisberg, 1954). For example secalonic acid A
and B and ergoflavin belong to the group of ergochromes. Frank (1984) considered secalonic
acid as even more toxic than ergotamine, but in contrast to the ergot alkaloids, secalonic acid
loses its activity already in a short period of storage (Wolff, 1992). Bourke et al. (2000) and
Ilha et al. (2003) deduced analogies between the pigments of ergot and the slowly
metabolized hypericine pigments which cause lethal hyperthermia in sheep. According to
these authors, these photodynamic compounds of ergot should be considered as a possible
contributory factor, at least for understanding the etiology of hyperthermia
Another slightly toxic ingredient is the ricinoleic acid, which is a specific fatty acid of the
ergot fat and which was suggested to irritate the intestine (Forth et al., 1992).
Thirty six percent of the fat of ergot is ricinoleic acid. Other main components of the ergot fat
are linoleic acid (11-15%), oleic acid (about 20%) and palmitic acid (23-28%) (Buchta and
Cvak, 1999).
Ricinoleic acid is released from the matrix mainly in the small intestine. In humans, an
amount of 10-30 g ricinoleic acid (about 290 mg/kg BW) resulted in an accumulation of water
by inhibiting the absorption of sodium and water from the intestine. This, in turn, leads to an
increasing influx of electrolytes and water to the lumen, and therefore to an increasing amount
of a softer stool, and may be followed by diarrhoea (Forth et al., 1992; Sogni et al., 1992).
Pharmacological attributes and toxic effects of ergot
Despite an absorption of about 66% after oral application, ergotamine undergoes “first-pass”
metabolism by the liver and only shows an oral bioavailability of less then 2% (Aellig and
Nuesch, 1977; Ibraheem et al., 1983; Tfelt-Hansen et al., 1995). Interestingly, the lysergic
acid amides and nicergoline had at least 20 times higher plasma levels at similar oral doses as
the ergopeptine alkaloids. This suggests that these alkaloids have a higher bioavailability and
thus a higher toxic potential than the ergopeptine alkaloids (Hill, 2005). Eckert et al. (1978)
also described the lysergic acid and its derivates to have a higher bioavailability than the
ergopeptides and to be less bioavailable than nicergoline and methergoline.
The pharmacological effects of ergot are manifold. Apart from nausea and/or vomiting,
probably due to a direct effect of the alkaloids on dopamine receptors in the area postrema of
5
Introduction
the brain, the ergobasine and ergotamine groups have a direct stimulatory action on smooth
muscle, and the polypeptide alkaloids have an inhibitory action on sympathetic functions of
the autonomic nervous system (Culvenor and Phil, 1974). Going more into details, they serve
as agonists, partial agonists or antagonists at receptors for serotonine, dopamine and
adrenaline as recently reviewed by Pertz and Eich (1999). Thus they induce, for example,
sympathoinhibition, leading to bradycardia and stimulation of smooth muscles, or
vasoconstriction which may be followed by gangrenes in the extremities. All alkaloids seem
to have some potential for vasoconstriction, but ergotamine is the most potent (Müller-
Schweinitzer, 1982; Tfelt-Hansen et al., 1995). The uterotonic effect of ergot alkaloids is a
result of the smooth muscle stimulation and might be followed by abortions or other
difficulties during pregnancy (Saameli, 1978).
Furthermore some of the alkaloids act as dopamine agonists and inhibit the prolactine release
of the pituitary gland (Forth et al., 1992), resulting in decreased milk production (von
Engelhardt and Breves, 2000). The immunological system is influenced by an increasing
cortisol concentration which has anti-inflammatory effects (Filipov et al., 2000). Browning et
al. (1997) showed that also the growth hormone is affected by ergot alkaloids. Its plasma
concentrations were transiently elevated by ergotamine and ergonovine injections.
Some ergot alkaloids act as biogenic amine agonists and affect neurotransmission.
Observed effects are convulsions and other central nervous symptoms mainly induced by the
lipophilic alkaloids which may enter the brain (Gloxhuber et al., 1994).
An elevation of the body temperature is observed in animals exposed to ergot and might be
due to the peripheral vasoconstriction which hampers evaporation and dissipation of
excessive body heat through the skin. But it might also be a result of the increased production
of heat by the energy-wasteful mixed-function oxidase system which has an important role as
an ergot alkaloid-detoxification system (Zanzalari et al., 1989).
Excretion of nicergoline and methergoline is mainly via the urine, whereas the ergopeptides
are excreted mainly with the bile (Eckert et al., 1978).
Carry over
Corresponding to the recent available data on the toxicokinetics of ergot alkaloids in target
animal species, the information regarding a potential carry over into edible tissues is scarce
(EFSA, 2005). Kalberer et al. (1970) administered 1 mg [3H] ergotamine/kg BW to rats and
observed an accumulation of this alkaloid after 2 hours in a decreasing order from the liver,
kidneys, lung, heart and brain to the blood. Similar results were described by Acramone et al.
6
Introduction
(1972) who administered rats with 20 mg [³H] nicergolin/kg BW and found highest amounts
of this alkaloid 30 min later in a decreasing order from liver, lung and kidneys to the heart,
blood, fat and brain. Until 12 hours after oral administration the analyzed residues were
rapidly decreasing.
Young and Marquardt (1982) fed poultry chicken with various concentrations of ergotamine
tartrate. But residual amounts of ergotamine in muscle (5 µg/kg) and liver (4 µg/kg) could
only be detected at the highest concentrations of 810 mg/kg feed. Possible presence of
metabolites has not been analysed.
Carry over research in pigs was conducted by Whittemore et al. (1976 and 1977) who did not
detect any residues after natural exposure to ergot alkaloids. Furthermore, Mainka et al.
(2006) did not find any carry over into edible tissue of growing-finishing pigs fed with
concentrates of 1, respectively 10 g ergot/kg diet.
The literature concerning carry over in cattle and dairy cows is scarce and in the few
published studies mostly only milk residues were analysed. Wolff et al. administered 3µg
ergot alkaloids/kg BW of the animals (as natural grown ergot), over a period of two weeks to
two dairy cows, but no residues could be detected in the milk (Wolff and Richter, 1995). In
another study, where very high and practically not relevant amounts of 125 mg ergot/kg BW
were fed to dairy cows, a milk contamination with alkaloids was found (Parkheava, 1979;
cited by (Wolff et al., 1995)).
Some authors analysed potential carry over of endophyte alkaloids into meat (Cunningham et
al., 1944; Realini et al., 2006), but unfortunately the only time that alkaloids have been
detected in beef tissue, exact data on alkaloid intake was not available (Realini et al., 2006).
Thus, although Gareis and Wolff (2000) considered a carry over of ergot alkaloids into edible
tissues as negligible, further research seems to be necessary. Additionally, kinetics,
metabolism and tissue deposition might depend on a variety of factors which were not
considered so far. Tfelt-Hansen et al. (1995) described a sudden occurrence of ergotism after
a long-term chronic exposure to ergot alkaloids after years. Hence, a temporary storage in
body tissues can not be excluded (Mainka et al., 2003).
Further hosts for Claviceps purpurea
Claviceptaceae may, besides rye and other cereals, also infect various species of wild grasses
which are often found around grain fields. Engelke (2002) analysed samples from the edge
and from the middle of 90 grain fields and compared them by their alkaloid contamination.
During the years 1987/88 the alkaloid contamination of the edge samples was twice as high
7
Introduction
and in 1998-2000 even fourfold higher than that of the middle samples. Rothacker et al.
(1988) published similar results in 1988. This might be explained by the influence of
surrounding biotopes around grain fields, which are of special interest for care of nature and
biodiversity (Kühne et al., 2000). These areas are preferred by flying insects which serve as
carriers for infectious honey dew (Guggisberg, 1954). But also they contribute to an
agglomeration of the inoculum, as not all of the hedges and grass districts may be cared for
accurately. Thus, in the next year primary infections may be caused (Engelke, 2002).
It does mainly depend on flowering time of the grasses and the strain of Claviceps purpurea,
if infections of the rye are associated with wild grasses. After Obst (1993) Lolium perenne
and Dactylus glomerata are the most important species to infect rye with Claviceptaceae.
Whereas Poa pratensis and rye do not belong to the same host circle and thus may not infect
each other (Mühle, 1971).
Endophyte alkaloids
Several important genera of pasture grasses including Festuca and Lolium may not only be
infected by Claviceptaceae but also by endophytic fungi of the genus Neotyphodium.
These fungi are found on 47 species of grasses in 12 genera (White et al., 1993).
They share a common phylogeny with Claviceptaceae (Figure 4) and even produce alkaloids,
but in different combinations.
Figure 4: Phylogeny of Claviceps purpurea and Acremonium coenophialium
(Glenn et al., 1996)
8
Introduction
The predominant ergot alkaloids produced by Neotyphodium are said to be ergovaline and
ergovalinine, followed by ergine, erginine and lesser amounts of ergosine (TePaske et al.,
1993; Porter, 1995). Additionally, the presence of indole terpenoides, such as lolitrem B, and
clavine alkaloids like chanoclavine, agroclavine, elymoclavine and penniclavine in infected
fescue is established. But the clavine alkaloids are obviously less toxic to mammalian species
(Porter, 1995). Another potentially toxic group of alkaloids detected in infected fescue are the
pyrrolizidine alkaloids as senecionine and lycopsamine (Westendorf et al., 1993).
Generally the main symptoms shown by cattle consuming endophyte infected feed are
reduced weight gain, lower conception rates, reduced milk production, hyperthermia, an
increased respiration rate and haircoat changes (Schmidt and Osborne, 1993; Paterson et al.,
1995; Stuedemann et al., 1998; Schultze et al., 1999; Oliver, 2005).
These symptoms are subsumed under the term “fescue toxicosis” which is mainly caused by
the vasoconstrictive effects of ergovaline which acts merely as a dopamine-receptor agonist
(Fink-Gremmels, 2005). The vasoconstriction may also result in a symptom called “fescue
foot” which refers to gangrenous lesions at the extremities (Botha et al., 2004; Tor-Agbidye et
al., 2006).
Schmidt and Osborne (1993) mentioned another form of disorder which has been shown to
occur in cattle grazing fescue. Bovine fat necrosis, also sometimes referred to as lipomatosis,
is characterized by the presence of masses of hard or necrotic fat, primarily in the adipose
tissue of the abdominal cavity. Dystocia and digestive disturbance are common signs of fat
necrosis manifestation caused by a lack of physical space within the abdominal cavity
(Stuedemann et al., 1985; Stuedemann and Hoveland, 1988; Hussein and Brasel, 2001).
Another syndrome caused by endophytal toxins is called “ryegrass staggers” and could be
reproduced largely by the application of lolitrem B (Cheeke, 1995; Fink-Gremmels, 2005).
Early clinical signs are head tremors, difficulties in rising and muscle fasciculation, later
followed by swaying while standing and, if stressed accessorily, end in titanic convulsions
(Cheeke, 1995).
Most of the outbreaks of ryegrass staggers and fescue foot have been reported predominantly
from the USA where tall fescue (Festuca arundinacea) is the major forage grass, and from
Australia and New Zealand, where perennial ryegrass is the most common pasture species
(Galey et al., 1991; Easton and Tapper, 2005; Fink-Gremmels, 2005; Reed et al., 2005).
Similarly in Africa outbreaks of fescue toxicosis are recorded. Fifty of 385 Braham cattle
grazing for 3 weeks on fescue pasture developed lameness and/or necrosis of the tail. The
9
Introduction
ergovaline concentrations in basal leaf sheets and grass stems collected during the outbreak
ranged from 1720-8170 μg/kg on a dry matter basis (Botha et al., 2004).
But also in Europe endophyte infected swards may occur. Oliveira (2002; cited by
Zabalgogeazcoa and Bony, 2005) collected wild ecotypes of perennial ryegrass in northern
Spain. Forty percent were infected with Neotyphodium and the average ergovaline content
even was 13.5 µg/g.
In Germany, Oldenburg (1997) detected infection with Neotyphodium endophyte in 33 of 38
ecotype populations of Lolium perenne originating from old grassland collected in 1997. The
frequency of individual infected plants of the different populations ranged mostly from 1% to
30% with a few populations showing higher infection levels up to 80%.
The interaction between thermal stress and fescue toxicosis, as recently reviewed by Spiers et
al. (2005), might be a reason for the scarcity of fescue toxicosis in Europe. Environmental
conditions are less stressful for farm animals as compared to the USA, for example.
Zabalgogeazcoa and Bony (2005) explain the lower incidence of clinical cases by the fact that
animals in most European production systems are not likely to be as heavily exposed as in the
USA or New Zealand. This is, according to their opinion, because extensive grazing systems
are not as widely used, and when they are, pastures are mainly natural or artificial mixtures of
many grass species and clover (Trifolium spp.), thus toxins contained in meadow fescue and
perennial rye grass are diluted.
Accordingly, research on ergotism caused by cereals infected with Claviceptaceae is of
greater interest in Germany, and toxicosis due to endophytal infection may for the time being
disregarded.
Interaction with other factors
Concerning ruminants some other factors than dosage and duration of toxin exposure might
influence the variability of toxin effects and of carry over (Seeling, 2005).
For example, the outside temperature obviously influences ergot-caused effects on ruminants.
The hyperthermic syndrome predominantly occurs during seasons with hot weather (Peet et
al., 1991; Schneider et al., 1996; Ilha et al., 2003). As already mentioned above, it has been
suggested that the clinical symptoms of respiratory distress and increased body temperature
are the results of peripheral vasoconstriction, and may be aggravated by high environmental
temperatures.
However, the gangrenous syndrome predominantly occurs in cold environmental conditions
(Burfening, 1973), since the vasoconstriction then is exaggerated by the cold and the tail, ears,
10
Introduction
and even the feet and limbs, may freeze more easily and slough off due to the lack of
circulation.
Both forms of the mycotoxicosis rarely occur simultaneously under spontaneous conditions
(Ilha et al., 2003).
However, Bourke et al. (2000) considered solar light radiation as more significant than
ambient temperature in producing hyperthermic ergotism in cattle.
Also the animal’s race plays an important role in influencing the variability of ergot effects in
cattle which, amongst others, is related to the different heat tolerances. For example, Braham
cattle are proven to be better adapted to resist or tolerate the hyperthermia described above
than Angus cattle (Schmidt and Osborne, 1993). Browning, Jr., and Thompson (2002)
compared several performance parameters of 7 Braham and 7 Hereford (heat sensitive) steers
given ergotamine tartrate iv. They also concluded a potential benefit of using heat tolerant
genetics to reduce the adverse effects of ergotism, although acute ergotamine exposure
generally resulted in similar effects on both breeds.
Ruminants are generally considered as less susceptible to mycotoxins (e.g., deoxynivalenol,
zearalenone) than monogastric animals, which is related to the potential degradation of these
substances by microorganism in the rumen (Kiessling et al., 1984; Hussein and Brasel, 2001).
However, with regard to the rumen development, differences should be considered between
fattening cattle, dairy cows, ruminant and pre- ruminant young calves (Seeling, 2005).
In young calves the rumen is not yet completely developed which might limit the metabolism
of the mycotoxins in the rumen and lead to an alteration of the metabolite profile and a
modification of the toxicity.
Exact data on the ruminal degradation of ergot alkaloids is lacking, but the fact that the
microbes interact with the toxins is mentioned by several authors (Kiessling et al., 1984;
Ayers et al., 2004 (cited by Hill; 2005)). The rumen is the main part of the forestomach
system to absorb ergot alkaloids due to its large surface area (Hill, 2005).
Calves are reported to react less sensitively to ergot than full-grown ruminants (Edwards,
1953; Coppock et al., 2006), which might be caused by the fact that in pre-ruminating calves
less toxins might be absorbed. On the other hand, Stuedemann et al. (1998) incubated
endophyte infected tall fescue in autoclaved rumen fluid and realised that the aqueous
concentration of ergot alkaloids increases with time when viable ruminal microbes
decompose the plant tissue. Conversely, the total alkaloid concentration in the fescue pellet
remained the same in the autoclaved ruminal fluid, but decreased when viable ruminal
11
Introduction
microbes were present. Keeping in mind that Stuedemann et al. (1998) used monoclonal
antibodies to analyse alkaloids in this study, which only recognize the intact lysergic moiety,
ergopeptine alkaloids could have been metabolized by the ruminal microflora into simpler
alkaloid forms via peptide cleavage or proline transformation within the peptide moiety
(Eckert et al., 1978). However, the microbes serve to liberate the toxins from the plant tissue
which is a disadvantage for the host, but, on the other hand, the toxins might be metabolised
by the microbes. This metabolism again might end in less active products but also in a higher
toxicity of the substances. Furthermore, increased water solubility as a result of microbial
action might increase the rate of excretion, but might also facilitate the absorption from the
intestine (Kiessling et al., 1984).
Further research is needed to understand the exact role of the rumen in the ergot alkaloid
metabolisation (Figure 5).
Figure 5: Potential fate and effects of ergot and its alkaloids in the ruminant
The ration composition may also be an important determinant in the relative toxin resistance
since, for example, high performance diets with small amounts of crude fibre may result in a
dysfunction of the rumen, and thereby in a lower detoxification capacity (Dirksen et al.,
2002).
Carry over into meat ?
Feed contaminated with ergot
Absorption ?
Detoxification ?
Carry over into milk ?
Microbes
Alkaloids
Liberation from plant tissue ? Modification ?
Excretion by urine and faeces?
Rumen:
Alteration of population
and/or activity ?
12
Introduction
Furthermore, concerning especially ruminants, the roughage is of special interest. Ruminants
compared to monogastric animals need a higher amount of roughage in their diet, which in
case of grass silage or hay, may form a second source for ergot entry (Landes, 1996; Mainka,
2006). Considering the worst case scenario, a permitted dose of 1000 mg ergot/kg unground
cereal grains (Council Directive 2002/32/EC of 7 May 2002) might be heavily boosted by a
possible ergot or endophyte contamination (with ergot being predominant in Europe) of the
roughage (mainly grass silage or hay) and thus might contribute to the toxic potential of a diet
for ruminants.
Also the passage rate and the level of feed intake need to be considered. The higher the feed
intake by a ruminant, the higher the rate of passage through the rumen, which is associated
with a decreased ruminal retention time, and thus with a shorter period of time to liberate or
metabolize ergot alkaloids. The ruminal retention time varies between animals, sexes and
species, but is additionally influenced by dietary components (Tamminga, 1979).
Although data on ergot studies with differing passage rates in ruminants are lacking, it might
be suggested that an increased passage rate may limit the metabolism of the mycotoxins in the
rumen and therefore alter the metabolite profile and modify the toxicity (Seeling, 2005).
Scope of the thesis The actual state of knowledge regarding the effects of ergot on cattle is very limited. No dose-
effect studies were carried out so far. In addition, literature on case reports is scarce and
mostly refered to weight based ergot sclerotia contamination and not to the actual food level
of toxic ergot alkaloids which may vary in wide ranges.
Furthermore information on carry over into edible tissue and milk as a potential risk for
humans is lacking.
The aim of this study was to answer following questions:
1. How do cattle of different ages react on increasing levels of ergot sclerotia in their
feed?
2. Which effects does ergot supplementation of the concentrate have on health,
performance and carcass characteristics depending on dosage and duration of toxin
exposure?
3. Is the given weight based upper limit of 1000 mg ergot/kg unground cereal justifiable?
4. Is there a measurable carry over of ergot alkaloids into edible tissue and milk after oral
ergot exposure?
13
Introduction
5. Which effects does ergot contaminated rye have on rumen fermentation?
6. Do variations in feed intake levels and passage rate in dairy cows make any difference
regarding ergot absorption and metabolism?
7. Which effect does ergot supplementation of the concentrate have on the daily rectal
temperature curve of dairy cows?
Three different experiments concerning effects of ergot on cattle were carried out to answer
the questions raised above. The batch of ergoty rye used in these experiments was sorted out
from the harvest 2002 by the Lochow Petkus GmbH and consisted of 45 % ergot and 55 %
rye grain. It had been stored in a deep freezer until usage and the analysed alkaloid content of
the ergot was 682 mg/kg TS.
First of all, a long term study with fattening bulls was initiated. A control group, a second
group with an ergot supplementation of the concentrate portion of 0.045%, and a third group
with a supplementation of 0.225%, were fed these so-prepared concentrates together with
maize silage for a period of approximately one year. After this period of time, and with an
approximate body weight of 550 kg, they were slaughtered and organ samples were taken to
examine carry over and pathological changes (Paper I).
Since no effects have been observed in the bulls, a second experiment with calves was carried
out since young animals are considered as especially sensitive to toxins. Additionally the
ergot levels during this experiment were raised. Beside the control group, two other feeding
groups were fed with 0.1% and 0.5% ergot sclerotia respectively over an entire test period of
84 days (Paper II).
Furthermore, in a third experiment varying levels of ergot contaminated and uncontaminated
rye were fed to ruminally and duodenally fistulated dairy cows to examine the effect on
ruminal nutrient fermentation, alkaloid metabolism, carry over into milk, several blood
parameters and rectal temperature. In this experiment, additionally varying amounts of
organic matter intake have been fed to analyse the interaction between ergot feeding and the
passage rate (Paper III).
14
Paper I
Paper I
Effects of different levels of ergot in concentrates on the growing
and slaughtering performance of bulls and on carry-over into
edible tissue
BARBARA SCHUMANN, SVEN DÄNICKE, ULRICH MEYER, KARL-HEINZ
UEBERSCHÄR, GERHARD BREVES
Institute of Animal Nutrition, Federal Agriculture Research Centre (FAL),
38116 Braunschweig
Archives of Animal Nutrition
In press
15
Paper I
Abstract
The aim of the present study was to examine long-term effects of low levels of ergot alkaloids
on growing bulls. Natural grown ergot with a mean total alkaloid concentrations of 633
mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent
alkaloids, was used. In a dose-response study 38 Holstein Friesian bulls were fed with three
different doses of this ergot (0, 0.45 and 2.25 g/kg concentrate corresponding to an average
total alkaloid concentration of the daily ration of 0, 69 and 421 µg/kg DM) over a period of
approximately 230 days. Live weight, feed intake and health condition were monitored over
the entire test period. The bulls were slaughtered at a live weight of approximately 550 kg.
Carcass composition and quality were recorded and samples of liver, muscle, kidneys, fat,
bile, urine and blood were analysed for ergot alkaloids. Liver enzyme activities and total
bilirubin were measured in the blood.
Statistically, no significant differences were detectable between the three feeding groups.
Mean live weight gain over all groups was 1.41 kg/day with a mean dry matter intake of 7.35
kg/day. No carry over into tissues could be proved out of the experiment. To derive a no-
effect level for beef cattle further research including higher ergot doses will be necessary.
Keywords: Ergot alkaloids, growing cattle, feed intake, weight gain, liver parameters, carcass
composition
1. Introduction
The solidified mycelium of the parasitic fungus Claviceps purpurea, called ergot, mainly
invades flowering grasses and develops instead of the host grains. Ergot is considered the
most enduring form of the fungus. The toxicity of ergot is due to its alkaloid content which
ranges from 900 to 2100 mg/kg ergot (Wolff 1989) or may even differ by a factor of ten
(EFSA 2005). Detailed information on toxicity, potential metabolism and excretion of the
alkaloids and possible effects on animals were reviewed by Landes (1996) and Kren and Cvak
(1999). Possible nutritional sources of ergot for ruminants may be the concentrate
(contaminated grain), but also the roughage as it may contain grasses infected with various
Claviceptaceae (Engelke 2002). A weight based upper limit of 1000 mg ergot per kg
unground cereal grains (Council Directive 2002/32/EC), not addressing the high variability of
the alkaloid content, is the only official protection for farm animals. Evaluating the literature
for ruminants, Landes (1996) considered this upper level as problematic, whereas Gedek
(2002) only recommended not exceeding 2500 mg ergot/kg of the daily ration for ruminants.
16
Paper I
Because of the confusing situation surrounding this species, information on dose-response
relationships with regard to ergot alkaloids is needed for an improved risk evaluation.
Furthermore literature about the carry-over of ergot into edible tissue is very scarce. Mainka
et al. (2005a), who fed growing-finishing pigs with diets containing 1 g or 10 g ergot/kg,
found that serum, bile, liver, meat and back fat did not contain any detectable amounts of
ergot alkaloids. Young and Marquardt (1982) only detected residual amounts of ergotamine
(10 µg/kg or less) in liver and muscle of chicken fed with 810 mg ergotamine tartrate/kg feed.
However, in these experiments only the parent compound (ergotamine) was analysed and the
presence of metabolites cannot be excluded (EFSA 2005). Whittemore et al. (1976), who fed
a diet containing 40 g ergot/kg to pigs, did not find any residues in tissues either. In cattle
there is a complete lack of carry-over data except for some data concerning milk (Wolff and
Richter 1995), and only one article which deals with the alkaloid carry over into meat
(Cunningham et al. 1944).
Ruminants are considered relatively insensitive to mycotoxins due to the potential microbial
modification. But it also might be possible that the anti-microbial properties of ergot may
derange the microbial digestion and could consequently have an impact on nutrient utilisation
and performance.
The aim of the present study was to examine the effects of increasing low level ergot amounts
in concentrates with defined alkaloid concentrations and patterns on performance, carcass
composition of bulls and carry-over into edible tissues.
2. Material and methods
2.1. Experimental design
A dose effect study with three different ergot proportions (0, 0.45 and 2.25 g/kg concentrate)
was carried out to comprise the weight based upper limit of 1 g ergot per kg unground cereal
grains (Council Directive 2002/32/EC of 7 May 2002). To get practical relevant results,
natural grown ergot of Claviceps purpurea with a mean total alkaloid concentrations of 633
mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent
alkaloids, was used. The ergot batch was sorted out from the harvest 2002 by the Lochow-
Petkus GmbH, Bergen. Altogether 38 Holstein Friesian bulls from the cattle herd of the FAL
Braunschweig were divided into three feeding groups designated as Control (n=12), Ergot 1
(n=13) and Ergot 2 (n=13), respectively.
17
Paper I
2.2. Fattening experiment and procedures
Bulls at the experimental station of the FAL were kept in group pens (4.85 x 8.00 m) with
slatted floors partly covered with rubber mats. The experiment started at a live weight of
approximately 227 kg with the change to the experimental diet. The concentrates, containing
increasing proportions of ergot, were offered by a self feeding station which was combined
with an animal scale (Type AWS HF, firm: Insentec, Marknesse, Netherlands). Animals were
individually identified using ear transponders. The composition of the diets is shown on Table
I. The daily concentrate allowance was adapted to live weight sections. Up to 250 kg live
weight (LW) bulls were offered 2 kg concentrate plus 100 g mineral and vitamin premix
(provided per kg concentrate: 7.5 g calcium, 2.6 g sodium, 1.2 g phosphorus, 1.1 g
magnesium, 15 000 IU vitamin A, 1 600 IU vitamin D3, 15 mg vitamin E, 120 mg zinc, 60
mg manganese, 15 mg copper, 0.6 mg selenium, 0.3 mg cobalt). Between 250 and 300 kg LW
concentrate allowance was increased on 2.5 kg, between 300 and 350 kg LW on 2.7 kg and
the amount of mineral and vitamin premix added was doubled, from 350 kg LW until
slaughtering bulls were fed 3.0 kg concentrate. Maize silage and water were offered for ad
libitum consumption through transponder sensitive automatic roughage feeders (Type: RIC
HF 50gr, firm: Insentec, Marknesse, Netherlands). There were 4 roughage feeders in each pen
and a maximum of 7 animals were allocated per pen. The day before the study started a blood
sample was taken from the Vena jugularis of the bulls (approx. 10 ml into tubes with heparine
for the plasma extraction and 10 ml into small serum tubes), centrifuged approximately 1-2
hours later at 3000 g and 15° C for 10 min. and then frozen.
Performance parameters are evaluated for particular fattening periods. These periods are from
the beginning of the experiment to 300 kg LW, from 300 to 400 kg LW and from 400 kg LW
to slaughtering.
Achieving a LW of approximately 550 kg, the bulls were slaughtered in the slaughter house
of the experimental station Braunschweig following at least 7 hours of fasting. After
anaesthesia with bolt shot bulls were immediately bleed while hanging and opened
afterwards. Samples of blood (at the moment of bleeding), urine from the urinary bladder, bile
from the gall bladder, samples of the liver, kidneys, the longissimus muscle close to the 13th
rib and abdominal fat from the kidney cavity were taken and shrink-wrapped for deep
freezing. Additionally, the health, physical condition, and weight of each bull as well as of
every organ and slaughtering product were recorded.
18
Paper I
Table I. Composition of the concentrates [g/kg as fed], mean values of dry matter [g/kg], nutrient composition [g/kg DM], energy concentration of the concentrates [MJ ME/kg DM] and alkaloid pattern of ergot and rye, [µg/kg DM] sorted out from the ergoty rye, and of concentrates (n = 8) containing increasing proportions of this ergoty rye (Percentage of total alkaloids in brackets) Group
Control Ergot 1 Ergot 2 Ergot Rye Components: Soy bean meal 227 227 227 Peas 150 149 145 Wheat 299 299 299
Sugar beet pulp 299 299 299
Ergoty rye* 0 1 5
Soy bean oil 20 20 20
Calcium carbonate 5 5 5 Nutrients and energy: Dry matter 891 890 890 928 884 Crude protein 194 199 197 216 112 Crude fat 39 37 38 354 19 Crude fibre 89 86 89 209 27 ADF 107 104 106 NDF 239 240 244 ME ° 12.6 12.5 12.6 Alkaloids: Ergometrine ≤ 11 12.2 (6) 63.1 (5) 56631 (8) 142 (20) Ergometrinine ≤ 11 2.0 (1) 13.3 (1) 8475 (1) 20 (3) Ergotamine ≤ 6 40.6 (19) 299.0 (24) 172305 (25) 92 (13) Ergotaminine ≤ 6 33.0 (16) 202.8 (16) 67798 (10) 43 (6) Ergocornine ≤ 6 11.6 (6) 61.9 (5) 34476 (5) 15 (2) Ergocorninine ≤ 6 6.9 (3) 35.0 (3) 19338 (3) 7 (1) Ergocryptine ≤ 6 13.6 (6) 66.7 (5) 42589 (6) 40 (6) Ergocryptinine ≤ 6 9.4 (4) 49.5 (4) 29584 (4) 28 (4) Ergocristine ≤ 6 33.1 (16) 196.7 (16) 105124 (15) 174 (25) Ergocristinine ≤ 6 16.3 (8) 81.4 (7) 32154 (5) 88 (12) Ergosine ≤ 6 19.0 (9) 117.6 (9) 91159 (13) 49 (7) Ergosinine ≤ 6 12.5 (6) 64.5 (5) 21387 (3) 9 (1) Total alkaloids ≤ 11 210.3 (100) 1251.5 (100) 681022 (100) 707 (100) * 45 % ergot ° calculated on the basis of the nutrient digestibilities measured with wethers
19
Paper I
2.3. Analyses
Ergot and feedstuffs were ground to 1 mm and than analyzed for ergot alkaloids (ergometrine,
ergocornine, ergotamine, α-ergocryptine, ergosine, ergocristine and their –inine isomers) by
HPLC based on the method of Wolff et al. (1988). The detection limit (defined as 20-fold of
the signal noise ratio) for all matrices was 11 ng/g dry matter (DM) for ergometrine and 6
ng/g DM for the other alkaloids. The recovery rates are detailed in Table II. Alkaloid
standards and ammonium carbonate were purchased by Sigma-Aldrich Chemie GmbH,
Buchs, Switzerland, and the remaining chemicals used for the following method were
produced by Carl Roth GmbH & Co.KG, Karlsruhe, Germany.
Approximately 3 g of each sample were dissolved in 100 ml extraction fluid (50 ml
dichlormethane + 25 ml ethylacetate + 5 ml methanol + 1 ml ammonium hydroxide (25%)).
One day later, after centrifugation, an aliquot was taken and evaporated to dryness. 2 ml
toluene / methanol (49 + 1) were used to dissolve the residue which was followed by
solubilisation per ultrasound. The fluid was mixed with 9 ml i-hexane and put on a 3 g
Extrelut® column (Merck, Darmstadt, Germany), which was acidified with 5 ml 2 %
aquaeous tartaric acid. 0.5 ml toluol / methanol + 4.5 ml i-hexane and 20 ml di-isopropylether
/ i-hexane (1+1) were used for the following elution. For 1-2 min air was sucked through to
dry the column before the alkaloids were assimilated in 25% ammonium gas, which was
detected by a colour reaction of phenolphthalein. The alkaloids were eluted with 25 ml
dichlormethane and evaporated to dryness at 35°C. Afterwards they were carefully blown off
with nitrogen. Finally the residue was filled up to a definite volume of 500 µl in the mobile
layer of the HPLC [acetonitrile/water (1+1); with ammonium carbonate adjusted on pH 8.4]
of which 20 μl were injected in the HPLC-apparatus, consisting of an isocratic pumping
system with a 250 x 4 mm column (5 μm, C 18 Gravity, Macherey-Nagel, Düren, Germany).
The HPLC operates at 44°C and is connected with a fluorescence detector (325 nm excitation
/ 418 nm emission wavelength).
The serum samples, bile, urine, liver, kidney, muscle and fat were freeze-dried and ground to
1 mm. Afterwards they were analysed with the same method.
Ergometrine, ergotamine, ergocristine, ergocornine and ergocryptine are referred as to “key
alkaloids” since standards are commercially available for their identification. These standards
may also be used for the identification of their –inine isomers. Ergosine and its isomer were
identified by their retention time (Baumann et al. 1985) and for quantification the mean
values of the standard alkaloids and their isomers (exclusive ergometrine and ergometrinine)
were used. The analytical results were not corrected by recovery.
20
Paper I
Contamination of the feedstuffs with deoxynivalenol (DON) was analysed by HPLC with
DAD (diode array detection) after cleaning-up with IAC (immunoaffinity column,
DONprepTM, R-Biopharm AG, Darmstadt, Germany) according to the slightly modified
procedure of the manufacturer (Oldenburg et al. 2007).
Zearalenone (ZON) was analyzed according to a modified VDLUFA (Verband Deutscher
Landwirtschaftlicher Untersuchungs- und Forschungsanstalten) method as described by
Ueberschär (1999).
One ml of each serum sample was analysed by the laboratory of the Cattle Clinic Hanover for
5 liver parameters with a fully automatic apparatus (Cobas-Mira, Fa. Hoffmann-La Roche &
Co. AG Diagnostika Basel, Switzerland). Photometric standard procedures were used for
gamma-glutamyl transferase (γ-GT, International Federation of Clinical Chemistry) and
aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and creatine kinase
(CK, German Federation of Clinical Chemistry). Total bilirubin was measured according to
the method of Jendrassik and Gróf (1938).
Pooled samples taken from the ergoty rye, maize silage and concentrates were examined for
dry matter, crude ash, crude protein, crude fat and crude fibre according to the methods of the
VDLUFA described by Naumann and Bassler (1993).
The digestibility of the concentrates and of the maize silage was measured in a balance
experiment using 4 wethers with a mean LW of 90 ± 7 kg. Therefore, the respective
concentrate was fed together with hay and by substracting the hay digestibilities determined
in a separate experiment, the digestibilities of the concentrates were estimated. The
experiment followed standard procedures as described by the GfE (1991).
21
Paper I
reco
very
) in
20 µ
l (n
= 2-
3)
Ergo
-cr
yptin
ine
Ergo
-cr
istin
e Er
go-
cris
tinin
e
Tab
le II
. Mea
n re
cove
ry ra
tes [
%] a
fter a
n ad
ditio
n of
0.5
ng
of th
e al
kalo
id (2
.5ng
for e
rgom
etrin
e
Er
go-
met
rine
Er
go-
met
rinin
e Er
go-
tam
ine
Ergo
-am
inin
e Er
go-
corn
ine
Ergo
-co
rnin
ine
Ergo
-cr
yptin
e Se
rum
10
7 10
3 94
99
89
10
2 10
0 92
89
97
Fat
107
88
117
96
95
73
103
Mus
cle
80
90
70
81
83
73
72
Live
r 87
75
63
68
73
64
60
52
91
65
65
75
64
52
64
55
Kid
ney
115
94
87
85
85
80
86
69
77
71
Bile
10
5 88
90
90
86
82
94
72
83
79
Urin
e 87
10
0 90
97
89
98
99
89
84
95
Feed
stuf
f 13
9 10
3 75
81
87
58
71
56
76
45
22
Paper I
2.4. Calculations and statistics
ME was calculated as described by the GfE (2001) using the results of the balance experiment
with the wethers.
Feed intake, live weight gain, ME to gain ratio and carcass compositional data were analysed
using a one-way factorial design of analysis of variance (ANOVA) with the following model:
yij = μ + ai + eij,
where yij = tested parameter of the bull "j" fed diet type "i"; µ = overall mean; ai = effect of
diet (i.e. Control, Ergot 1 and Ergot 2); eij = error term. The fixed effect of the group (ergot) and
for further evaluation of the dose effects the probabilities for orthogonal effects (linear,
quadratic) were estimated.
The multiple t-test was used for analysing mean value differences.
Variance of the clinical serum parameters was evaluated according to the restricted maximum
likelihood (REML) method for random effect variances and the Kenward-Roger-method for
calculation of degrees of freedom implemented in the SAS-software package (SAS Institute Inc.
2003, Version 9.1, procedure "mixed") according to the following model:
yijk = μ + ai + bj + eijk
where yijk = tested parameter of the bull "k" fed diet type "i"; µ = overall mean; ai = effect of
diet (i.e. Control, Ergot 1 and Ergot 2); bj = random effect of animal to account for repeated
measurements within the same individual; eijk = error term.
3. Results
3.1. Chemical composition of feedstuffs and ergot
Pooled concentrate and maize silage samples were analysed monthly over the whole
experimental period.
The maize silage comprised mean contents of crude protein of 83 g/kg DM, crude fibre of 201
g/kg DM, ADF of 223 g/kg DM and NDF of 424 g/kg DM.
The nutrient composition of the three different types of concentrate showed almost no
differences (Table I).
23
Paper I
No alkaloids were detected in the concentrate of the control group and in the maize silage
over the whole experimental period.
Total alkaloid contents of the ergot supplemented concentrates ranged from 102 to 294 µg/kg
DM in group Ergot 1, and from 905 to 1869 µg/kg DM in group Ergot 2. The ergot and
alkaloid exposure per kg DM of the daily ration was nearly constant (Table III). Ergotamine,
ergocristine and ergotaminine were the most prominent alkaloids in the ergot and in the
concentrates (Table I) comprising together between 50 % and 56 % of the total alkaloids.
In all of the feedstuffs, no β-zearalenol was detected. The detection limit for β-zearalenol was
5 ng/g dry matter. In pooled samples of the maize silage 4.2 ng α-zearalenol/g and 220.2 ng
zearalenone/g on a dry matter basis were detected.
At a detection limit of 1 ng/g DM, no α-zearalenol was detected in the concentrates.
Zearalenone was detected with 8.2 ng/g DM in the concentrate of the control group, with 6.3
ng/g DM in the concentrate of the Ergot 1 group and with 3.7 ng/g DM in that of the Ergot 2
group.
On a DM basis, deoxynivalenol was detected in the maize silage with a concentration of 2107
ng/g, and with a concentration of 135 ng/g in the concentrate of the control group, of 102 ng/g
in the concentrate of the Ergot 1 group and of 81 ng/g in that of the Ergot 2 group.
3.2. Performance and carcass composition
During the winter, some problems with light coughing were noted in each group, but this was
found not to be permanent and was not treated. Furthermore there were two bulls in the
control group which needed some medical treatment. One suffered of bronchitis and the other
had a serious panaritium. In the Ergot 1 group no bull was conspicuous over the entire test
period, and in the Ergot 2 group one of the bulls got an analgesic because of a distortion of the
front limb.
The study took its course without any unusual incidents. Only one of the bulls needed to be
euthanized with a body weight of 508 kg due to a muscle disruption after being straddled.
This bull later was excluded from the evaluation. It took the animals approximately 230 days
to reach the final live weight of 550 kg. None of the performance parameters was significantly
influenced by increasing ergot concentrations.
24
Paper I
Table III. Ergot and alkaloid exposure and the effects of ergot contaminated concentrate on live weight gain (LWG), dry matter intake (DMI) and ME to gain ratio of growing bulls
Ergot exposure Alkaloid exposure g/kg
concentrate g/kg DM of
the diet° mg/d*kg
LW
µg/kg DM of the diet°
µg/d*kg LW
LWG
kg/day
DMI & [roughage
percentage] kg/day
ME to gain ratio
MJ/kg LWG
0.00 0.00 0.00 0.0 0.00 1.43 7.34 [66] 60.0 0.45 0.17 2.07 69.4 1.40 1.40 7.36 [67] 61.5 2.25 0.86 12.53 421.3 8.56 1.41 7.34 [66] 60.1
ANOVA:
Ergot 0.722 0.997 0.780 linear 0.500 0.982 0.980
quadratic 0.645 0.947 0.482
PSEM* 0.04 0.36 3.0 ° Alkaloid concentrations which were lower than the detection limits were considered with a zero concentration * Pooled standard error of means
Live weight gain (LWG) of the control group varied between 1.10 and 1.65 kg/day in the first
period, between 1.10 and 1.69 kg/day in the second and between 1.10 and 1.70 kg/day in the
third period. Group Ergot 1 showed a variation in LWG in Periods 1 and 2 from 1.15 to 1.68
kg/day and in Period 3 from 1.16 to 1.69 kg/day. In group Ergot 2, LWG ranged from 1.19 to
1.48 kg/day in the first period, from 1.23 to 1.65 kg/day in the second and from 1.23 to 1.70
kg/day in the last period.
Dry matter intake (DMI) and ME to gain ratio were independent of the ergot supplementation
of the concentrate over the entire experimental period (Table III).
The assigned amount of concentrate was taken up by the bulls more or less completely
independent of the ergot supplementation. Average maize silage intake increased from 3.9 kg
DM/day in the first third of the experiment, and 4.8 kg DM/day in the second third, up to 5.8
kg DM/day in the last third, and was not influenced by the ergot supplementation of the feed.
At the day of slaughtering mean live weights of the three feeding groups were supposed to be
almost at one level and actually varied between 534 and 577 kg. At the weights of the warm
carcass as percent of live weight, which varied between 50 and 55 %, there is a trend to
significance for the effect of ergot feeding (linear: p = 0.081).
Further carcass compositional data and organ weights are detailed on Table IV.
25
Paper I
Eleven of the bulls (3 of the control group, 4 of group Ergot 1 and 4 of group Ergot 2) showed
slight inflammation of the urinary bladder, partly with uroliths. There were abscesses in one
of the livers and two reticula (caused by a small piece of wire). The lung of one bull showed
slight emphysema.
3.3. Clinical chemical serum parameters
The activity range of the AST was significantly influenced by age (p = 0.048), but
independent of ergot feeding, with values varying over the three feeding groups between 53
and 108 U/l at the first experimental day and accordingly between 43 and 100 U/l
approximately one year later.
The GLDH ranged between 6.5 and 97.7 U/l at the beginning of the study, and between 6.42
and 35.3 U/l at the day of slaughtering, and was also significantly influenced by age
(p<0.001), but remained unaffected by ergot feeding.
The γ-GT activity varied between 12 and 38 U/l on the first, and between 10 and 35 U/l on the
last experimental day, only showing a trend to significance for the effect of age (p = 0.096).
Activities of the CK varied between143 and 495 U/l at the beginning, and 118 and 907 U/l at
the end of the experiment and remained unaffected by dietary treatments, but were
significantly influenced by age (p<0.001).
Total bilirubin diversified in a range between 1.30 and 5.32 μmol/l at the first experimental
day and between 1.21 and 5.34 μmol/l at the last day of the study and was not significantly
influenced by the mentioned factors.
Ergot alkaloid were not detected in the blood samples.
3.4. Carry over
Neither the analysed tissue samples, nor the samples of blood, bile or urine, contained
detectable amounts of alkaloids.
26
Paper I
s ea
rt Th
ymus
Pan
crea
s Sp
leen
Te
stic
les
Tab
le IV
. Eff
ects
of e
rgot
con
tam
inat
ed c
once
ntra
te o
n ca
rcas
s com
posi
tion
and
orga
n w
eigh
ts o
f gro
win
g bu
lls
EB
W°
Dre
ssin
g†
Live
r Lu
ng
Kid
ney
HLi
ve
wei
ght*
A
bdom
inal
fa
t‡ G
astro
in-
test
inal
tract
Er
got (
g/kg
co
ncen
trate
) [k
g]
[kg]
[%
] [k
g/10
0 kg
EB
W]
[g
/1
00 k
g EB
W]
0.00
55
5 49
2 52
.0
8.45
4.
78
15
06
701
230
406
105
114
218
383
0.45
55
5 49
7 52
.4
8.21
4.
60
15
24
731
241
2.25
55
3 49
7 52
.8
8.19
4.
65
15
13
720
226
AN
OV
A (p
roba
bilit
ies)
Ergo
t 0.
758
0.46
3 0.
211
0.89
8 0.
329
0.
923
0.57
1 0.
258
lin
ear
0.49
6 0.
280
0.08
1 0.
676
0.30
2
0.87
8 0.
500
0.64
1 q
uadr
atic
0.
784
0.53
0 0.
928
0.83
7 0.
270
0.
712
0.40
9 0.
119
421
101
115
231
380
421
101
105
232
398
0.67
4 0.
915
0.32
7 0.
273
0.64
8 0.
436
0.71
5 0.
229
0.14
6 0.
475
0.66
4 0.
832
0.39
0 0.
467
0.56
6 PS
EM
2 3
0.3
0.43
0.
09
31
20
7
14
8 5
7 15
* A
fter a
t lea
st 7
h fa
stin
g ° E
mpt
y bo
dy w
eigh
t: di
ffer
ence
bet
wee
n liv
e w
eigh
t and
the
wei
ght o
f the
con
tent
s of t
he g
astro
-inte
stin
al tr
act
and
of th
e ur
inar
y bl
adde
r † W
eigh
ts o
f the
war
m c
arca
ss a
s per
cent
of l
ive
wei
ght
‡ Sum
of t
he fa
t of t
he k
idne
y ca
vity
and
the
fat c
over
ing
the
gast
ro-in
test
inal
trac
t
27
Paper I
4. Discussion
4.1. Chemical composition of feedstuffs and ergot
In spite of the commercial availability of individual alkaloid standards, naturally grown ergot
was used as alkaloid source to study the effects of a defined, and for Germany practically
relevant, pattern. Each alkaloid has a specific impact on the organism (Buchta and Cvak,
1999). And depending on the alkaloid pattern of the ergot, varying effects might be caused in
the animal due to potential synergistic, but also antagonistic effects between individual
alkaloids (Buchta and Cvak, 1999) (Gareis and Wolff, 2000) which limit the validity of the
data to the specific alkaloid pattern tested.
The nutrient composition and energy content of the concentrates were comparable (Table I).
Thus, it can be assumed that feeding conditions were equal for each bull, and that potential
effects might be due to the ergot supplementation of the concentrate.
In Germany, the alkaloid concentration in ergot was reported to range between 818 and 1635
mg/kg ergot DM sampled from the harvest 2004 (Mainka et al. 2006a+b); in 2003 from 42 to
343 mg/kg ergot DM (Mainka et al. 2006a+b), and in previous years between 900 and 2100
mg/kg ergot (Wolff 1989). In Canadian rye, ergot alkaloids varied between 100 and 4500
mg/kg (Young 1981b).The ergot used in this experiment fits into the discussed range of
alkaloid variation as an averaged alkaloid concentration of 681 mg/kg DM has been detected.
The percentage alkaloid composition of the two concentrates differs slightly (Table I), which
might be explained by the error of sampling. In fact, the amount of alkaloid content, and the
composition, depends on the current sample taken and is considered never to be uniform
(Young 1981a+b; Young and Chen 1982; Filipov et al. 2000).
During the entire experimental period, no alkaloids were detected in the maize silage, which
facilitates the interpretation of the present results. If ruminants are additionally exposed to
ergot from roughage containing grass infected by various Claviceptaceae, the effects might be
exponentiated as noted by the EFSA (2005). The actual alkaloid exposure in each group is
shown in Table III.
The deoxynivalenol contamination of the maize silage of 2107 ng/g DM (or 1854 ng/g at a
dry matter content of 88%) corresponds to an approximate contamination of the total diet of
1427 ng/g DM (or 1256 ng/g at a dry matter content of 88%). The contamination is
significantly lower than the European orientation value for bulls of 5000 ng/g (Amtsblatt der
Europäischen Union 2006) and thus may not cause any negative effects. The zearalenone
contamination of the maize silage of 220 ng/g DM (or 194 ng/g at a dry matter content of
88%) relates to 147 ng/g DM of the total diet (or 129 ng/g at a dry matter content of 88%). In
28
Paper I
Europe for zearalenone no orientation value applies for bulls. But even those for dairy cows
and calves of 500 ng/g are more than three times as high as the zearalenone contamination in
the current study.
4.2. Performance and carcass composition
Live weight gain and dry matter intake, which were in normal ranges for bulls of this age
(DLG 1995; Meyer and Lebzien 2004; Meyer et al. 2003), were not influenced by the ergot
supplementation of the concentrate (Table III).
Dinnusson et al. (1971) recommended that any diet containing 600 mg ergot/kg or more
should be considered as potentially toxic, particularly for long term feeding. In the current
study ergot contents of 860 mg/kg DM of the diet were reached. Thus corresponding to
Dinnusson et al. toxic effects might already be caused.
But these authors also mentioned that beef type cattle seem to be more sensitive to ergot
feeding than cattle of crossbred types (Dinnusson et al. 1971), which might add to explain the
circumstance that the bulls in the present study did not show any effects on the ergot
supplementation of their feed.
Skarland and Thomas (1972) tested the effect of barley ergot on young heifers over a period
of nine months with a three week break at mid-term. During the first part of the experiment,
the daily intake of ergot ranged up to about 60 mg/kg LW, which corresponded to 150 μg
total alkaloids/kg LW. In the second part of the experiment, the ergot intake ranged up to 40
mg/kg LW, which was approximately 100 μg total alkaloids/kg LW. These dosages, which
were more than 10-fold higher than in the current study, were followed by a significant
decrease in feed intake and a reduction of LWG (Skarland and Thomas 1972).
In the year 1989, Ross et al. published a study with 8 Hereford steers. Half of them were fed
with grain containing 5 g ergot/kg over a period of 21 days. With a feed intake of 3.17 kg/day,
and a live weight of 250 kg at the beginning of the experiment, this would correspond to a
dose of 63.4 mg ergot/kg LW which was four times higher than in the current study. Ross et
al. (1989) reported that the 4 Hereford steers fed with ergot showed a significant drop in feed
intake and loss of weight. But nothing is stated about the alkaloid content of the ergot used in
their experiments.
In 1996, a review on ergot contamination in feedstuff for cattle was published by Landes. This
literature contained a table with reports on field outbreaks and stated that 0.1% ergot in the
total diet of cattle might cause toxic effects. The results of the current study correspond to a
29
Paper I
maximum dose of 0.23% ergot in the concentrate which, related to the total diet and not on
dry matter basis, is approximately 0.03% ergot.
In 2003, ergot research had progressed and studies concerning ergot intake and its effects on
livestock mostly included an alkaloid analysis. Al-Tamimi et al. (2003) tested the
thermoregulatory response of dairy cows fed ergotized barley. A ten-day administration of 10
μg ergopeptine alkaloids/kg LW (only ergopeptine alkaloids have been analysed) to Holstein
cows did not cause significant effects on DMI. But due to the observed marginal symptoms of
ergot toxicosis reflected in body temperature and milk production, the author suggested this
alkaloid dose as a level for minimal induction of the ergot toxicity response (Al-Tamimi et al.
2003). Considering that Al-Tamimi et al. (2003) used ergotized barley, which probably also
contained ergometrine and its isomere, the only alkaloids analysed in the current study which
do not belong to the ergopeptine group, the total alkaloid administration might have been a
little higher. The current study revealed a percentage of 93 % ergopeptine alkaloids of the
total alkaloid amount (Table I).
As the average total daily alkaloid exposure in the current experiment did not exceed 9 µg per
kg LW (8.4 μg ergopeptine alkaloids/kg LW), it might have been low enough to be
completely tolerated by the animals, but following Al-Tamimi et al. (2003) this dose would
then be very close to the no-effect level.
The calculated ME-to-gain ratio is lower compared with the data of GfE (1995), but according
to new data which consider the currently used genotypes of fattening bulls (Meyer and
Lebzien 2004; Meyer et al. 2003), the recommendations of the GfE need to be adapted. The
ME-to-gain ratio was not influenced by the ergot supplementation of the concentrate.
The carcass composition also was not different between the feeding goups (Table IV).
Dänicke et al. (2002) found some effects on carcass composition in a study with 56 Holstein-
Friesian bulls after an exposure to mycotoxins (DON and ZON). Similar effects are described
in swine and chicken exposed to ergot (Mainka 2005b). Mean heart and spleen weights and
carcass dressing percentage were significantly higher in a group of growing-finishing pigs fed
with a total daily amount of alkaloids up to 130 μg/kg LW (Mainka et al. 2005a).
However, in the current study organ weights and carcass compositions of the bulls fed with
ergotized concentrate were completely inconspicuous which might be due to the low doses of
ergot administrated to the bulls.
30
Paper I
The pathological findings described above (uroliths, abscess, etc.) could not be ascribed to the
dietary treatment as they were almost equally distributed to the three groups.
4.4. Clinical chemical serum parameters
The metabolization of the ergot alkaloids mainly takes place in the liver (Cheeke 2006;
Moubarak and Rosenkrans 2000). Thus the liver might also be expected to be a target organ
for the toxic effects of the alkaloids. It could be anticipated that some useful information on
ergot toxicosis can be provided by analyzing specific liver enzyme activities in the serum
(Reichling and Kaplan 1988).
Studies of hepatic enzyme analyses after ergot administration to cattle are scarce.
Some authors published relations between the prolonged consumption of endophyte-infected
tall fescue and an increase of total bilirubin (Oliver et al. 2000) or of CK (Schultze et al.
1999) in cattle, but articles about experimental administration of ergot from Claviceps
purpurea are lacking.
The current study did not reveal any correlations between ergot supplementation of the
concentrate and the liver enzyme values in the serum.
4.3. Carry over
Carry over has been described for endophytic produced ergot alkaloids (Cunningham et al.
1944; Realini et al. 2006). But no experiments have been puplished so far concerning carry
over research of ergot alkaloids of Claviceps purpurea in cattle. In the present study, in none
of the samples taken, alkaloids were detected.
However, bulls had fasted for at least 7 hours. The reason for that was that fasting is very
common under practical conditions because it eases the slaughtering procedure greatly. This
experiment was meant to be as near to practice as possible.
In earlier studies, bile was reported to be the most important route of alkaloid elimination for
ergotamine (Nimmerfall and Rosenthaler 1976). Recent research showed that approximately
94% of endophyte alkaloids are excreted in the urine (Stuedemann et al. 1998; Hill et al.
2000). The excretion path seems to be determined by the molecular weight (Nimmerfall and
Rosenthaler 1976). Since ergotamine was the most prominent alkaloid of the ergot used in the
present study, the bile samples were of great interest. But no alkaloids could be detected,
neither in bile nor in urine, which might be due to the fact that the alkaloid administration was
quite low. Other explanations might be, that the alkaloids might have been metabolized prior
31
Paper I
to the elimination by bile or urine and thus were not detectable by the method used, or that
most of the alkaloids were already excreted after 7 hours of fasting.
5. Conclusions
In the current study, no effects on performance or slaughtering parameters caused by the ergot
supplementation of the concentrate were detectable. Negative effects on health and
performance, as reported in the literature for higher doses of ergot (Landes 1996), could not
be evoked by feeding diets containing up to 2.25 g ergot or 421 µg alkaloids/kg DM.
To establish a no-effect level for cattle, further experiments with higher levels of ergot
administration have to be arranged. It is also necessary to consider other age groups, since
especially young animals are known to be sensitive to toxic impacts (Burfening 1973) and
also dairy cows are assumed to be more sensitive than fattening bulls (Clar 1995). Even the
breed is reported to be of importance as bulls of the beef-type show stronger reactions than
crossbreeds (Dinnusson et al. 1971).
In addition, further studies dealing with the effects of ergotized feed should rather be based on
alkaloid concentration than on ergot content because of the highly variable percentage of
alkaloids in different ergot sclerotia (Filipov et al. 2000; Young 1981b).
Acknowledgement
The assistance of the co-workers of the Institute of Animal Nutrition of the Federal
Agricultural Research Centre (FAL) Braunschweig for animal care and performing of
analyses is gratefully acknowledged.
32
Paper I
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GfE. 2001. Empfehlungen zur Energie- und Nährstoffversorgung der Milchkühe und Aufzuchtrinder 8. DLG-Verlag, Frankfurt am Main.
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Ilha MRS, Loretti AP, Barros CSL. 2003. Hyperthermic syndrome in dairy cattle associated with consumption of ergots of Claviceps purpurea in southern Brazil. Vet Hum Toxicol 45:140-145.
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Mainka S, Dänicke S, Böhme H, Ueberschär KH, Liebert F. 2006b. On the alkaloid content of ergot (Claviceps purpurea). in preparation
Mainka S, Dänicke S, Böhme H, Ueberschär KH, Polten S, Hüther L. 2005a. The influence of ergot-contaminated feed on growth and slaughtering performance, nutrient digestibility and carry over of ergot alkaloids in growing-finishing pigs. Arch Anim Nutr 59:377-395.
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Young JC, Chen Z. 1982. Variability in the content and composition of alkaloid found in canadian ergot. 3. Triticale and barley. J Environ Sci Heal 17:93-107.
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36
Paper II
Paper II
Effects of different levels of ergot in concentrate on the health and
performance of male calves
Barbara Schumann, S. Dänicke, Sonja Hübner, K.-H. Ueberschär , U. Meyer
Institute of Animal Nutrition, Federal Agriculture Research Centre (FAL), Bundesallee 50,
38116 Braunschweig
Mycotoxin Research
2007
in press
Printed with kind permission of
The Society for Mycotoxin Research
37
Paper II
Abstract A number of studies dealing with the effects of ergot and ergot alkaloids on the health and
performance of poultry and pigs were reported in the past, but only a few studies and field
reports are available for ruminants. Therefore, a dose-response study was carried out with
calves since young animals are considered to be especially sensitive to ergot.
A total of 35 male Holstein calves were randomly assigned to three feeding groups after one
month of feeding milk replacer. The mean initial live weight of the calves was 49.4 ± 5.7 kg.
One control group was fed an ergot-free concentrate (n = 12), one group an ergot proportion
of 1000 mg/kg in the concentrate (n = 11), and another group was fed a concentrate
containing 5000 mg/kg ergot (n = 12). Hay, grass silage and water were available for ad
libitum consumption, whereas the daily concentrate portion was restricted to 2 kg. Live
weight, health parameters and feed intake were monitored over the experimental period of 84
days. In addition, blood samples were taken from the Vena jugularis at the beginning and at
the end of the experiment and analysed for ergot alkaloids and liver parameters.
Total dry matter intake, live weight gain and feed-to-gain ratio were not significantly
influenced by increasing ergot proportions when the whole experimental period was
considered, although there was a trend for an ergot-related decrease in concentrate intake
during the first 6 weeks of the experiment. After this period of time, it seemed that calves got
used to the presence of ergot in the concentrate and were able to adjust their intake to the level
of the control group. Moreover, health and liver parameters, such as total bilirubin, aspartate
aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase and creatine
kinase in the serum were not significantly influenced by dietary treatments. Concentrations of
the individual ergot alkaloids in serum were lower than the detection limits of the applied
HPLC-method.
In conclusion, it can be assumed that an ergot contamination of the concentrate up to 5000
mg/kg resulted in a transient depression of concentrate intake by the calves. However, no
significant effects on health and performance could be detected when the entire test period of
84 days was considered.
Keywords: Ergot alkaloids, calves, liver parameters, feed intake, growth
38
Paper II
Introduction Ergot refers to the solidified mycelium of the parasitic fungus Claviceps purpurea invading
mainly grasses. Ergot develops at the site of grain development instead of the host grains and
is considered as the outlasting form of the fungus. The primary toxic constituents of ergot are
the alkaloids which may occur in concentrations varying between 900 and 2100 mg/kg ergot
(34). Up until now, farm animals have been protected from these ergot alkaloids with only a
weight-based upper limit of 1000 mg ergot per kg unground cereal grains (Council Directive
2002/32/EC of 7 May 2002) despite the variation in the toxic alkaloids mentioned. Therefore,
this upper limit was considered to be problematic when the literature was evaluated for
ruminants (17), whereas Gedek (9) recommended not exceeding 2500 mg ergot/kg of the
daily ration for ruminants. Thus the situation in cattle is not clear, and information on dose-
response relationships with regard to ergot alkaloids is needed for an improved risk evaluation
of ergot for this species.
Therefore the aim of the present study was to examine the effects of increasing proportions of
ergot with a defined alkaloid concentration and pattern in concentrates for calves on their
health and performance as young animals generally are considered to be sensitive to
mycotoxins.
Material and methods Experimental design
Three doses of ergot were prepared by mixing increasing proportions of ergoty rye (~45 %
ergot) into concentrates for calves to give ergot concentrations of 0, 1000 and 5000 mg/kg
concentrate (Tables 1 and 2) and fed to 3 groups of calves designated as Control, Ergot 1 and
Ergot 2 group, respectively.
Table 1. Experimental design Group Ergot (mg/kg concentrate) Calves/group
Control 0 12 Ergot 1 1000 11 Ergot 2 5000 12
39
Paper II
Table 2. Composition of the concentrates (g/kg as fed) Group Control Ergot 1 Ergot 2 Components: Soy bean meal 300 300 300 Barley 180 180 180 Wheat 170 167.8 158.9 Oats 305 305 305 Ergoty rye1) 0 2.2 11.1 Soy bean oil 15 15 15 Calcium carbonate 10 10 10 Mineral and vitamin premix 2) 20 20 20
1) 45% ergot 2) provided per kg concentrate: 3.2 g calcium, 2 g sodium, 1.6 g phosphor, 0.6 g magnesium, 14 000 IU vitamin A, 1 600 IU vitamin D3, 8 mg vitamin E, 20 mg iron, 16 mg copper, 80 mg manganese, 100 mg zinc, 1 mg iodine, 1 mg selenium, 0.6 mg cobalt Table 3. Dry matter in g/kg, nutrient composition in g/kg DM, energy concentration of the concentrates in MJ ME/kg DM, and alkaloid pattern of ergot and rye in µg/kg (Percentage of total alkaloids in brackets) sorted out from the ergoty rye and of concentrates containing increasing proportions of this ergoty rye
Group Control Ergot 1 Ergot 2
Ergot Rye
Nutrients and energy: Dry matter 890 887 888 928 884 Crude protein 215 210 217 216 112 Crude fat 39 41 43 354 19 Crude fibre 71 71 71 209 27 ME 12.0 12.0 12.1 Alkaloids: Ergometrine 5 (12) 34 (9) 94 (6) 52667 (8) 125 (20) Ergometrinine 1 (3) 10 (3) 44 (3) 7882 (1) 18 (3) Ergotamine 11 (26) 93 (25) 401 (27) 160244 (25) 81 (13) Ergotaminine 5 (12) 43 (11) 183 (12) 63052 (10) 38 (6) Ergocornine 2 (5) 21 (6) 78 (5) 32063 (5) 13 (2) Ergocorninine 1 (2) 9 (2) 33 (2) 17984 (3) 6 (1) Ergocryptine 2 (4) 20 (5) 72 (5) 39608 (6) 35 (6) Ergocryptinine 2 (4) 17 (5) 58 (4) 27513 (4) 25 (4) Ergocristine 5 (12) 50 (13) 207 (14) 97765 (15) 153 (25) Ergocristinine 2 (6) 22 (6) 84 (6) 29903 (5) 77 (12) Ergosine 4 (11) 41 (11) 173 (12) 84778 (13) 43 (7) Ergosinine 2 (4) 17 (4) 70 (5) 19890 (3) 8 (1) Total alkaloids 43 (100) 378 (100) 1496 (100) 633350 (100) 622 (100)
40
Paper II
Growth experiment and procedures Altogether 35 male calves of the breed Holstein Friesian from the milk cattle herd of the FAL
Braunschweig were used. After the birth (seasonal calving from October till March) the
calves stayed with their mother for 1 day. After that they were kept in small stables (87 x 175
cm) on straw and received six litres of colostrum per day in two equal portions in the morning
and afternoon for seven days. Thereafter calves were kept in group boxes with straw bedding
and were fed with a commercial milk replacer (skim milk powder 41.9 %, whey powder 35.9
%, vegetable oil, refined and homogenised 13.4 %) for another 3 weeks using automatic self
feeders. Each calf started individually with the test depending on its day of birth. The test
commenced with the transfer of the calf into the experimental unit and with the change to the
experimental diet which consisted of roughage (grass silage, hay) and concentrates (Tables 2
and 3).
The experimental unit comprised 3 group boxes (5.20 x 7.40 m). Each box was divided into
an area with straw litter and an area close to the feeding equipment consisting of a slatted
floor. The concentrate, containing increasing proportions of ergot, was offered by a self
feeding station which was combined with an animal weighing machine (Type AWS HF 2ST,
firm: Insentec, Marknesse, Netherlands). Animals were identified individually by using ear
transponders. The daily concentrate allowance was restricted to 2 kg in order to stimulate
roughage intake by the calves. Grass silage, hay and water were offered for ad libitum
consumption through transponder-sensitive automatic roughage feeders (Type: RIC HF 2PL,
firm: Insentec, Marknesse, Netherlands).
On the day before the experiment started, and on the 84th day of the test period, a blood
sample was taken from the Vena jugularis of the calves (approximately 10 ml into pipes with
heparin for the plasma extraction, and 10 ml into small serum pipes), centrifuged
approximately 1-2 hours later at 3000 g and 15°C for 10 min and then frozen.
Analyses
Ergot alkaloids in ergot, feedstuffs and serum
Ergot and feedstuffs were analyzed for ergot alkaloids (ergometrine, ergocornine, ergotamine,
α-ergocryptine, ergosine, ergocristine and their –inine isomers) by HPLC based on the
method of Wolff al. (36).
Approximately 3 g of each sample were mixed with 100 ml extraction fluid (50 ml
dichlormethane + 25 ml ethylacetate + 5 ml methanol + 1 ml ammonium hydroxide (25%))
and the next day, after centrifugation, an aliquot was taken and evaporated to dryness. The
next step was to dissolve the residue in 2 ml toluene / methanol (49 + 1) and solubilize it
41
Paper II
using ultrasound. The fluid was mixed with 9 ml i-hexane and added to a 3 g Extrelut®
column (Merck, Darmstadt, Germany), which was acidified with 5 ml 2 % aquaeous tartaric
acid. For the following elution, 0.5 ml toluol / methanol + 4.5 ml i-hexane and 20 ml di-
isopropylether / i-hexane (in equal shares) were used. For 1-2 min air was sucked through to
dry the column before the alkaloids were assimilated in 25 % ammonium gas which was
detected by colour reaction of phenolphthalein. The alkaloids were eluted with 25 ml
dichlormethane which was evaporated to dryness at 35°C and carefully blown off with
nitrogen. Finally the residue was filled up to a definite volume of 20 μl and was injected in the
HPLC-apparatus. The HPLC consists of an isocratic pumping system with a 250 x 4 mm
column (5 μm, C 18 Gravity, Macherey-Nagel, Düren, Germany), operates at 44°C and is
connected with a fluorescence detector (325 nm excitation / 418 nm emission wavelength).
The serum samples were analysed with the same method.
The detection limit was 5 ng/g, except for ergometrine where it was 10 ng/g. Ergometrine,
ergotamine, ergocristine, ergocornine and ergocryptine are referred to as “key alkaloids”
since standards are commercially available for their identification. Ergosine and its isomer
were identified by their retention time (1).
Further mycotoxins
Contaminations with zearalenone (ZON) were analyzed according to a modified VDLUFA
(Verband Deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten) method
according to Ueberschär et al. (32). Contaminations with deoxynivalenol (DON) were
analysed by HPLC with DAD (diode array detection) after cleaning-up with IAC
(immunoaffinity column, DONprepTM, R-Biopharm AG, Darmstadt, Germany) according to
the slightly modified procedure of the manufacturer.
Clinical-chemical serum parameters
A 1 ml serum sample was sent to the laboratory of the Clinic for Cattle of the Veterinary
School Hanover. It was analysed for five liver parameters with a fully automatic apparatus
(Cobas-Mira, Fa. Hoffmann-La Roche & Co. AG Diagnostika, Basel). Photometric standard
procedures were used for gamma-glutamyl transpeptidase (International Federation of
Clinical Chemistry) and aspartate aminotransferase, glutamate dehydrogenase and creatine
kinase (German Federation of Clinical Chemistry). Total bilirubin was tested according to the
method of Jendrassik & Gróf.
42
Paper II
Crude nutrients
Pooled samples taken from the ergoty rye, grass silage, hay and concentrate were examined
for dry matter (DM), crude ash, crude protein, crude fat and crude fibre according to the
methods of the VDLUFA (21).
Fatty acids
Fatty acids were extracted in a cold process developed by Nürnberg et al. (22) and then
methylised according to the method of Schulte and Weber (26). Afterwards the samples were
detected by flame ionisation after gas chromatography.
Calculations and statistics
Performance data
Feed intake, live weight gain and feed-to-gain ratio were evaluated according to a one-way
factorial design of analysis of variance (ANOVA) corrected for initial live weight using the
following model:
yij = μ + ai + βy.x⋅(xij -⎯x..) + eij
where yij = tested parameter of the calf "j" fed diet type "i"; µ = overall mean; ai = effect of
diet (i.e., Control, Ergot 1 and Ergot 2); βy.x = regression coefficient of y on x; xij = co-variable;
⎯x.. = mean value for x; eij = error term.
Clinical serum parameters
Variance of these data was analyzed by using the restricted maximum likelihood (REML)
method for random effect variances and the Kenward-Roger-method for calculation of degrees
of freedom implemented in the SAS-software package (SAS Institute Inc. 2003, Version 9.1,
procedure "mixed") according to the following model:
yijk = μ + ai + bj + eijk
where yijk = tested parameter of the calf "k" fed diet type "i"; µ = overall mean; ai = effect of
diet (i.e., Control, Ergot 1 and Ergot 2); bj = random effect of animal to account for repeated
measurements within the same individual; eijk = error term.
43
Paper II
Results Chemical composition of feedstuffs and ergot
The concentrates were analysed monthly over the whole experimental period. The average
total alkaloid concentration of the Control, Ergot 1 and Ergot 2 concentrate was 43 µg/kg (0-
157 μg/kg), 378 µg/kg (310-567 μg/kg), and 1496 µg/kg (1302-2189 μg/kg), respectively.
The mean total alkaloid concentrations of the ergot sorted out from the ergoty rye and of the
separated rye were 633 mg/kg and 622 µg/kg, respectively. Ergotamine, ergocristine and
ergosine were the most prominent alkaloids in the ergot and in the concentrates. The
proportions of ergotamine and ergocristine varied between 25-27 % and 12-15 %,
respectively (Table 3).
Alkaloid contents in hay and grass silage were lower than the indicated detection limits.
The nutrient compositions of the concentrates and of the roughage used in the current study
are shown in the Tables 3 and 4. The crude protein and fat content of the ergot sorted out from
the ergoty rye was 216 g/kg DM and 354 g/kg DM, respectively (Table 3). The proportions of
fatty acids amounted to 31.9 % palmitic acid, 21.7 % oleic acid, 19.2 % linoleic acid, 8.2 %
stearic acid, 7.9 % ricinoleic acid and 11.1 % residual fatty acids.
Table 4. Nutrient composition of the roughage in g/kg DM and energy concentration in MJ ME/kg DM Hay Gras silage Dry matter (g/kg) 803 405 Crude protein 125 147 Crude fat 21 33 Crude fibre 300 296 ME 9,6 9,9
The concentrations of ZON and β - zearalenol in all feedstuffs were lower than the detection
limit of 1 ng/g and concentrations of α – zearalenol lower than 4 ng/g respectively. DON
concentrations analysed on a basis of 88 % DM were 0.053 µg/g in the grass silage; 0.034
µg/g in the concentrate of the Ergot 1 group; 0.033 µg/g in that of the Ergot 2 group, and
0.032 µg/g were detected in the concentrate of the control group. DON concentrations in the
hay used in the current study were lower than the detection limit of 0.03 µg/g.
Performance
Four calves died during the first third of the experiment. One had to be euthanized due to a
fracture, while the other three died for unknown reasons. A relationship to dietary treatments
could not be deduced since mortality was evenly distributed among the three feeding groups.
44
Paper II
Calves consumed approximately 0.6 kg concentrate DM per day in the first week and
increased this amount in an exponential related fashion almost up to the daily allowance of 2
kg concentrate per day at Week 7 of the experiment. Thereafter, the daily maximum
concentrate amount was consumed more or less completely for the remaining 5 weeks, which
can be deduced from the decrease in standard deviation (Figure 1). Concentrate intake was not
significantly influenced by increasing ergot proportions in the concentrate, although in the
first half of the experiment the consumption was 2.6 % and 3.4 % lower in calves of groups
Ergot 1 and 2, respectively (Figure 1, Table 5).
0.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 12
Con
cent
rate
inta
ke(k
g D
M/d
ay)
Experimental week
0.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 12
Con
cent
rate
inta
ke(k
g D
M/d
ay)
0.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 120.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 12
Con
cent
rate
inta
ke(k
g D
M/d
ay)
Experimental week
Figure 1. Concentrate dry matter intake during the 12 experimental weeks (●= control group, ■= ergot 1, ▲=ergot 2) Only in the third week of the study was there a trend for a decreased concentrate intake
(p=0.076). The roughage intake, which comprised approximately 15 % hay and 85 % grass
silage on average, was characterized by an initial delay or slight increase until Week 6 of the
experiment, and thereafter increased more steeply in a linearly related manner (Figure 2).
45
Paper II
0.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 12
Rou
ghag
ein
take
(kg
DM
/day
)
Experimental week
0.0
0.5
1.0
1.5
2.0
2.5
1 2 3 4 5 6 7 8 9 10 11 12
Rou
ghag
ein
take
(kg
DM
/day
)
Experimental week
Figure 2. Roughage dry matter intake during the 12 experimental weeks (●= control group, ■= ergot 1, ▲=ergot 2) Table 5. Daily intake of concentrate and roughage (kg dry matter)
Concentrate intake Roughage intake Ergot (mg/kg concentrate) d 1-28 d 29-56 d 57-84 d 1-84 d 1-28 d 29-56 d 57-84 d 1-84
0 1.153 1.655 1.779 1.516 0.219 0.651 1.309 0.708 1000 1.099 1.689 1.773 1.541 0.244 0.596 1.363 0.761 5000 1.028 1.637 1.751 1.464 0.240 0.683 1.485 0.794
ANOVA (probabilities) Ergot 0.535 0.569 0.502 0.411 0.897 0.430 0.217 0.176 Linear 0.269 0.699 0.263 0.370 0.699 0.623 0.089 0.066 Quadratic 0.941 0.326 0.734 0.314 0.799 0.232 0.715 0.812
PSEM 0.079 0.033 0.018 0.071 0.039 0.045 0.071 0.057 Table 6. Daily live weight gain (kg) and feed to gain ratio (kg dry matter/kg live weight gain)
Live weight gain Feed to gain ratio Ergot (mg/kg concentrate) d 1-28 d 29-56 d 57-84 d 1-84 d 1-28 d 29-56 d 57-84 d 1-84
0 0.597 0.936 0.992 0.828 2.578 2.519 3.183 2.700 1000 0.596 0.901 1.057 0.872 2.326 2.582 2.982 2.512 5000 0.548 0.917 0.988 0.811 2.528 2.583 3.304 2.704
ANOVA (probabilities) Ergot 0.821 0.877 0.568 0.435 0.787 0.929 0.189 0.431 Linear 0.575 0.780 0.957 0.707 0.885 0.737 0.464 0.980 Quadratic 0.787 0.671 0.292 0.215 0.502 0.859 0.094 0.199
PSEM 0.062 0.046 0.048 0.058 1.262 0.698 0.602 0.545
46
Paper II
Live weight development showed an exponential increase from about 50 to 110 kg, and was
obviously influenced by the initial live weight (Figure 3). Therefore, the initial live weight
was considered as a co-variable in evaluating the variance of the performance parameters
(Table 5 and 6). The mean live weight gain over all treatments and the entire experimental
period amounted to 0.84 kg per day. A significant effect of dietary treatments on this
parameter could not be detected in any of the experimental periods (Table 6). Similarly, feed
to gain ratio, which was approximately 2.7 kg DM/kg live weight gain over the whole
experimental period and over all treatments, remained unaffected by the ergot presence in the
concentrates (Table 6).
Live
wei
ght(
kg
)
30
50
70
90
110
130
150
0 1 2 3 4 5 6 7 8 9 10 11 12Experimental week
Live
wei
ght(
kg
)
30
50
70
90
110
130
150
0 1 2 3 4 5 6 7 8 9 10 11 12Experimental week
Figure 3. Live weight during the 12 experimental weeks (● = control group, ■ = ergot 1, ▲ =ergot 2)
Animal health
Many of the calves experienced some health problems before the experiment commenced. A
post experimental evaluation of veterinary treatments revealed that six animals later assigned
to the control group, nine assigned to the Ergot 1 group and seven to the Ergot 2 group needed
to be treated by a veterinarian for different reasons, but mainly because of diarrhoea.
These problems disappeared to a large extent when the solid feed supply was increased.
After starting the experiment, none of the calves in the control group suffered from diarrhoea.
At Day 40 of the experiment, two animals of this group were treated because of inappetence
47
Paper II
and umbilical hernia. Two animals in the Ergot 1 group were treated because of diarrhoea and
bronchitis from the beginning of the experiment until Day 13. After that the animals were
clinically inconspicuous. Five calves of the Ergot 2 group required a prolonged treatment for
bronchitis, diarrhoea and inappetence until Day 50 of the experiment (Figure 4).
0
1
2
3
4
5
0 10 20 30 40 50 60 70 80 9
Experimental time (d)
Num
ber o
f tre
atm
ents
0
Figure 4. Distribution of treatments during the test (● = control group, □ = ergot 1, ▲ = ergot 2)
Clinical chemical serum parameters
Aspartate aminotransferase (AST) varied between 36 and 147 U/l on the day before the
experiment started, and accordingly between 44 and 75 U/l at experimental day 84. It was
neither influenced by dietary treatments nor by the age of the calves (Figure 5). Activities of
the creatine kinase (CK) diversified in a range between 68 and 284 U/l at the beginning, and
67 and 296 U/l at the end of the experiment and remained unaffected irrespective of ergot
presence in the concentrate or age of calves. Total bilirubin was significantly influenced by
age (p<0.001) but independent of ergot feeding with values varying between 2.3 and 14.8
μmol/l before the first, and between 1.4 and 4.9 μmol/l at the last experimental day. The
activity range of the gamma-glutamyl transpeptidase (γ-GT) over the three feeding groups
diversified between 12 and 91 U/l the day before the experiment started and between 8 and
24 U/l at experimental day 84 with the effect of age being significant (p<0.001), and of ergot
being not significant. The glutamate dehydrogenase (GLDH) activity showed a variation
between 7.5 and 306 U/l at the beginning, and between 14.2 and 110 U/l at the end of the
experiment and was not significantly influenced by the mentioned factors.
Serum ergot alkaloid concentrations which were measured at the end of the experiment were
lower than the indicated detection limits.
48
Paper II
Figure 5. Mean AST- (a), CK- (b), γ-GT- (d) and GLDH values (e) in U/l, as well as total bilirubin (c) in µmol/l and their standard deviations at the beginning of the experiment (Week 5 of age, pictured in white) and at the end of the experiment (Month 4 of age, pictured in black) with an ergot addition of 0, 1000, respectively 5000 mg/ kg concentrate (no significant treatment effects; effect of age significant for γ-GT and total bilirubin)
Discussion Experimental design
The doses of 0, 1000 and 5000 mg ergot/kg concentrate were chosen to cover the weight
based upper limit of 1000 mg ergot per kg unground cereal grains (Council Directive
2002/32/EC of 7 May 2002) and also to exceed it. Following a previous experiment with
Ergot (mg/kg concentrate)
0
20
40
60
80
100
120
0 1000 5000
)A
ST (U
/l
Ergot (mg/kg concentrate)
0
50
100
150
200
250
0 1000 5000
CK
(U/l)
Ergot (mg/kg concentrate)
0
2
4
6
8
10
12
0 1000 5000
ubi
mTo
tal b
ilir
n(µ
ol/l)
Ergot (mg/kg concentrate)
0
10
20
30
40
50
60
70
0 1000 5000γ-
GT
(U/l)
Ergot (mg/kg concentrate)
0
50
100
150
200
250
0
20
40
60
80
100
120
Ergot (mg/kg concentrate)0 1000 5000
)A
ST (U
/l
0 1000 5000
CK
(U/l)
Ergot (mg/kg concentrate)
0
10
20
30
40
50
60
70
0
2
4
6
8
10
12
ol/l)
Ergot (mg/kg concentrate)0 1000 5000
ubi
mn
(µTo
tal b
ilir
0 1000 5000γ-
GT
(U/l)
Ergot (mg/kg concentrate)
Ergot (mg/kg concentrate)
0
20
40
60
80
100
120
140
160
0 1000 5000
GLD
H (U
/l)
Ergot (mg/kg concentrate)
0
20
40
60
80
100
120
140
160
0 1000 5000
GLD
H (U
/l)
49
Paper II
fattening bulls, in which no effects on live weight gain (LWG) or dry matter intake (DMI)
could be detected (28), the ergot supplementation rate was doubled and an age group
considered as more sensitive was chosen in order to provoke potential ergot effects.
Chemical composition of feedstuffs and ergot
The alkaloid content of the three concentrates showed some variation over the course of the
experiment. The coefficient of variation in total alkaloid concentration amounted to 152 % in
the concentrate of the Control group, and to 16 % of the Ergot 1 as well as in the Ergot 2
group. However, this variation was not tendential, which is in contrast to earlier findings
where a continuous decrease in alkaloid concentration was reported during prolonged storage
times (24, 35). The variation observed in the present study might be explained in part by the
error of sampling. Depending on which particular sclerotia are in the sampled concentrate, the
content and the cluster of the alkaloids is considered never to be uniform (37-39). Filipov et
al. (7) found that the total alkaloid content was highly variable between sclerotia from the
same ear, crop area or region. The individual alkaloid composition was uniform throughout a
single sclerotium or in different sclerotia from the same ear, somewhat uniform for averages
in different fields throughout a region, but highly variable from ear to ear in a given field (38).
The alkaloid concentration in ergot from Germany was reported to vary between 818 and
1635 mg/kg ergot DM sampled from the harvest 2004 (19); from 42 to 343 mg/kg in 2003
(19) and in previous years between 900 and 2100 mg/kg (34). Ergot alkaloids from Canadian
rye ranged between 100 and 4500 mg/kg (38).The ergot which was used in this experiment
contained a moderate alkaloid concentration of approximately 633 mg/kg and fits into the
discussed range of alkaloid variation.
As the concentrate intake was restricted to 2 kg per day, this would correspond to an ergot
intake of 10 g per calf per day and to an alkaloid intake of 4378 µg per calf per day if the
maximum alkaloid concentration of 2189 µg/kg of the Ergot 2 concentrate is considered
(worst case scenario). The roughage was also analysed and the alkaloid content was less than
the respective detection limits. This level must be kept in mind since it may become critical
for ruminants (as noted by the EFSA (6)) if they are additionally exposed to ergot from
roughage containing grass infected with various Claviceptaceae.
As the concentrations of DON in the feedstuffs all were very low, and also the distribution
among the three different concentrates was almost equal, effects caused by DON are of little
importance.
50
Paper II
Performance
LWG during the first three weeks of the experiment increases only slightly (Figure 3). This
might be due to the change from milk to solid feed which needed some adaptation time. But
the proportion of concentrate to roughage at the beginning of the experiment was also quite
high and might have caused some digestion problems such as ruminal acidosis or
fermentative diarrhoea which again may have negatively influenced LWG.
The slight differences in concentrate DMI (Table 5, Figure 1) by calves of the three
experimental groups over the first six weeks of the experiment were not the result of a
variation in nutrient composition of the concentrates as they were proven to be isoenergetic
and isonitrogenic (Table 3), but might be due to the ergot presence. As already described for
swine, ergot-contaminated feed may reduce DMI and LWG (19). So it may be assumed that
the temporary decrease in total DMI of group Ergot 2, which only resulted from a decreased
intake of the concentrate (Figures 1 and 2), is an effect of the ergot supplementation. Since the
DMI of the three groups was comparable from the 6th week on, an adaptation to the ergot-
contaminated concentrate might have occurred. Similar findings were reported for pigs and
chicks (19, 25).
Calves might be especially sensitive to ergot since the formation of their forestomach system
is not yet established. The vasoconstrictive effects of the alkaloids might delay forestomach
development and consequently reduce the rumen activity. However, since roughage intake did
not differ between the groups it seems to be more likely that the calves developed a temporary
taste aversion to the ergot supplemented concentrate.
Furthermore, it needs to be considered in interpreting the ergot effects on feed intake that the
concentrate portion was restricted to a maximum of 2 kg per calf and day. All groups
approached to this limit up to the 6th experimental week and consumed this amount nearly
completely for the remaining experimental period. Therefore, the effect of ergot on feed
intake could only be detected at the beginning of the experiment where the amount of offered
concentrate was higher than calves could voluntarily consume, i.e., ad libitum or semi-ad
libitum feeding conditions.
The roughage dry matter intake shows a steeper increase from the 6th week on, which
coincided with the period when all calves consumed their concentrate portion completely.
This means that calves might have been forced to consume more roughage as they were not
able to increase their concentrate intake.
51
Paper II
Animal health
In proportion to the roughage intake, the concentrate intake was favoured by the experimental
design. These feeding conditions may promote ruminal acidosis and fermentative diarrhea and
might have been one reason for health problems in this experiment. Furthermore, it has to be
kept in mind that the number of animals used in this study is not very high and therefore the
results show only a possible linkage. Further experiments are needed to obtain concrete
conclusions.
However, after the change to solid experimental feed the control group had no further
diarrhoea problems. The two veterinary treatments around Day 40 are probably not related to
the experimental diet. An umbilical hernia is heritable. Inappetence might have many causes,
but since only one calf was affected and only one treatment was necessary, the significance is
arguable.
One calf in the Ergot 1 group needed some more time to cope with diarrhoea, but also the
anamnesis of this group has to be kept in mind, as it was the one with the most health
problems before the start of the experiment.
In the Ergot 2 group there were three calves attracting attention and one of them had to be
treated until Day 50. It might be assumed that there could be a relation to the higher ergot
level as ergot is reported to influence the immune system by suppressing the induction of
antibody formation by the B lymphocytes (30) and by stimulating the natural killer cells with
its alkaloid glycosides (16).
Loken (18) found that three of four lambs dosed for two months with 120 to 750 mg sklerotia
per kg body weight (4.2 g and 26.3 g in total) reacted after 2 to 6 days with inappetence and
diarrhoea among others. Greatorix and Mantle (10, 11) and Cunningham (4) demonstrated
similar findings in experimentally induced ergotism. Moreover, severely inflamed
gastrointestinal lesions were described by Osborne [cited by (10)] in dead sheep after an
ergotism field outbreak.
Another factor might be the ricinoleic acid, a specific fatty acid of the ergot fat suggested to
irritate the intestine (8). As the ergot was comprised of 28 g ricinoleic acid per kg, the
calculated ricinoleic acid concentrations of the Ergot 1 and Ergot 2 concentrates amounted to
28 and 140 mg/kg, respectively, only considering the amount contained in the ergot. This
would correspond to a dosage of approximately 2.3-2.8 mg ricinoleic acid/kg body weight
(BW) when a concentrate intake of 1 kg at the beginning of the experiment, and of 2 kg at the
end, was related to the respective live weights of approximately 50 kg and 120 kg.
52
Paper II
Kelly et al. (14) showed that very high dosages of castor oil (60 ml) given orally to dogs
caused diarrhoea. 60 ml corresponded to approximately 3.6 g ricinoleic acid/kg BW,
assuming a content of 85% ricinoleic acid in the castor oil. It must be stressed that such a
ricinoleic acid exposure would hardly occur via ergot as the dosage in the cited report with
dogs was more than thousand-fold higher than that in the present experiment.
Ricinoleic acid is released from the matrix mainly in the small intestine and in humans an
amount of 10-30 g ricinoleic acid (about 290 mg/kg BW) resulted in an accumulation of water
by inhibiting the absorption of sodium and water from the intestine, which in turn leads to an
increasing influx of electrolytes and water to the lumen, and therefore to an increasing amount
of a softer stool (8, 29).
Again, compared to the literature, the dosage of ricinoleic acid in the present study was very
low, but it might be assumed that the higher ricinoleic acid content of the Ergot 2 concentrate
could have contributed to the incidence of diarrhoea in this group.
Clinical chemical serum parameters
As the metabolization of the ergot alkaloids takes place mainly in the liver (3), it might be
expected that the liver is also a target organ for the toxic effects of the alkaloids and that
analyzing enzyme activities in serum specific for the liver can provide some useful
information on ergot toxicosis (23). Beside the possible toxic effects of the ergot alkaloids on
the liver, the age of cattle was reported to be a main factor causing variance in liver enzymes
(2, 13, 27). Therefore, the variance in liver enzymes was analyzed by considering both the age
of the calves and the ergot exposure in the present experiment. The AST values determined in
our study were generally higher than the reference values by Schulte-Langforth et al. (27),
which are given as lower than 18 U/L at an age of 3 weeks, and lower than 30 U/L at 18
weeks of age. Berry et al. (2) published a value of lower than 30 U/L at an age period between
2 and 4 months. On the other hand, the values of the present study fall into the ranges given
by Walser et al. (33), Kraft and Dürr (15) and by the Clinic for Cattle of the Veterinary
School Hanover. However, the latter reference values are not specifically recommended for
application to calves. Moreover, the AST values measured in the serum of calves in our study
were independent of age, which is in contrast to the discussed increases in AST with age.
Thus it might be concluded that AST is influenced by other factors besides a non-reproducible
age effect and that it seems not to be a useful indicator for ergot alkaloid effects.
The γ-GT values and total bilirubin are in the reference ranges as reported in the cited
literature. Schulte-Langforth et al. (27) described in his study an age dependent decrease
53
Paper II
between the 3rd week of age and the 18th week of age. In our experiment both parameters
decreased significantly with age, too. However, this decrease was on the same order of
magnitude for all groups, which resulted in a non-significant ergot effect. In interpreting the
age effect, it needs to be considered that the first blood sample coincided not only with the
beginning of the experiment, which implied a feed change, but also with the transfer of the
calves to the experimental unit. Thus the apparent effects of age on these parameters might be
biased to a greater or lesser extent by the above-mentioned environmental changes.
GLDH values were generally higher than published reference values (lower than 9 U/L
Schulte-Langforth, lower than 20 U/L Berry). However, since this was already the case before
the beginning of the experiment, no conclusions should be made on the influence of feeding
conditions.
CK activity in serum might be used as an indicator for a loss of muscle cell integrity and its
increase could result from a disturbance in muscle perfusion. This in turn could be mediated
by the vasoconstrictive effects of the ergot alkaloids. However, supplementing the
concentrates with increasing proportions of ergot did not result in such supposed effects. The
reference values for the CK activity are also lower than those measured in the present
experiment (lower than 33 U/L Schulte-Langforth, lower than 95 U/L Berry). From the
perspective that CK determination is considered to be strongly laboratory dependent (15), the
discussed disagreement with the reported reference values should not be stressed excessively.
The focus should be on comparisons between dietary treatments within the present
experiment. Assuming that the latter statement applies also to the other discussed parameters,
it can be concluded that reference values are not always useful in interpreting dietary effects.
The concentrations of ergot alkaloids in the serum of calves were all lower than the indicated
detection limits. Ergot alkaloids are quickly metabolized within the body. For example,
ergotamine in blood, liver and kidney was found to climax approximately 2 hours after
feeding, followed by slow decreases (12). Calves in the present study had free access to their
concentrate until the maximum amount of 2 kg per day was reached. Thus, it can not be said
exactly how many hours they fasted before the blood samples were taken. Therefore, it might
be possible that most of the metabolites were already excreted. Stuedemann et al. (31)
reported rapid urinary alkaloid excretion in steers. 67 % were excreted via the urine after 24
hours. Moubarak et al. (20) similarly observed rapid disappearance of ergotamine from the
blood administered by intravenous injection to steers (14 µg/kg BW). Serum concentrations
54
Paper II
of ergosine and ergine also were highest immediately after injection of these alkaloids (~25
ng/ml) and dropped significantly within 30 min (~4 ng/ml).
Similar to the present findings in calves, where the maximum alkaloid concentration in the
concentrate supplemented with 5000 mg ergot/kg amounted to 1.5 mg total alkaloids/kg,
Mainka et al. (19) were unable to detect ergot alkaloids in the serum of fattening pigs fed diets
containing 1000 and 10000 mg ergot/kg, which was equivalent to 0.6 and 4.66 mg total
alkaloids/kg, respectively.
Conclusions In conclusion it can be assumed that an ergot contamination of the concentrate up to 5000
mg/kg, which corresponded to a total alkaloid concentration of 1.5 mg/kg, resulted in a
transient depression of concentrate DMI by the calves. However, no significant effects on
performance and clinical chemical parameters could be detected when the entire test period of
84 days was considered. The tested range in ergot supplementation up to 5000 mg/kg of the
concentrate was not high enough to derive a no-effect level.
With regard to critical ergot concentration in diets for farm animals, Gedek (9) recommended
not exceeding 2500 mg ergot/kg in the daily ration of cattle. However, she pointed out that
critical ergot concentrations can only be taken as a rough orientation because of the variation
in the alkaloid concentration of ergot. Indeed, if the cited range of alkaloid concentration in
European ergot between 900 and 2100 mg/kg is considered, it can be estimated that the
critical ergot concentration would be just 1100 mg/kg feed when the maximum alkaloid
concentration applies (5).
Therefore, further studies should rather be based on the alkaloid concentration than on ergot
content.
Acknowledgement The assistance of the co-workers of the Institute of Animal Nutrition of the Federal
Agricultural Research Centre (FAL) Braunschweig for animal care and performance of
analyses is gratefully acknowledged.
55
Paper II
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1 Baumann U, Hunziker HR, Zimmerli B (1985) Mutterkornalkaloide in schweizerischen Getreideprodudukten. Mitt Gebiete Lebensm Hyg 76: 609-630
2 Berry CN (2005) Untersuchung von Blutparametern bei Jungrindern der Rassen Deutsche Holsteins und Deutsches Braunvieh. Dissertation TiHo Hannover
3 Cheeke PR (2006) The role of the liver in the detoxification of poisonous plants. Plant-associated toxins Cab International Chapter 51: 281-286
4 Cunningham IJ, Swan JB, Hopkirk CEM (1944) The symptoms of ergot poisoning in sheep. N Z J Sci & Tech 26a: 124
5 Dänicke S, Mainka S (2004) Mutterkorn. FAL Agricultural Research Special Issue 294: 116-130
6 EFSA (2005) Opinion of the scientific panel on contaminants in food chain on a request from the commission related to ergot as undesirable substance in animal feed. The EFSA Journal 225: 1-27
7 Filipov NM, Thompson FN, Stuedemann JA, Elsasser TH, Kahl S, Stanker LH, Young CR, Dawe DL, Smith CK (2000) Anti-inflammatory effects of ergotamine in steers. Proc Soc Exp Biol Med 225: 136-142
8 Forth W, Henschler D, Rummel W, Starke K (1992) Allgemeine und spezielle Pharmakologie und Toxikologie. Wissenschaftsverlag Mannheim-Leipzig-Wien-Zürich 6.Auflage: 485
9 Gedek B (2002) Pilzkrankheiten der Haustiere. In: Medizinische Mikrobiologie, Infektions- und Seuchenlehre Hrsg : Rolle, M & Mayr, A , Enke Verlag Stuttgart613-632
10 Greatorex JC, Mantle PG (1974) Effect of rye ergot on the pregnant sheep. J Reprod Fert 37: 41
11 Greatorex JC, Mantle PG (1973) Experimental ergotism in sheep. Res Vet Sci 15: 346
12 Kalberer F (1970) Absorption,distribution and excretion of [3H]ergotamine in the rat. Internal report Sandoz Ltd , Basel
13 Kaske M, Kunz HJ (2003) Handbuch Durchfallerkrankungen der Kälber. Kamlage Verlag Osnabrück
14 Kelly DG, Kerlin P, Sarr MG, Phillips SF (1981) Ricinoleic acid causes secretion in autotransplanted (extrinsically denervated) canine jejunum. Digest Dis Sci 26: 966-970
15 Kraft W, Dürr UM (2005) Klinische Labordiagnostik in der Tiermedizin. Schattauer GmbH 6. Auflage
16 Kren V, Fiserova A, Auge C, Sedmera P, Havlicek V, Sima P (1996) Ergot alkaloid glycosides with immunomodulatory activities. Bioorgan Med Chem 4: 869-876
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17 Landes E (1996) Mutterkorn in Futtermitteln für Rinder. Übers Tierernährung 24: 92-101
18 Loken T (1984) Ergot from meadow grass in Norway - chemical composition and toxicological effects in sheep. Nord Vet Med 36: 259-265
19 Mainka S (2006) Zum Einfluß von Mutterkornalkaloiden im Futter auf Gesundheit und Leistung von Schwein und Huhn. Dissertation Georg-August-Universität Göttingen
20 Moubarak AS, Piper EL, Johnson ZB, Flieger M (1996) HPLC method for detection of ergotamin, ergosine and ergine after intravenous injection of a single dose. J Agr Food Chem 44: 146-148
21 Naumann C, Bassler R (1993) Die chemische Untersuchung von Futtermitteln. Darmstadt, VDLUFA-Verlag
22 Nürnberg K, Kuhn G, Ender K, Nürnberg G, Hartung M (1997) Characteristics of carcass composition, fat metabolism and meat quality of genetically different pigs. Fett-Lipid 99: 443-446
23 Reichling JJ, Kaplan MM (1988) Clinical use of serum enzymes in liver disease. Dig Dis Sci 33: 1601-1614
24 Richter WIF, Röhrmoser G, Komusinski S, Rindle C, Wolff J (1990) Einfluß der Lagerdauer von Mutterkorn kontaminiertem Futtergetreide und dessen Rationsanteil auf Zuwachs und Futterverwertung von Ferkeln. Das wirtschaftseigene Futter 36: 236-245
25 Rotter RG, Marquardt RR, Crow GH (1985) A comparison of the effect of increasing dietary concentrations of wheat ergot on the performance of leghorn and broiler chicks. Can J Anim SCI 65: 963-974
26 Schulte E, Weber K (1989) Schnelle Herstellung der Fettsäuremethylester aus Fetten mit Trimethylsulfoniumhydroxid oder Natriummethylat. Fat Sci Technol 91. Jahrgang: 181-183
27 Schulte-Langforth M (1990) Die Entwicklung physiologische Merkmale von Kälbern innerhalb des ersten Lebensjahres. Dissertation TiHo Hannover Rinderklinik: 21-41
28 Schumann B, Dänicke S, Meyer U (2006) Effects of different levels of ergot in the concentrate on growing and slaughtering performance of bulls and on the carry over into edible tissue. submitted
29 Sogni P, Chaussade S, Kue-Gohe K, Nepveux P, Homerin M, Couturier D, Guerre J (1992) [Comparative effects of ricinoleic acid and senna on orocecal and oroanal transit time in healthy subjects. Application of the salacylazosulfapyridine method]. Gastroen Clin Biol 16: 21-24
30 Sterzl J, Rehacek Z, Cudlin J (1987) Regulation of the immune response by ergot alkaloids. Czech Med 10: 90-98
31 Stuedemann JA, Hill NS, Thompson FN, Fayrer-Hosken RA, Hay WP, Dawe DL, Seman D.H., Martin SA (1998) Urinary and biliary excretion of ergot alkaloids from steers that grazed endophyte-infected tall fescue. J Anim Sci 76: 2146-2154
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32 Ueberschär K-H (1999) Einfluß von Zearalenon auf Wachstum und Rückstände in den Geweben von Mastkaninchen. VDLUFA-Kongreßband 1999, Halle/Saale, VDLUFA-Schriftenreihe 52/1999: 425-428
33 Walser K, Bostedt H (1990) Neugeborenen- und Säuglingskunde der Tiere. Enke Verlag
34 Wolff J (1989) Mutterkorn in Getreide. In: Bayerisches Staatsministerium für Ernährung Landwirtschaft und Forsten, Referat Landmaschinen und Energiewirtschaft (eds) Getreidekonservierung und Futterschäden durch Getreide. Heft 30 edn. Grub, pp 28-36
35 Wolff J (1992) Mutterkorn in Getreide und Getreideprodukten. In: Ocker HD (ed) Rückstände und Kontaminanten in Getreide und Getreideprodukten. Behr`s Verlag, Hamburg, pp 113-137
36 Wolff J, Neudecker C, Klug C, Weber R (1988) Chemical and toxicologic studies of native corn in flour and bread. Zeitschrift für Ernährungswissenschaft 27: 1-22
37 Young JC (1981) Variability in the content and composition of alkaloids found in Canadian ergot 2. Wheat. J Environ Sci Heal 16: 381-393
38 Young JC (1981) Variability in the content and composition of alkaloids found in Canadian ergot. 1. Rye. J Environ Sci Heal 16: 83-111
39 Young JC, Chen Z (1982) Variability in the content and composition of alkaloid found in Canadian ergot. 3. Triticale and barley. J Environ Sci Heal 17: 93-107
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Paper III
Effects of the level of feed intake and ergot contaminated
concentrate on ruminal fermentation, ergot alkaloid metabolism
and carry over into milk, and on physiological parameters in cows
BARBARA SCHUMANN1, SVEN DÄNICKE1*, PETER LEBZIEN1, KARL-HEINZ
UEBERSCHÄR1, JOACHIM SPILKE2
1Institute of Animal Nutrition, Federal Agriculture Research Centre (FAL), Bundesallee 50,
38116 Braunschweig 2Biometrics and Informatics in Agriculture Group, Martin-Luther University, Halle/Saale,
Germany
Food Additives and Contaminants submitted
59
Paper III
Abstract The aim of the present study was to examine the effects of ergot contaminated concentrate at
differing levels of feed intake on ruminal fermentation, ergot alkaloid metabolism and carry
over into milk, as well as on various physiological parameters in dairy cows. Twelve double
fistulated (in the rumen and the proximal duodenum) Holstein Friesian cows were fed either
the control diet (on a dry matter (DM) base: 60 % maize silage, 40 % concentrate) or the
contaminated diet (concentrate contained 0.23 % ergot, which caused an alkaloid
concentration of the daily ration between 504.9 and 619.5 µg/kg DM) over a period of four
weeks. Daily feed amounts were adjusted to the current performance which resulted in a dry
matter intake (DMI) variation between 6.0 and 18.5 kg/day. The actual alkaloid exposure
varied between 4.1 and 16.3 µg/kg body weight when the ergot contaminated concentrate was
fed.
Isovalerate, propionate and NH3-N concentrations in the rumen fluid were significantly
influenced by ergot feeding, and the amount of ruminally undegraded protein, as well as the
fermentation of NDF, tended to increase with the ergot supplementation at higher levels of
feed intake, which might indicate a shift in the microbial population. Other parameters of
ruminal fermentation such as ruminal pH, fermented organic matter as a percentage of intake,
or the amount of non-ammonia N (NAN) measured at the duodenum were not significantly
influenced by ergot feeding.
Approximately 67 % of the alkaloids fed were recovered in the duodenal digesta, and
approximately 24 % of the intake were excreted via the faeces. No alkaloid residues could be
detected in the blood or milk samples, and the activities of liver enzymes in the serum were
independent of ergot feeding. The rectally measured body temperature of the cows
significantly increased after ergot administration (p = 0.019). Thus, body temperature can be
regarded as a sensitive parameter to indicate ergot exposure of dairy cows.
Keywords: Dairy cow, feed intake, rumen fermentation, ergot alkaloids, carry over into milk,
body temperature
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Paper III
Introduction
The solidified mycelium of Claviceps purpurea is called ergot and its presence in feed might
worsen performance and can even lead to intoxification symptoms (Landes 1996).
Full-grown ruminants are considered less susceptible to mycotoxins due to the detoxifying
potential of their rumen microbes. These microbes again may be influenced in their activity
by differing organic matter intakes and passage rates through the rumen (Seeling et al. 2005).
The level of organic matter intake of a cow normally shows a high variation over the year,
dependent on the stage of lactation. With an increasing intake of organic matter, the retention
time in the rumen decreases and the passage rate in turn increases, which might influence
digestibility and the time available for the metabolization of mycotoxins such as ergot
alkaloids. Moreover, the pH and probably the proteolytic and cellulolytic activities in the
rumen are thought to be influenced by the level of feed intake, which might in turn influence
the alkaloid metabolism and/or absorption (Wolff 1992, Wirth and Gloxhuber 1994, Buchta
and Cvak 1999).
The present study was conducted to examine the effects of an ergot contaminated concentrate
on ruminal fermentation at differing levels of feed intake, and to measure alkaloid
concentrations and profiles at the proximal duodenum to investigate the ruminal ergot
alkaloid metabolism and to assess the potential absorption of these alkaloids. Furthermore,
blood and milk samples were collected to prove whether alkaloids reach the systemic
circulation and the milk.
As the body temperature was shown to respond to the ergot presence in feed (Zanzalari et al.
1989, Schmidt and Osborne 1993, Jones et al. 2000, Mcleay et al. 2002, Cross 2003, Ilha et
al. 2003) this parameter was also to be measured in the present experiment.
Material and methods
Experimental design and animals
The study was conducted with twelve dairy cows of the “Holstein Friesian” breed (Table 1),
fitted with large rubber cannulas in the dorsal sac of the rumen (inner diameter: 10 cm) and
simple T-shaped plastic cannulas at the proximal duodenum close to the pylorus (inner
diameter: 2 cm). The cows had a mean body weight of 610 (± 82) kg at the beginning of the
study and were housed in a tethered-stall with neck straps. Each cow box was equipped with
an individual trough and had free access to water and a salt block containing sodium chloride.
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The diets consisted of 40 % concentrate and 60 % maize silage on a dry matter basis. All
animals were fed the same diet, whereas the daily amount was adjusted according to the
current performance and production stage in order to cover a wide range in dry matter intake.
Maize silage was given in two equal portions at 5:00 and 15:00 h. The concentrate was evenly
distributed over four feeding times per day at 5:00, 8:00, 15:00 and 17:00 h.
The composition of the concentrates is shown in Table 2. Lactating cows (n = 8) were milked
at 5:00 and 16:00 h.
Table 1. Experimental design
Group Ergot (g/kg concentrate) Cows/group
Control 0 12 Ergot 2.25 12
Table 2. Composition of the concentrates (g/kg as fed) Group Components: Control Ergot Soy bean meal 200 200 Barley 100 100 Wheat 260 255 Dried sugar beet pulp 230 230 Peas 150 150 Calcium carbonate 18 18 Soy bean oil 10 10 Urea 12 12 Mineral and vitamin premix¹ 20 20 Ergoty rye² 0 5
1) provided per kg concentrate: 175 g Ca, 100 g Na, 50 g P, 30 g Mg, 1.5 g Fe, 2 g Mn, 6 g Zn, 1.2 g Cu, 30 mg I, 20 mg Co, 40 mg Se, 1 000 000 IU vitamin A, 100 000 IU vitamin D3, 2 000 IU vitamin E 2) 45 % ergot
Sample collection and measurements
The experiment was split into two treatments (Control treatment and Ergot treatment). Each
cow passed through both treatments. One cow had to be used twice in both treatments to
complete the animal numbers, and another had to be replaced between the treatments due to
experimental conditions.
Each treatment period consisted of four weeks. Two weeks of adaptation to the diets were
followed by duodenal chyme collection. Over a period of five days four 100 ml-chyme
samples were taken at two hour intervals through the duodenal cannula. Immediately after the
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collection, pH-values were measured with a glass electrode (digital pH measurement device,
pH525, WTW) in the samples taken per cow. The one with the lowest pH was added to the
daily pooled sample of each cow as described by Rohr et al. (1984).
Cr2O3 was used as a marker, given every 12 hours into the rumen beginning 10 days before
the duodenal sampling period, and every 6 hours during the sampling period to estimate the
duodenal flow.
At Week four of each treatment period rumen fluid was sampled for NH3-N analyses through
the rumen fistula before feeding at 5:30, and again at 6:00, 6:30, 7:00, 7:30, 8:30 and 10:30 h
using a hand vacuum pump. The pH- values were determined as described above. Short chain
fatty acids were analysed in the rumen fluid at 8:30 h.
Blood samples (heparin and serum tubes) were taken once per treatment period during Week
four at 10:00 h to cover the peak of the serum alkaloid curve, which occurs approximately
two hours after the start of feeding (Kalberer 1970). The tubes were centrifuged at 2000g and
15°C for 10 min approximately two hours after sampling and afterwards stored in a deep
freezer. The cows were weighed on the day before the experiment started and on the last day.
Milk yield was recorded at each milking time and samples for ergot alkaloid analyses were
taken through four consecutive morning and evening milking at Week four of each treatment
period.
Urine (spontaneous micturition) and faeces (rectally removed) of each cow were sampled
once in Week four. Body temperatures of the cows were measured anally in a 6-hour interval
during the duodenal chyme sampling period using a clinical thermometer (type: DIGItempTM,
firm: servoprax GmbH, Wesel). The outside air temperature was recorded by the
meteorological station of the FAL Braunschweig as a potential co-variable.
Samples of maize silage, concentrates and feed refusals, if occurring, were collected daily
during the digesta sampling weeks, pooled and dried at 60°C. Samples of the faeces, the milk
and the duodenal digesta were freeze-dried and faeces and digesta samples were, just as the
dried samples of the feedstuffs, ground to 1 mm for analysis.
Analysis
The chemical composition of the feedstuffs was analysed according to the methods of the
VDLUFA (Verband Deutscher Landwirtschaftlicher Untersuchungs- und
Forschungsanstalten) (Naumann and Bassler 1993). Ergot and feedstuffs were analyzed for
ergot alkaloids (ergometrine, ergocornine, ergotamine, α-ergocryptine, ergosine, ergocristine
and their –inine isomers) by HPLC based on the method of Wolff et al. (1988).
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Approximately 3 g of the samples were mixed with 100 ml extraction fluid (50 ml
dichlormethane + 25 ml ethylacetate + 5 ml methanol + 1 ml ammonium hydroxide (25%))
and the day after centrifugation an aliquot was taken and evaporated to dryness. The next step
was to dissolve the residue in 2 ml toluene / methanol (49 + 1) and to solubilise it using
ultrasound. The fluid was mixed with 9 ml i-hexane and added to an 3 g Extrelut® column
(Merck, Darmstadt, Germany), which was acidified with 5 ml 2 % aquaeous tartaric acid. For
the following elution, 0.5 ml toluol / methanol + 4.5 ml i-hexane and 20 ml di-isopropylether /
i-hexane (1+1) were used. For 1-2 min air was sucked through to dry the column before the
alkaloids were assimilated in 25 % ammonium gas, which was detected by colour reaction of
phenolphthalein. The alkaloids were eluted with 25 ml dichlormethane, which was evaporated
to dryness at 35°C and carefully blown off with nitrogen. Finally the residue was filled up to a
definite volume of 500 µl and 20 μl was injected in the HPLC-apparatus. The HPLC consists
of an isocratic pumping system with a 250 x 4 mm column (5 μm, C 18 Gravity, Macherey-
Nagel, Düren, Germany), operates at 44°C and is connected with a fluorescence detector (325
nm excitation / 418 nm emission wavelength).
The serum samples, milk, urine and faeces were analysed with the same method.
The detection limit was 6 ng/g dry matter, except for ergometrine where it was 11 ng/g dry
matter. Ergometrine, ergotamine, ergocristine, ergocornine and ergocryptine are referred as to
“key alkaloids”, since standards were commercially available for their identification. Ergosine
and its isomer were identified by their retention time (Baumann et al. 1985).
Contaminations with zearalenone (ZON) were analyzed according to a modified VDLUFA
method according to Ueberschär (1999) as described by Dänicke et al. (2001) and in a version
more adapted to physiologic samples by Goyarts et al. (2006).
Kjedahl nitrogen (N) was analysed in thawed chyme samples, whereas all other duodenal
digesta analyses were done with freeze-dried and ground samples.
The Cr2O3 in marker and digesta samples was determined using atomic absorption
spectrophotometry as described by Williams et al. (1962), and was used to estimate the daily
digesta flow.
Near infrared spectroscopy (NIRS) according to Lebzien and Paul (1997) was used to
estimate the microbial N-proportion of non-ammonia-N in freeze-dried duodenal digesta
samples.
For NDF analysis, the digesta samples were pooled using aliquots from each day according to
the calculated daily dry matter flow. NDF was determined by the method of Goering and Van
Soest (1970).
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Ruminal fluid was analysed for ammonia-N immediately after sampling according to DIN
38406-E5-2 (1998). Short chain fatty acids (SCFA) were analysed using a gas chromatograph
(Hewlett Packard 5580, Avondale, PA, USA) equipped with a flame ionization detector as
described by Geissler et al. (1976).
The infrared milk analyser MILCOSCAN was used to determine milk fat concentrations
(Milcoscan FT6000, Foss Electric, Hillerod, Denmark).
One ml of each serum sample was analysed by the laboratory of the Cattle Clinic Hanover for
five liver parameters with a fully automatic apparatus (Cobas-Mira, Fa. Hoffmann-La Roche
& Co. AG Diagnostika Basel, Switzerland). Photometric standard procedures were used for
gamma-glutamyl transferase (γ-GT, International Federation of Clinical Chemistry) and
aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and creatine kinase
(CK, German Federation of Clinical Chemistry). Total bilirubin was measured according to
the method of Jendrassik and Gróf (1938).
Calculations and statistics
For the calculation of the dry matter flow (DMF) the following formula was used:
DMF (kg/day) = 1000/)/(
)/(gDMmgionconcentratchromiumduodenal
dmgnapplicatiochromium
The daily duodenal flows of organic matter (OM) and nutrients were estimated by the
multiplication of their chyme concentrations with the DMF.
The fraction of microbial N of non-ammonia N (NAN) at the duodenum estimated with NIRS
was multiplied by the NAN flow to get the flow of microbial N and protein respectively (N *
6.25).
The mean ammonia N proportion was assumed to be 5 % of duodenal total N (Riemeier
2004). Subtracting this amount of ammonia N from total N at the duodenum, NAN was
calculated.
Ruminally fermented OM (FOM) was calculated by:
FOM (kg/d) = OM-intake – (duodenal OM flow – microbial OM)
where microbial OM = 11.8 * microbial N (Schafft 1983)
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The utilizable crude protein (uCP) at the duodenum was estimated according to Lebzien and
Voigt (1999):
uCP = crude protein flow at the duodenum – endogenous protein (EP)
where EP = endogenous N (EN) * 6.25 and EN (g) = 3.6 * kg DM flow at the duodenum
(Brandt and Rohr 1981)
The undegradable feed crude protein (UDP) was estimated as follows:
UDP = 6.25 * [g NAN at the duodenum – (g microbial N + g EN)].
The ruminal N balance (RNB) was calculated by the following formula:
RNB = (CP – uCP) / 6.25.
Fat corrected milk (FCM) was estimated as following:
FCM (kg/d) = ((% milk fat * 0.15) + 0.4) * kg milk yield (Helfferich and Gütte 1972).
ME in the feedstuffs was calculated as described by the GfE (2001) using the prediction
equation:
ME (MJ) = 0.0312 x g DXL + 0.0136 x g DXF + 0.0147 x g (DOM – DXL – DXF) + 0.00234 x
g XP
The daily amount of faecal organic matter was estimated by the amount of organic matter
intake and the digestibilities given by the DLG (1997). The daily alkaloid excretion with the
faeces could thus be calculated by multiplying the estimated amount of faecal OM with the
alkaloid content in the sample-OM.
Statistical analysis
Data were analysed using the SAS software package (Version 9.1.3, procedure mixed, SAS
Institute, Cary, NC, USA.). Fixed effects were “GROUP” (Control treatment = Group 1, and
Ergot treatment = Group 2); and additionally “LAKST” (1 = first 100 days post partum (p.p.),
2 = days 101 – 200 p.p. and 3 = > 200 days p.p.) for FCM evaluation.
As fixed regressive components, the effect of increasing organic matter intake (OMI) within
the treatment (group) “OMI(GROUP)” and, if quadratic relations were calculated by ML-
method, additionally its square “OMI_2(GROUP)”, were considered.
66
Paper III
Furthermore the effect of repeated measurements within the same individual was considered
as the random effect “ANI”.
Variances were evaluated according to the restricted maximum likelihood (REML) method for
random effect variances and the Kenward-Roger-method for calculation of degrees of freedom
according to the following model also used by Seeling et al. (2005):
PROCEDURE MIXED METHOD = REML; CLASS GROUP LAKST ANI; MODEL Y = GROUP LAKST OMI(GROUP) OMI_2(GROUP) / DDFM = KENWARDROGER; RANDOM ANI; RUN; For the analysis of pH and NH3-N ("PH", "NH3"), the effects “ANI” and “GROUP” were
considered as described above. Furthermore, the time (in minutes) after feeding was
considered as the fixed effect “MINUTES” and additionally implemented in the model:
PROC MIXED METHOD = REML; CLASS GROUP MINUTES ANI; MODEL PH or NH3= MINUTES GROUP OMI(GROUP) OMI_2(GROUP) / DDFM=KENWARDROGER; RANDOM ANI; RUN; For evaluating of the body temperature variance, the model was modified to consider possible
day-time effects (“TIME”, time of the measurement: 3:00, 9:00, 15:00, 21:00 h) the effects of
the day on experimental diet (“DAY”). The latter two effects were handled as fixed effects
beside the fixed effect of “GROUP” as described above, whereas animal related variance
caused by repeated measurements was considered as the random effect “ANI” (see above).
In addition, the outside temperature corresponding to the day-time measurements was
considered as fixed regression component ("OUTSIDE_TEMP") in the model:
PROC MIXED METHOD=REML; CLASS GROUP DAY TIME ANI; MODEL TEMPERATURE = GROUP TIME DAY GROUP*TIME GROUP*DAY OUTSIDE_TEMP DDFM= KENWARDROGER; RANDOM ANI; RUN;
67
Paper III
Results
Chemical composition of the feedstuffs
The nutrient composition of the feedstuffs pooled over the course of the study was almost
equal (Table 3).
The ergot used in the present experiment had a mean alkaloid concentration of 681 mg/kg
DM with ergotamine, ergocristine and ergosine together amounting to more than 50 % (data
not shown).
The total alkaloid content of the ergotized concentrate varied between 930 and 1484 µg/kg
DM with ergotamine, ergocristine, ergosine and its isomers being the most prominent
alkaloids (Table 3). The alkaloid content of the maize silage over the entire experimental
period was lower than the indicated detection limits, whereas the concentrate of the Control
treatment showed a mean alkaloid content of 7 µg/kg DM (0 – 53 µg/kg DM).
Table 3. Mean values (n = 6) of dry matter (g/kg), nutrient composition (g/kg DM), energy concentration (MJ ME/kg DM) and alkaloid pattern of the concentrates (µg/kg DM; Percentage of total alkaloids in brackets) Concentrate Maize Control Ergot silage
Nutrients and energy (g/kg DM): Dry matter [g/kg] 878.0 882.4 336.7 Crude protein 222.6 229.9 77.1 Crude fat 28.2 29.3 29.8 Crude fibre 76.5 81.2 181.5 ME [MJ/kg] 12.8 12.9 10.7 Alkaloids [µg/kg DM (%)]: E-metrine 2.2 (29) 88 (6) 0 E-metrinine 0 18 (1) 0 E-amine 1.7 (22) 347 (24) 0 E-aminine 0.8 (11) 191 (13) 0 E-cornine 0 72 (5) 0 E-corninine 0 39 (3) 0 E-cryptine 0 67 (5) 0 E-cryptinine 0 60 (4) 0 E-cristine 1.2 (16) 231 (16) 0 E-cristinine 0.4 (5) 103 (7) 0 E-sine 0.9 (12) 143 (10) 0 E-sinine 0.4 (5) 70 (5) 0 Total alkaloids: 7.6 1430 0
68
Paper III
In all of the feedstuffs, the content of β-zearalenol was lower than the detection limit of 5 ng/g
dry matter. In pooled samples of the maize silage 2.3 ng α-zearalenol/g and 139.4 ng
zearalenone/g on a dry matter basis were detected. The concentrates showed α-zearalenol
contents which were lower than the detection limit of 1 ng/g DM. Zearalenone was detected
with 5.6 ng/g DM in the Control concentrate and with 4.6 ng/g DM in the ergotized
concentrate.
On a DM basis, deoxynivalenol was detected in the maize silage with a concentration of 1858
ng/g. The deoxynivalenol contents of the concentrates were lower than the detection limit of
34 ng/g DM.
Animal study
The experiment proceeded without any mentionable incidents.
Significant relations between most of the analysed parameters and the amount of OMI were
detected (Table 4 and 5), but since this relationship has been described before (Seeling et al.
2005), this study was focused on the effects of ergot feeding.
Furthermore, as most of the evaluated data were proven to be insignificant (P> 0.05) for the
fixed “GROUP” effect and for the difference between the regression coefficients, only
significant effects or trends to significance are particularly stated in the following results.
Rumen fermentation variables
Concentrations of the short chain fatty acids measured three hours after morning feeding in
the rumen fluid were mostly influenced by OMI and are shown on Table 4. Molar proportion
of acetate, isobutyrate, butyrate and valerate were independent of the ergot supplementation
of the concentrate. Isovalerate decreased significantly with increasing organic matter intake
(p(linear) = 0.035; p(quadratic) = 0.023). Furthermore, a significant difference between the linear
regression coefficients of the Control treatment and of the Ergot treatment was evaluated (p =
0.011), which was caused by the faster OMI-related decrease in the Control treatment
compared to the Ergot treatment.
Propionate concentrations significantly increased with ergot feeding (p = 0.026) and in a
steeper fashion with increasing OMI than after feeding the control concentrate (p = 0.006).
Ruminal pH measured three hours after morning feeding decreased with increasing organic
matter intake (r² = 0.44; Figure 1). Ruminal pH and SCFA concentration were negatively
correlated (r² = 0.37; data not shown).
69
Paper III
CE
CE
CE
CE
CE
CE
5.7
5.9
66.2
69.7
16.2
12.9
1.4
1.1
13.2
13.5
2.2
1.9
5.9
6.4
69.3
68.4
13.6
13.4
1.2
1.3
12.3
13.6
2.3
2.3
9.2
9.6
67.3
6714
.214
.81.
11.
314
.314
1.9
1.9
9.6
9.6
65.1
62.2
18.5
19.9
10.
812
.314
.41.
91.
510
.811
.456
.564
.214
.916
.60.
51.
123
.113
.81
3.4
11.4
11.6
5359
.716
.216
.20.
60.
627
.519
.51.
30.
713
.113
.651
.460
.321
.616
.40.
60.
520
.920
.60.
60.
813
.514
.259
.251
.516
.629
.70.
40.
620
.213
.61.
30.
913
.614
.252
.952
.320
.125
.80.
20.
320
.814
.60.
40.
414
.314
.454
.549
.126
.724
.50.
50.
414
.418
.50.
70.
414
.914
.558
.257
.720
.322
.60.
40.
617
.216
.50.
61.
117
.617
.155
.353
15.6
19.5
0.6
0.8
2422
.31.
51.
658
.759
.718
.319
.60.
70.
818
.516
.01.
31.
4
¹ Pro
babi
lity
unde
r H0
that
a F
-dis
tribu
ted
rand
om v
aria
ble
exce
eds o
bser
ved
F, fo
r org
anic
mat
ter i
ntak
e² P
roba
bilit
y un
der H
0 th
at a
F-d
istri
bute
d ra
ndom
var
iabl
e ex
ceed
s obs
erve
d F,
for q
uadr
atic
org
anic
mat
ter i
ntak
e³ P
roba
bilit
y un
der H
0 th
at a
t-di
strib
uted
rand
om v
aria
ble
exce
eds o
bser
ved
|t|, fo
r the
diff
eren
ce o
f the
line
ar re
gres
sion
coe
ffici
ents
bet
wee
n C
Tab
le 4
. Con
cent
ratio
n of
tota
l and
indi
vidu
al sh
ort c
hain
fatty
aci
ds (S
CFA
) in
the
rum
en fl
uid
(mea
sure
d 3
hour
s afte
feed
in
CE
CE
0.9
171
861.
31.
110
892
1.3
1.1
106
111
1.3
1.3
9611
14
0.9
132
711.
43.
394
106
51.
614
011
02.
33.
810
711
15.
66.
616
918
83.
27.
171
135
3.2
1.6
113
673.
22.
714
012
62.
72.
611
310
8
ontro
l and
Erg
ot tr
eatm
ent
4 Pr
obab
ility
und
er H
0 th
at a
t-di
strib
uted
rand
om v
aria
ble
exce
eds o
bser
ved
|t|, fo
r the
diff
eren
ce o
f the
qua
drat
ic re
gres
sion
coe
ffici
ents
bet
wee
n C
ontro
l and
Erg
ot tr
eatm
ent
r the
beg
inni
ng o
f the
mor
ning
g)
dur
ing
Con
trol (
C) a
nd E
rgot
(E) f
eedi
ng in
rela
tion
to th
e or
gani
c m
atte
r int
ake
P4 = 0
.008
P³ =
0.1
27P³
= 0
.011
P³ =
0.6
35P³
= 0
.006
P³ =
0.1
84P²
= 0
.023
P¹ =
0.0
53P¹
= 0
.035
P¹ =
0.0
09P¹
= 0
.026
P¹ =
0.0
14LS
Mea
ns
But
yrat
e
P³ =
0.8
48P³
= 0
.873
P¹ =
0.0
33P¹
= 0
.149
(Mol
%)
Isov
aler
ate
(Mol
%)
SCFA
Val
erat
e
(M
ol %
)(m
mm
ol/l)
Org
anic
mat
ter
inta
ke (k
g/d)
Ace
tate
(Mol
%)
Prop
iona
te
(Mol
%)
Isob
utyr
ate
(Mol
%)
70
Paper III
CE
CE
CE
CE
CE
CE
5.7
5.9
4.1
5.4
56.7
60.1
713.
358
0.6
153.
611
8.5
83.2
92.5
5.9
6.4
5.1
5.1
67.8
74.5
491.
650
5.7
106.
711
4.5
89.6
85.4
9.2
9.6
4.7
4.5
60.3
61.1
577
783.
912
6.4
183.
193
.984
.19.
69.
63.
53.
168
.370
.384
5.8
932.
319
1.3
209.
983
.581
.510
.811
.43.
83.
968
.367
.411
91.1
1037
243.
223
1.1
89.2
84.6
11.4
11.6
3.3
3.7
71.8
65.2
1031
.715
16.3
245.
429
4.2
7892
.113
.113
.62.
43.
763
.762
.711
83.3
1358
.825
8.9
301.
587
83.3
13.5
14.2
3.6
1.7
54.5
6111
95.7
1742
.627
2.8
384.
583
.979
13.6
14.2
2.6
263
.766
1402
.412
76.6
316
289.
680
.683
.814
.314
.42
251
60.7
1364
.312
19.1
295.
828
2.9
87.9
84.4
14.9
14.5
2.9
2.6
64.2
64.4
1201
.514
3327
9.2
329.
384
80.5
17.6
17.1
3.6
2.7
60.1
71.3
1483
.216
89.5
332.
742
0.5
8771
3.3
3.3
62.6
65.6
1068
.411
62.6
237.
426
0.3
87.5
83.4
Tab
le 5
. Ace
tate
/pro
pion
ate
ratio
in th
e ru
men
flui
d an
d so
me
sele
cted
par
amet
ers m
easu
red
in th
e du
oden
al d
iges
tC
ontro
l
CE
CE
226.
416
8.7
58.2
61.8
122.
810
6.7
41.6
31.1
104.
313
6.1
50.6
47.4
131.
213
8.6
55.1
40.6
162.
513
4.9
45.2
47.6
125.
920
2.2
3749
.914
0.5
161
60.1
64.2
163.
920
8.9
62.3
74.2
166.
113
6.4
6561
.818
913
9.3
83.8
62.9
125.
515
3.4
60.3
62.9
144.
813
9.7
6241
.415
0.1
152.
056
.754
¹ Pro
babi
lity
unde
r H0
that
a F
-dis
tribu
ted
rand
om v
aria
ble
exce
eds o
bser
ved
F, fo
r org
anic
mat
ter i
ntak
e³ P
roba
bilit
y un
der H
0 th
at a
t-di
strib
uted
rand
om v
aria
ble
exce
eds o
bser
ved
|t|, fo
r the
diff
eren
ce o
f the
line
ar re
gres
sion
coe
ffici
ents
bet
wee
n C
ontro
l and
Erg
ot tr
eatm
ent
a of
dai
ry c
ows a
nd a
naly
sed
durin
g (C
) and
Erg
ot (E
) fee
ding
in re
latio
n to
the
orga
nic
mat
ter i
ntak
e
P¹ =
0.0
07P¹
= 0
.705
P¹ <
0.0
01P¹
< 0
.001
P¹ =
0.3
97LS
Mea
nsP¹
= 0
.297
P³ =
0.0
31P³
= 0
.874
P³ =
0.2
77P³
= 0
.070
P³ =
0.3
70P³
= 0
.531
P³ =
0.8
24P¹
= 0
.801
ND
F at
the
duod
enum
(% o
f in
take
)
Mic
robi
al p
rote
in
flow
at t
he
duod
enum
(g/d
)
Non
am
mon
ia N
flo
w a
t the
du
oden
um (g
/d)
Deg
rade
d fe
ed
prot
ein
(% o
f int
ake)
Mic
robi
al p
rote
in
at th
e du
oden
um
(g/k
g FO
M)
Ace
tic
acid
/pro
pion
ic
acid
Ferm
ente
d or
gani
c m
atte
r (%
of i
ntak
e)
Org
anic
mat
ter
inta
ke
(k
g/d)
71
Paper III
Organic matter intake(kg/d)
PH-v
alue
1
1
7 11
12
2
3
8
6
9
5 4
7 1
1
10
4
12
8
9
36
5
2
5.2
5.6
6.0
6.4
6.8
6 8 10 12 14 16 18 20
Organic matter intake(kg/d)
PH-v
alue
1
1
7 11
12
2
3
8
6
9
5 4
7 1
1
10
4
12
8
9
36
5
2
5.2
5.6
6.0
6.4
6.8
6 8 10 12 14 16 18 20
Figure 1. Relationship between organic matter intake and pH-value in rumen fluid (measured three hours after morning feeding) in the Control treatment (o) and the Ergot treatment (▲). [Numbers over the symbols denote individual cows; r2 = 0.44; y = 7.018 – 0.091x] NH3-N concentrations in the rumen fluid also were significantly influenced by organic matter
intake (p(linear) = 0.010; p(quadratic) = 0.003) and ergot supplementation of the concentrate (p =
0.047). Mean NH3-N concentrations significantly differed over time (p = 0.025) and were
characterized by an initial increase and a final decrease (Figure 2) which occurred at lower
levels when the organic matter intake decreased.
The acetate/propionate proportion was significantly influenced by both parameters and is
detailed in Table 5.
Nutrient flow at the duodenum
Organic matter intake diversified in a range from 5.7 and 17.6 kg/d in the Control treatment
and between 5.9 and 17.1 kg/d in the Ergot treatment. OM flows from 3.8 to 9.8 kg/d in the
Control, and from 3.5 to 8.1 kg/d in the Ergot treatment, were measured, which corresponded
to mean percentages of 54.8 % and 53.1 % of OMI respectively.
The ruminally fermented organic matter ranged between 3.2 and 10.6 kg/d in the Control
treatment and between 3.6 and 12.2 kg/d in the Ergot treatment and increased significantly
with increasing OMI (p < 0.001; data not shown). Relationships between the
72
Paper III
acetate/propionate ratio in the rumen fluid, microbial protein and non-ammonia N at the
duodenum, percentages of fermented OM, degraded protein and NDF at the duodenum, the
efficiency of microbial protein synthesis and the amount of OMI are shown in Table 5. Only
the acetate/propionate ratio was significantly influenced by the presence of ergot in the
concentrate. Microbial protein and non-ammonia N at the duodenum significantly increased
with increasing OMI (p < 0.001).
Figure 2. NH3-N (mmol/l) concentrations in the rumen depending on organic matter intake
(OMI; kg/d) and time after the morning feeding (min) in the Control treatment (o) and the
Ergot treatment (▲)
Ruminally undegraded protein (UDP) ranged between 75.2 and 370.7 g/d in the Control
treatment, and between 62.9 and 721.5 g/d in the Ergot treatment (Figure 3). UDP was
influenced by OMI (p = 0.048) in a quadratic manner and was slightly influenced by the
ergotized feeding (p = 0.062). The quadratic regression coefficients describing the parabolic
shape of increasing OMI differed significantly between both treatments (p = 0.023).
Cows consumed between 1.97 and 6.04 kg NDF/day in the Control treatment, and between
2.00 and 5.95 kg NDF/d in the Ergot treatment. In the former, between 0.86 and 4.19 kg (37 -
84 %), and in the latter between 0.85 and 3.54 kg (31 - 74 %) of it were not degraded in the
rumen (Table 5; Figure 4), which was slightly influenced by OMI (p = 0.079) and by the ergot
feeding (p = 0.062). The treatment caused significantly different courses of OMI related NDF
flow as indicated by the significantly different quadratic regression coefficients (p=0.043).
73
Paper III
Organic matter intake (kg/d)
Und
egra
ded
Prot
ein
(g/d
ay)
11 7
1112
2
38
6
9
5
71
110 4
12
8
936
5
2
11 7
1112
2
38
6
9
5 4
71
110 4
12
8
936
5
2
0
200
400
600
800
1000
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
Und
egra
ded
Prot
ein
(g/d
ay)
11 7
1112
2
38
6
9
5
71
110 4
12
8
936
5
2
11 7
1112
2
38
6
9
5 4
71
110 4
12
8
936
5
2
0
200
400
600
800
1000
6 8 10 12 14 16 18 20
Figure 3. Relationship between organic matter intake and undegraded protein flow at the duodenum of dairy cows (numbers over the symbols denote individual cows) in the Control treatment ( O ) and the Ergot treatment ( ▲ )
Organic matter intake (kg/d)
ND
F (k
g/d)
11
711 12
2
386
9
5
4
7
1
110
4
12
8
9
365
2
0
1
2
3
4
5
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
ND
F (k
g/d)
11
711 12
2
386
9
5
4
7
1
110
4
12
8
9
365
2
0
1
2
3
4
5
6 8 10 12 14 16 18 20
Figure 4. Relationship between organic matter intake and NDF flow at the duodenum of dairy cows (numbers over the symbols denote individual cows) in the Control treatment ( ) and the Ergot treatment ( ▲ )
74
Paper III
In the Control group between 83.7 and 270.0 g, and in the Ergot group between 73.2 and
217.2 g, endogenous protein per day was in the collected samples. In relation to the OMI this
corresponds to 1.5 and 1.4 % respectively, resulting in 582.9 to 1809.5 g utilisable crude
protein (uCP) per day in the Control group, and in 642.5 to 2411.0 g uCP/day in the Ergot
group. The amount of uCP was significantly influenced by OMI (p < 0.001).
The mean ruminal nitrogen balance (RNB) during the Control treatment was 0.53±0.26 g/MJ
ME, and during the Ergot treatment 0.45±0.29 g/MJ ME, which was significantly influenced
by OMI (p = 0.034). The amount of uCP and the RNB were independent of ergot feeding (p >
0.05; data not shown).
Alkaloid metabolism and excretion
The alkaloid exposure of the cows fed the ergot supplemented concentrate ranged between
504.9 and 619.5 µg/kg DM of the ration which corresponded to a daily exposure of between
4.1 and 16.3 µg/kg live weight (LW) (Table 6). Mean recovery rates in the duodenal samples
ranged between 78 and 111 %, and in the faeces between 63 and 108 %.
The mean absolute amount of alkaloid intake was 7088 ± 1974 µg/day. Approximately 67 ±
13 % of it were recovered in the duodenal digesta and approximately 24 ± 8 % were excreted
with the faeces. The mean percentage of –in isomers in total alkaloids was 73 ± 7 % in the
concentrate, 48 ± 3 % in the duodenal chyme and 68 ± 8 % in the faeces (Figure 5).
Table 6: Alkaloid exposure, content of the duodenal digesta and excretion via faeces Alkaloid exposure Alkaloid flow to the duodenum Alkaloid flow to the faeces
(µg/day) (µg/day*kg LW) (µg/day) (% of intake) (µg/day) (% of intake)3583.3 4.1 2594.1 72.4 600.9 16.8 4200.0 5.5 2288.5 54.5 904.7 21.5 5593.2 7.8 4642.3 83.0 1899.7 34.0 5888.9 10.2 2848.0 48.4 1252.4 21.3 6481.5 10.6 4988.9 77.0 1305.6 20.1 8576.4 13.9 5911.3 68.9 2066.5 24.1 7695.0 14.0 5445.7 70.8 1458.3 19.0 8268.0 14.2 3722.3 45.0 1975.9 23.9 8325.8 14.6 7271.9 87.3 1543.4 18.5 8760.0 15.2 5940.0 67.8 3350.4 38.2 7468.4 15.3 4381.5 58.7 966.2 12.9 10213.1 16.3 7052.5 69.1 3290.3 32.2
75
Paper III
The mean alkaloid proportions in feedstuff, duodenal chyme and faeces, and the relationship
between alkaloid content of the duodenal chyme and the faeces, are shown in Figure 5 and 6.
Ergot residues and carry over
Mean recovery rates in the milk samples ranged between 62 and 86 %, and in the blood
between 77 and 106 %. Neither the analysed blood samples, nor the pooled milk samples of
the lactating cows contained detectable amounts of alkaloids.
Milk yield
FCM in lactating cows varied between 14.8 and 24.3 kg/day in the Control group, and
between 15.0 and 23.6 kg/day in the Ergot group, and showed no significant effects of
increasing OMI or of ergot supplementation of the concentrate. Only the stage of lactation
“LAKST” significantly influenced the milk yield (p = 0.044; data not shown).
Serum activities of hepatic enzymes
Aspartate aminotransferase (AST) activities ranged between 40 and 256 U/l in the Control
group, and between 42 and 122 U/l in the Ergot group. The serum activities of the γ-
glutamyltransferase (γ-GT) varied between 12 and 87 U/l in the Control and between 10 and
100 U/l in the Ergot group. The glutamate dehydrogenase (GLDH) values showed a
diversification between 4 and 82 U/l in the Control group and, respectively, between 4 and
147 U/l in the Ergot group. Serum activities of the creatinine kinase (CK) were measured in
the Control group with values between 66 and 405 U/l and in the Ergot group between 48 and
114 U/l. The alkaline phosphatase (AP) activities differed in range from 39 to 127 U/l and
from 40 to 110 U/l in the Control and in the Ergot group respectively.
No significant effects of increasing OMI or of ergot supplementation of the concentrate were
found for the serum levels of these enzymes.
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Paper III
0
5
10
15
20
25
30
E-metrine E-metrinine E-amine E-aminine E-cornine E-corninine E-cryptine E-cryptinine E-cristine E-cristinine E-sine E-sinine
Figure 5. Mean alkaloid proportions (% of total alkaloids) in feedstuff (striped), duodenal chyme (white) and faeces (spotted) of dairy cows
1
23
64
5
98
10
7
1 12
800
1600
2400
3200
2500 4500 6500 8500
Alkaloid flow at the duodenum (µg/d)
Faec
alal
kalo
idex
cret
ion
(µg/
d)
1
23
64
5
98
10
7
1 12
800
1600
2400
3200
2500 4500 6500 8500
Alkaloid flow at the duodenum (µg/d)
Faec
alal
kalo
idex
cret
ion
(µg/
d) Figure 6. Relationship between alkaloid flow at the duodenum and to the faeces [Numbers over the symbols denote individual cows; r2 = 0.44; y = 74.165 + 0.346x]
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Body temperature
Body temperature (BT) values measured anally showed a physiological course over the day
and were significantly influenced by ergot intake (Figures 7 and 8). Lowest BTs were
measured at 9 a.m., then increased over the course of the day by approximately 0.5 °C, and
finally declined during the night (Figure 7). Mean BTs in the Ergot group were 0.07 °C higher
than in the Control group (p = 0.019). Additionally the outside air temperature had a
significant influence on the BT of the dairy cows (p = 0.027, Figure 8).
Time (h)
Bod
y te
mpe
ratu
re(°
C)
38.1
38.2
38.3
38.4
38.5
38.6
03:00 09:00 15:00 21:00
Time (h)
Bod
y te
mpe
ratu
re(°
C)
38.1
38.2
38.3
38.4
38.5
38.6
03:00 09:00 15:00 21:00
Figure 7. Pooled daily body temperature course during the Control treatment (o) and the Ergot treatment (▲)
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Figure 8. Relationship between body temperature (measured at 3 o´clock in the morning), outside air temperature and alkaloid exposure of dairy cows during the Control treatment (o) and the Ergot treatment ( )
Discussion
Chemical composition of the feedstuffs
The nutrient composition and energy content of the concentrates, as shown on Table 3, were
almost equal and thus potential effects may be attributed to the differing OMI and
respectively to the ergot supplementation of the concentrate.
The ergot used in this experiment fits into the recently reported ranges of alkaloid variation
(42 – 2100 mg/kg ergot DM) in Germany (Wolff 1989, Mainka et al. 2006a, Mainka et al.,
2006b).
Since ergot alkaloids were not detectable in the maize silage throughout the entire
experimental period, the actual daily alkaloid exposure of the cows resulted only from the
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Paper III
concentrate contamination and ranged from 4.1 – 16.3 µg/day*kg LW considering the
differences in OMI and the body weight of the cows.
The zearalenone contamination of the maize silage of 139.4 ng/g DM relates to 75.6 ng/g DM
of the total ration at a dry matter content of 88%. The European orientation value for dairy
cows and calves of 500 ng/g (Amtsblatt der Europäischen Union, 2006) is more than six times
higher than the zearalenone contamination in the current study. The deoxynivalenol
contamination of the maize silage of 1858 ng/g DM corresponds to an approximate
contamination of the total ration of 981 ng/g at a dry matter content of 88%. The
contamination of deoxinivalenol is also significantly lower than the European orientation
value of 5000 ng/g (Amtsblatt der Europäischen Union, 2006) and thus may not cause any
negative effects.
Rumen fermentation variables and nutrient flow at the duodenum
Experiments with similar designs have been analysed before in the Institute of Animal
Nutrition of the FAL Braunschweig, and the influence of level of feed intake on ruminal
digestibility and efficiency of microbial protein synthesis (g microbial protein/kg FOM), was
recently reviewed by Seeling et al. (2005). The compiled and self-revealed results of this
study were generally confirmed by the current experiment. OM, NDF and protein digestibility
decreased with increasing OMI, which might be explained by the increased passage rate
which in turn is followed by a lower retention time and less contact to the rumen microbes.
However, the efficiency of microbial protein synthesis (g microbial protein/kg FOM) was
independent of the OMI (r² = 0.002) which is in contrast to the results of Seeling et al. (2005)
who described a quadratic dependency of the efficiency of microbial protein synthesis on
OMI. As neither ergot contamination nor OMI influenced the efficiency of microbial protein
synthesis in the present experiment, it might be assumed that rumen microbes were not
hampered in their protein synthesis efficiency by these experimental factors.
Regarding the effect of the ergot contaminated concentrate in the current study, only three of
the analysed parameters were significantly different between the two treatments (NH3-N
concentrations in the ruminal fluid and the proportion of isovalerate and propionate in the
total SCFA) and two others showed a trend to significance (NDF and UDP flow to the
duodenum).
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The changes in the composition of the fatty acids in the rumen at constant total SCFA
amounts might indicate a shift in microbial population.
The dynamics of the ruminal NH3 release suggested a more pronounced NH3 peak
concentration after feeding the Ergot concentrate, especially at high levels of OMI (Figure 2),
whereas feeding of the control diet resulted in smoother kinetics. However these significant
differences did obviously not have an impact on the efficiency of microbial protein synthesis
as discussed above. On the other hand, the flow of undegraded protein at the duodenum
increased in a quadratic fashion as OMI increased in the presence of Ergot contaminated
concentrate whereas this increase was less pronounced during the Control treatment (Figure
3).
Further support for Ergot related effects on ruminal protein turnover comes from the
significantly slower OMI related decrease of isovalerate. The iso-acids mainly derive from
dietary protein degradation (Miura et al. 1980) and thus if less protein is degraded, less
isovalerate is a result.
Taken together, these Ergot related changes in ruminal protein turnover were not large enough
to alter the flow of microbial protein at the duodenum (Table 5).
Balance studies with ruminants after ergot feeding which include the analysis of rumen fluid
and duodenal digesta are lacking, but there are some reports about experiments with
endophytic alkaloids which share a common phylogeny with those of Claviceps purpurea
(Glenn et al. 1996) and also have some similar pharmacological effects (Schmidt and Osborne
1993, Paterson et al. 1995, Stuedemann et al. 1998, Schultze et al. 1999, Oliver 2005). Thus,
they might serve conditionally for comparison, if no other studies using ergot alkaloids of
Claviceps purpurea are available.
Westendorf et al. (1993) reported a decrease in dry matter (DM) and ruminal NDF
digestibilities after a daily exposure to 945 mg endophyte alkaloids (ergovaline and its
isomere) per sheep (which corresponds to approximately 16 mg/kg LW). But with an
exposure to 2346 mg of these alkaloids per day the digestibilities increased again.
In two other studies 16 Hereford x Angus steers were assigned to increasing diet
concentrations of ergovaline (0, 158, 317 and 475 mg/kg) for 36 days and apparent DM
digestibility as well as total tract NDF digestibility were not influenced by alkaloid
concentrations. But ADF digestibility decreased linearly with a higher ergovaline intake
(Stamm et al. 1994). Consuming ad libitum rations of diets containing increasing amounts of
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lolitrem B, another alkaloid produced by endophytes, DM and OM digestibilties, ruminal pH,
total SCFA and NH3-N concentrations in the rumen were not affected and ADF digestibility
decreased. Additionally a quadratic influence was observed for acetate and isovalerate
proportions in the rumen fluid (Fisher et al. 2004).
An experiment with ten wether lambs revealed a greater N-retention after a consumption of
highly infected tall fescue hay (909.6 mg/kg). Unfortunately the alkaloid pattern is not
reported. Abomasal total and bacterial N-flow and ruminal digestion of cell wall components
were not affected, but total tract digestion of DM, NDF and ADF were lower for the highly
infected hay (Fiorito et al. 1991).
Since the alkaloid exposure in the current study did not exceed 619 µg/kg DM of the ration
and 16.3 µg/day*kg LW, it was much lower than in all of the above mentioned experiments
and thus a direct comparison is difficult.
However, Hannah et al. (1990) conducted experiments with sheep which were administrated
0, 1.5 and 3 mg ergovaline/kg diet in two different environmental conditions. These doses
corresponded rather closely to those of the present study. Lower ruminal and total tract OM,
and NDF digestibilities were revealed at the highest ergovaline concentration. But they also
observed an interaction between the outside temperature and ruminal fibre digestion, and a
higher fibre content in the infected tall fescue seeds, which may have influenced the digestion
results.
Recapitulating all of the above mentioned results, it can be concluded that alkaloid-
contamination of the feed might have negative effects on fibre digestibility and may also
influence the SCFA proportions in the rumen fluid.
In the current study with higher OMI (which means also a higher alkaloid intake in the Ergot
period) more UDP and a higher NDF digestibility were analysed in the duodenal digesta after
ergot supplementation. Higher UDP values might also be caused by an increased endogenous
N-loss due to the irritant effect of ricinoleic acid on the mucosal tissue (Forth et al. 1992).
Ricinoleic acid is part of the ergot and in the present study contents of 28 g ricinoleic acid per
kg concentrate were analysed.
The higher NDF fermentation might reflect a shift in microbial species and/or microbial
activity due to ergot effects. Since no studies concerning the effects of ergot alkaloids on
rumen microbes in vitro are available, further research will be necessary to evaluate this
assumption.
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Alkaloid metabolism and excretion
The fact that only 67 % of the ergot alkaloids get to the duodenum leads to the question:
which part in alkaloid metabolism and absorption is allotted to the rumen.
Studies about this subject concerning ergot alkaloids of Claviceps purpurea are lacking, but
there are some reports on alkaloid metabolism and absorption in the rumen of endophyte-
produced alkaloids. Westendorf et al. (1993) incubated different combinations of the
pyrrolizidine alkaloids N-formyl and N-acetyl loline in ruminal fluid for 0, 24 or 48 hours.
Their disappearance increased over time and significant amounts of both alkaloids were
metabolised and converted to loline. In a second experiment he used abomasally cannulated
sheep which were administered doses of 945 mg and 2346 mg ergot alkaloids/day (ergovaline
and ergovalinine). Analysing pooled samples of abomasal fluid and faeces, he recovered 50 –
60 % of the alkaloids in the abomasal contents, but only 5 % in the collected faeces. From the
in vitro experiment he concluded that alkaloids in general may be degraded in ruminal
fermentation. But as the in vivo recoveries in abomasal fluid and faeces of the indicated
pyrrolizidine alkaloids were much lower than of the ergot alkaloids, a relative stability of the
ergot alkaloids towards ruminal bioconversion might be assumed (Westendorf et al. 1993).
Nevertheless an absorption by the ruminal tissue might be presumed which agrees with the
results of Hill et al. (2001), who placed sheep ruminal and omasal tissues in parabiotic
chambers. He found the ergot alkaloid transport to be an active process and measured a much
greater transport potential for lysergic acid and lysergol than for the ergopeptines
(ergonovine, ergotamine and ergocryptine). In spite of a 25 % higher transport capability of
the rumen compared with the omasum (600 % higher compared with the reticulum),
ergopeptines tended rather to be absorbed by omasal tissue than by ruminal tissues.
Stuedemann et al. (1998) also postulated that ergot alkaloids must be absorbed in the
forestomach system because of their rapid excretion.
Since mean recovery rates ranged between 78 and 111 % in the duodenal digesta, and
between 63 and 108 % in the faecal samples, it might be assumed that in the current study
nearly 30 % of the alkaloids were absorbed or metabolised prior to the duodenum, and nearly
70 %, or maybe a bit less, in the total tract, which agrees with the results of the above
mentioned studies.
Ruminants are considered to be less sensitive to mycotoxins due to the potential
transformations by their rumen microbes (Hussein and Brasel 2001). Toxic agents may be
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biotransformed and excreted as non-detectable metabolites. These metabolites might be
degraded and thus less toxic, but might also be converted to more toxic compounds, as it is
known, for example, for nitrite, which is a toxic nitrate metabolite during nitrate poisoning
(Dirksen et al. 2002). The complexity of potential metabolites of the alkaloids has recently
been reviewed by Kren (1999).
But not only the microbes, also the pH-value might influence the toxicity of the ergot
alkaloids. Hence, a change of pH may cause a conversion to their –inine isomers which are
pharmacologically less active and thus less toxic (Wolff 1992, Wirth and Gloxhuber 1994,
Buchta and Cvak 1999). In the current study, the –inine fraction increased posterior to the
forestomachs, which might be caused by the low pH-values in the abomasum. As this
conversion is reversible (Wolff 1992), this would also explain that the –inine fraction in the
faeces is lower again (higher pH-values in the gut).
Ergot residues in the blood
In preceding experiments maximum doses of 9.1 µg/kg LW were fed to growing bulls over a
period of approximately 230 days and the alkaloid residues in the analysed blood samples
were lower than the indicated detection limits (Schumann et al. 2007b). Also in calves
exposed to maximum doses of 35.5 µg/day*kg LW over a period of 84 days, no alkaloid
residues were detectable in the blood (Schumann et al. 2007a). It has to be considered, that in
these experiments, due to the feeding management the exact time between the last ergot intake
and sampling of the blood was not known. Thus, most of the alkaloids might have already
been excreted or metabolised into non-detectable metabolites. Ergot alkaloids are quickly
metabolized within the body. Approximately 2 hours after feeding, ergotamine was found to
climax in the blood, followed by slow decreases (Kalberer 1970). Although in the current
study the moment of sampling was exactly 2 hours after the morning feeding, no detectable
residues were found. It might be suggested that analysis methods with lower detection limits
would be necessary to demonstrate potential alkaloid absorption.
Carry over into milk
Cunningham et al. (1944) fed two dairy cows with 17.5 g and 24 g total alkaloids per day and
caused lameness. No alkaloids could be detected in the milk by analysis or an animal feeding
experiment, in which rats were fed the organs and tissues of the sickened cows. But it has to
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be kept in mind that Cunningham’s paper (1944) is very old. He used a colorimetric
procedure with p-dimethylaminobenzaldehyde as a chemical assay according to an extraction
based on Kluge (1934). This method is featured for a detection limit of 500 ng/g, which is a
lot higher than the indicated detection limits of the method used in the current study.
After an administration of silage contaminated with 0.1 % ergot (0.132 % total alkaloids in
the ergot), milk samples of severely affected cows were negative for ergot alkaloids (McKoen
and Egan 1971).
Similarly, a mean alkaloid exposure of 3 µg/day*kg LW over a period of five weeks did not
cause any detectable amounts of ergot alkaloids in the milk of four dairy cows, which lead the
author to the assumption that the rate of excretion is less than ten per cent, if there is carry
over into the milk and that an accumulation of the alkaloids might be nearly excluded (Wolff
et al. 1995).
In contrast to these findings, Parkheava (1979) [cited by Wolff et al. (1995)] published a carry
over of ergot alkaloids into the milk of dairy cows not associated with any apparent toxicosis
symptoms after an ergot exposure of approximately 125 mg ergot/kg LW. This is
approximately 100 mg ergot /kg LW more than in the current study (≈ 23.9 mg ergot/kg LW).
The highest alkaloid exposure in the present experiment was 16.3 µg/day*kg LW which is
closer to the experimental conditions described by Wolff et al. (1995). Thus the fact that no
alkaloids could be detected in the milk confirms the earlier published results.
Milk yield
Long ago, agalactia was observed in sows fed 0.5 or 1.0 % barley ergot (Nordskog and Clark
1945), but reports on ergot administration to lactating dairy cows considering the milk yield
can hardly be found in the literature.
Ross et al. (1989) published a severe loss of milk production in cattle fed a diet with a mean
ergot concentration of 3.75 g/kg. Similar observations were published by several authors and
have been attributed to decreased concentrations of plasma prolactin which might be caused
by the dopamine-like activity of certain ergot alkaloids (Ilha et al. 2003, Cross 2003).
Dopamine inhibits the prolactin release at the pituitary gland Neill (1994) [cited by Ilha et al.
(2003)].
Salobir et al. (1980) observed a slight decrease of the milk yield in different groups (n = 3)
after a daily alkaloid intake of 59.4 µg/kg LW0.75. No significant effects on milk production
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were revealed after 10 days of ergopeptine (10 µg/kg LW/day) administration (Al-Tamimi et
al. 2003), but the authors assumed a slight non-significant depression in the afternoon milk
yield level. Ergotized barley, which was used in this study, probably also contained
ergometrine and its isomere, the only alkaloids analysed in the current study which do not
belong to the ergopeptine group. Thus the total alkaloid administration in the experiment of
Al-Tamimi et al. (2003) might have been a little higher than reported. However, in the current
study, 93 % of the total alkaloid amount were ergopeptine alkaloids (Table 3), which leads to
a maximum exposure of 15.2 µg ergopeptines/kg LW, and thus is comparable to the study of
Al-Tamimi et al. (2003). However, no effects of ergot feeding on milk yield could be
observed in the present experiment. But it has to be considered that the number of lactating
cows used in this study was very small (n = 8) and that the duration of exposure (3 weeks)
and milk amount recording (12 days) was rather short. Thus, the present insignificant results
should not be overvalued.
Serum activities of hepatic enzymes
As ergot alkaloids are mainly metabolised in the liver (Moubarak and Rosenkrans 2000,
Cheeke 2006), and early cases of liver disease are clinically inapparent, analyzing specific
liver enzyme activities in the serum might provide some useful information (Reichling and
Kaplan 1988).
Some studies about liver enzyme activity changes after ergot exposure of humans (Teychenne
et al. 1979) and swine (Dignean et al. 1986, Mainka et al. 2005a, Mainka et al. 2005b) are
published, but data about hepatic enzyme analyses after ergot administration to cattle are
scarce.
Oliver et al. (2000) observed an increase of total bilirubin after prolonged consumption of
endophyte-infected tall fescue. Schultze et al. (1999) compared serum chemistry profiles of
cattle (n = 8-16) grazing on endophyte-infected tall fescue and endophyte-free tall fescue.
They revealed a significant decrease in AP activity, also a slight decrease in AST activity but
no detectable changes of total bilirubin in the serum. Some other authors reported similar
results about endophyte-caused enzyme changes in cattle (Bond et al. 1984).
But there is a lack of data concerning experimental administration of ergot from Claviceps
purpurea. In the current study, no relationship between ergot contamination of the concentrate
and the activity of the analysed serum enzymes could be revealed.
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Body temperature
Ergot alkaloids and derivates are known to change body temperature (BT) in various animals
and humans. In their review, Clark and Clark (1980) detailed increases in BT up to 2.0°C
after subcutaneous injection of 4 mg ergotamine/kg in rats and up to 5.3°C after i.v. injection
of 1 mg ergotoxine/kg in rabbits. Also cases with decreasing BT were reported. For example
7-12 mice showed a decrease in BT of approximately 3°C after an administration of 2 mg
dihydroergotamine/kg i.v. (Clark and Clark 1980).
The alkaloid exposure in the current study ranged from 4.1 to 16.3µg/kg LW which is lower
than in most of the reports cited by Clark and Clark (1980). Only a cat which was
administered ergotamine by intracerebral injection at a dose of 20-100 µg/kg developed an
increase of BT of 0- 0.2°C. The BT of 11 guinea pigs stayed the same after an intracerebral
injection of 30 µg/kg LW, and more than 6 rats showed a slight decrease in BT after
intracerebral injection of ergotamine. There were no reports in this paper of per oral
administration and the lowest dose cited by Clark and Clark (1980), which caused an increase
in BT when given i.m. and i.v. to humans and cats was 0.5 mg/kg .
Ross et al. (1989), who fed mean doses of 50.9 mg ergot/kg LW to steers, examined a
significant BT increase after a period of 23 days, which reached up to 41°C in the afternoon
and returned to normal overnight.
Many authors reported hyperthermia in cattle after grazing endophyte infected grasses
(Zanzalari et al. 1989, Schmidt and Osborne 1993, Cross 2003). The most important alkaloid
of these endophytes is ergovaline (Porter 1995). Mcleay et al. (2002) compared effects of 20
nmol ergopeptines/kg (ergotamine = 25 µg/kg and ergovaline = 10 µg/kg) after i.v. injection
to sheep. Upon administration, the BT was increased slightly but continued to increase
thereafter, with greater increases developing after ergovaline injection (3-3.5°C) than after
ergotamine injection (0.6-3.4°C). These changes in BT remained at least 10 hours (Mcleay et
al. 2002). As effects on BT were less distinct after ergot administration, hyperthermia caused
by endophytes may be difficult to compare with the current study. Furthermore reports on
endophyte caused hyperthermia contain only in a few cases exact data on alkaloid exposure,
but these reports are quite interesting to get a general idea of potential alkaloid-caused effects.
Studies on experimental low dose exposure to ergot alkaloids of Claviceps purpurea are
scarce. Al-Tamimi et al. (2003) observed increases in BT of 0.14°C after 10 days of feeding
ergotized barley during summer heat stress. As already mentioned above, cattle in his study
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received 10 µg total ergopeptine alkaloids/kg LW/day. The author described this dose as a
level for minimal induction of the ergot toxicity response.
The amplitude of BT elevation is strongly influenced by the time of the day and by the
outside temperature (Ilha et al. 2003), which agrees with the results of the current study
(Figure 8). An explanation for the ergot-related increase might be the alkaloid-mediated
peripheral vasoconstriction, which prevents heat loss and evaporation through the skin.
Additionally, it might be boosted by an increased production of heat by the energy wasteful
mixed- function oxidase system which has an important role as an ergot alkaloid-
detoxification system (Zanzalari et al. 1989).
Ross et al. (1989) reviewed that some alkaloids of ergot with central effects may induce
hyperthermia in the midbrain or by boosting the prolactin release which may affect fluid
exchange across the gut and thus could be a further factor in the pathogenesis of
hyperthermia. The current experiment also might lead to the assumption that there might be a
relationship between the amount of alkaloid exposure and the increase of the BT (Figure 8).
Finally, to reveal a no-effect-level, further information on dose-response studies with ergot
contaminated feed and exact data on alkaloid intake is needed.
Conclusions The amount of organic matter intake exerted a significant effect on ruminal metabolism. But
due to the change in NH3-N, propionate and isovalerate concentrations in the rumen fluid, as
well as to the increased ruminally undegraded protein, and the higher NDF fermentation in
the present study, it might also be concluded that feeding ergot-contaminated rations might
cause a shift in the microbial community and/or activity of the rumen.
Furthermore, the slight but significant increase in the body temperature of the ergot-fed cows
might suggest that an ergot exposure between 4.1 and 16.3 µg/kg LW already induces a
physiological response in dairy cows, and that body temperature can be viewed as a sensitive
ergot intoxication indicator. Further dose-response studies are necessary to titrate the no-
effect level of ergot alkaloids in dairy cows.
In dairy cows fed with alkaloid doses not exceeding 16.3 µg/kg LW, a carry over into the
milk in doses toxic for humans might be excluded.
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Acknowledgement The assistance of the co-workers of the Institute of Animal Nutrition of the Federal
Agricultural Research Centre (FAL) Braunschweig for animal care and performing the
analyses is gratefully acknowledged.
89
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94
General discussion
General discussion Studies dealing with the effects of experimental ergot administration to cattle are very scarce,
an exact no-effect level (NOEL) has not been described so far and for ruminants only a
weight based upper limit of 1000 mg ergot per kg unground cereal grains applies (Council
Directive 2002/32/EC of 7 May 2002). Since the alkaloid content in the ergot varies in wide
ranges (Mainka et al., 2006) and the alkaloids mainly determine the toxicity of ergot
(Gloxhuber et al., 1994) it was the aim of the present experiments to study the effects of ergot
with a defined alkaloid pattern on growing calves, fattening bulls and dairy cows. In spite of
the commercial availability of individual alkaloid standards, naturally grown ergot was used
as alkaloid source to study the effects of a defined, and for Germany practically relevant,
pattern. Therefore it needs to be considered in interpreting of the observed results that the
overall effects might be influenced by potential synergistic, but also antagonistic effects
between individual alkaloids (Buchta and Cvak, 1999) (Gareis and Wolff, 2000) which limit
the validity of the data to the specific alkaloid pattern tested.
Figure 1: Alkaloid pattern of the ergot (left side) used in the three feeding experiments and of
the rye (right side) separated from the ergot
Ergoty rye with an alkaloid content of 45 % was used as ergot source. This ergot was
separated from the rye in order to analyse the alkaloid pattern of the ergot and of the
respective rye (Figure 1). Approximately 0.1 % of the amount of alkaloids analysed in the
ergot were found in the rye grains. The alkaloid pattern also was different. It might be
suggested that these alkaloids originated from the ergot and that due to differing chemical
properties, some might have passed more easily into the surrounding grains than others.
Also the alkaloid pattern of the separated ergot and of the ergot supplemented concentrates
used in the feeding experiments differed. This in turn may have other causes, such as for
example, that the manufacturing process or a prolonged storage might cause an isomerisation
ErgometrineErgometrinineErgotamineErgotaminineErgocornineErgocorninineErgocryptineErgocryptinineErgocristineErgocristinineErgosineErgosinine
ErgometrineErgometrinineErgotamineErgotaminineErgocornineErgocorninineErgocryptineErgocryptinineErgocristineErgocristinineErgosineErgosinine
ErgometrineErgometrinineErgotamineErgotaminineErgocornineErgocorninineErgocryptineErgocryptinineErgocristineErgocristinineErgosineErgosinine
ErgometrineErgometrinineErgotamineErgotaminineErgocornineErgocorninineErgocryptineErgocryptinineErgocristineErgocristinineErgosineErgosinine
95
General discussion
of the alkaloids (Wolff, 1992). Thus, to ensure that the time of storage in the present study
was not followed by a decreasing alkaloid content of the concentrates over the course of the
experiment, the concentrates sampled each month have been compared. As shown in “Figure
2” the alkaloid content showed some variations over time but these again showed no trend,
and might rather have been caused by the error of sampling as described in Paper II.
0.0
500.0
1000.0
1500.0
2000.0
2500.0
1 2 3 4 5 6
Month
Alk
aloi
d co
ncen
trat
ion
(µg/
kg)
Control
Ergot 1
Ergot 2
Figure 2: Chronological sequence of the alkaloid concentration in the concentrate (µg/kg on a dry matter basis of 88 %) used in the calf-experiment in the Control group, the Ergot 1 group (1 g ergot/kg concentrate) and the Ergot 2 group (5 g ergot/kg concentrate) The separated rye was additionally analysed for its nutrient composition. The contents of
crude protein (11.2 % DM), crude fibre (2.7 % DM), crude fat (19.1 % DM) and the other
components were comparable to the values of the DLG (1997). However, a modification of
chemical and physical properties of the feedstuff in response to fungal invasion, as it is shown
for other mycotoxins (Matthäus et al., 2004), should be unfounded in the case of ergot. Ergot
mainly develops on one specific grain and does not afflict the whole ear.
Wolff et al. (2001, cited by Mainka, 2006) recommended analysing the six alkaloids and their
isomers mainly found in ergot (ergotamine, ergometrine, ergocornine, ergocryptine,
ergocristine and ergosine) at detection limits not exceeding 10 µg/g to get representative
results. The present study followed this proposal, and in the following, the sum of the six
alkaloids cited above is considered as the total alkaloid content. Nevertheless, reviewing the
literature the analysis profile varies. Some authors (Mainka, 2006) prefer only analysing the
96
General discussion
five alkaloids (key alkaloids) for which standards may be produced (ergotamine, ergometrine,
ergocornine, ergocryptine, ergocristine). Thus for a good comparison it is important to know
which alkaloids have been analysed. Approximately 15 % of the total ergot alkaloids in the
present experiment were ergosine and its isomer (not belonging to the key alkaloids). Thus,
just considering the five key alkaloids, a daily alkaloid exposure (in the highest ergot
supplemented groups) would arise between 6.8 and 7.7 µg/kg LW in fattening bulls (Paper
I), between 23.7 and 25.8 µg/kg LW in calves (Paper II) and between 3.5 and 13.9 µg/kg
LW in dairy cows (Paper III).
As detailed in Table 1, the actual alkaloid exposure per kg LW and day (inclusive ergosine
and its isomer), calculated with the analysed alkaloid contents of each period/month and the
weight and DMI of the respective animals, was quite low in the first experiment with
fattening bulls. Due to the lower LW and the raised ergot content of the experimental
concentrate, the alkaloid exposure of the calves was approximately three times higher.
Whereas the dairy cows were exposed to varying alkaloid doses, which might be classed
between those of the bulls and the calves.
Since ergotism often occurs without any clinical symptoms and with unspecific effects
preponderating (Ross et al., 1989b), in the present study physiological parameters, such as the
body temperature and serum enzyme activities have been additionally considered. Also feed
intake and live weight gain are described to be influenced spontaneously and directly by ergot
intake as the first clinical symptoms (Dinnusson et al., 1971; Whittemore et al., 1976).
Effects on dry matter intake and performance
The current experiments revealed a slight depression in dry mater intake (DMI) of growing
calves when fed the ergot supplemented concentrate only at a daily alkaloid exposure of more
than 30 µg/kg LW (Paper II). At lower exposures no effects on feed intake were detectable in
bulls and cows (Paper I and III). But it has to be considered that Paper II was conducted
with young animals in which the rumen development not has been finished yet. The rumen is
important for the potential toxin degradation due to its microbial population (Hussein and
Brasel, 2001). Thus these animals might have been more sensitive to mycotoxins.
After a period of approximately six weeks the DMI of these calves normalised again, which
might be due to the proceeding rumen development, but might also indicate an adaptation to
the potentially adverse taste of the ergot supplemented concentrate.
97
General discussion
Tab
le 1
: Alk
aloi
d an
d er
got e
xpos
ure
of th
e bu
lls, c
alve
s and
dai
ry c
ows
Tim
e (d
)W
eigh
t (kg
)C
ontro
lEr
got 1
Ergo
t 2C
ontro
lEr
got 1
Bul
lsPe
riod
1-3
000
69.4
413
01.
7(P
aper
I)Pe
riod
230
0 - 4
000
71.5
438
01.
3Pe
riod
340
0 - 5
500
67.3
413
01.
2C
alve
sPe
riod
1Ja
n 28
39.3
367
1374
18.
6(P
aper
II)
Perio
d 2
29 -
5635
.432
5.4
1213
.21
9.6
Perio
d 3
57 -
8428
.125
6.1
931.
50.
87.
5D
airy
cow
s El
sche
n (1
a)76
112
.357
5.4
0.1
(Num
bers
Gut
e (7
)87
10.
061
9.5
0.0
used
for
Elsc
hen
(1b)
717
1.7
556.
00.
0th
e fig
ures
)A
mbr
a (1
0)57
958
2.3
Elli
(9)
618
0.0
538.
70.
0(P
aper
III)
Rog
ate
(4)
613
12.3
576.
00.
3Li
ska
(6)
548
0.0
504.
90.
0U
rte (8
)58
312
.257
7.1
0.3
Fich
te (5
)57
00.
054
4.2
0.0
Wol
ke (3
)57
61.
755
5.5
0.0
Div
isio
nµg
/kg
DM
of t
he ra
tion
µg/d
*kg
LEr
got 2
Ergo
t 1Er
got 2
9.1
2.5
13.3
8.6
1.9
12.6
81.
811
.730
.412
.644
.635
.514
.152
.127
.911
40.9
4.1
6.0
5.5
8.1
7.8
11.4
10.2
15.0
10.6
15.5
13.9
20.4
14.0
20.6
14.2
20.8
14.6
21.4
15.2
22.3
Rei
la (1
2)48
90.
060
7.1
0.0
15.3
22.4
Wal
li (2
)62
61.
754
9.7
0.0
16.3
23.9
Gol
ine
(11)
608
0.0
0.0
Wm
g er
got/d
*kg
LW
98
General discussion
Furthermore, this period coincided with the restriction of the daily concentrate allowance
which in turn induced a more forced roughage intake (Figure 1 and 2, Paper II) and could
have masked the possible ergot-related regulation in feed intake.
None of the experiments revealed a loss in live weight gain. Up to an alkaloid exposure of
30.4 µg/d*kg LW, all effects seemed to be subclinical. Although after a long term
administration to low doses of alkaloids no significant changes in carcass composition or on
individual organs of the bulls could be observed (Paper I), after a slightly increased exposure
several physiological parameters (body temperature, digestibility, DMI) in dairy cows and
calves were influenced (Paper II and III).
Reviewing the literature, many cases are reported where an ergot intake was followed by a
depression in DMI and a decreasing LWG, but most of them were field outbreaks without
exact data on ergot intake or alkaloid exposure (Woods et al., 1966; McKoen and Egan, 1971;
Jessep et al., 1987; Peet et al., 1991; Ilha et al., 2003). The ergot was mainly detected in the
roughage or published as a percent of the ration, which means higher intakes than in the
present study, where the ergot was part of the concentrate.
Dinnusson et al. (1971) recommended that any ration containing 600 mg ergot/kg or more
should be considered as potentially toxic, particular for long-term feeding.
Skarland and Thomas (1972) observed a significant decrease in feed intake and a reduction in
LWG after an administration of 100 – 150 µg alkaloids/kg LW to young heifers and Ross et
al. (1989) used 8 Hereford steers and obtained similar results with a dose of 50.9 mg
ergot/d*kg LW (Paper I). In the current study, calves consumed maximum doses of 52.1 mg
ergot/d*kg LW (Table 1). Thus the initial depression in DMI of the calves (Paper II) would
fit to the results of Ross et al. (1989). That no effects on LWG were observed, might be
caused by differing alkaloid contents of the ergot, since nothing is stated about the ergot
composition by Ross et al. (1989).
Effects on animal health parameters
Diarrhoea associated with ergotism has been described by several authors (Cunningham et al.,
1944b; Greatorex and Mantle, 1974; Greatorex and Mantle, 1973; Loken, 1984).
A higher diarrhoea incidence in the calves of the Ergot group might lead to the assumption
that either the immunological system or the gastro-intestinal mucosa was affected by the
alkaloids (Paper II). Ergot Alkaloids are supposed to decrease α- and γ-globuline production
99
General discussion
(Sterzl et al., 1987; Schultze et al., 1999; Oliver et al., 2000) and to stimulate the natural killer
cells (Kren et al., 1996).
Additionally, the ricinoleic acid, which is also part of the ergot, might have irritated the
intestine (Forth et al., 1992). The ergot used in the present experiments was comprised of 28 g
ricinoleic acid per kg which leads to a calculated maximum ricinoleic acid exposure of the
calves of approximately 2.3 – 2.8 mg/d*kg LW just considering the amount contained in the
ergot (Paper II).
Compared to doses in the literature (approximately 3.6 g/kg LW) used to examine
experimental ricinoleic acid-caused diarrhoea (Kelly et al., 1981), the dose of the present
study with calves seemed to be too low to assume an association to the diarrhoea. However,
as synergistic effects can not be excluded, the ricinoleic acid might have sensitised the
mucosa for the toxic alkaloids or vice versa. An increased endogenous N-loss caused by the
mucosal irritation might also be an explanation for the higher undegraded protein flow to the
duodenum of cows fed with higher doses of ergot (Forth et al., 1992) (Paper III).
Post mortem gross macroscopic inspection of the intestinal mucosa of bulls showed no
obvious alterations (Paper I), but compared to the calves, the alkaloid exposure of the bulls
was three times lower and a histological analysis would be necessary for definite information.
As the absorbed alkaloids are mainly metabolised by the liver and/or might exert toxic effects
on liver tissue (Cheeke, 2006), enzyme activities and other clinical-chemical parameters
indicating such alterations have been analysed (GLDH, γ-GT, AST, total bilirubin, CK and
AP). But no significant changes were detected between the feeding groups in any of the
experiments. In contrast the enzyme activities within one group varied in quite wide ranges
and did not always agree with given reference values (Paper II). Therefore it should be
questioned if these enzyme activities in the serum are an adequate detection tool for ergot
caused liver damage. A histological analysis might here also provide some useful information.
Seeling et al. (2005) observed an OMI-related increase in AST, γ-GT and GLDH activities
(Figure 3) while analysing the influence of Fusarium-contaminated concentrate on these
enzymes. This OMI-related increase was not significant in the present study (Paper III), but
at least a visual trend could be assumed (Figure 4).
100
General discussion
Figure 3: Relationship between organic matter intake and serum activities as reported by Seeling et al. (2005) of AST (y = 30.3 - 1.8*x + 0.2*x²), γ-GT (y = 21.1 - 1.6*x + 0.2*x²) and GLDH (y = 16.8 - 4.9*x + 0.4*x²) in the Control treatment ( ) and the Ergot treatment (▲) of the present study. [Numbers over the symbols denote individual cows]
Figure 4: Relationship between organic matter intake and serum activities of AST (y = 20.3 + 4.9*x; r² = 0.12), γ-GT (y = 6.0 + 2.5*x; r² = 0.12) and GLDH (y = (-20.0 + 3.9*x; r² = 0.15) in the Control treatment (o) and the Ergot treatment (▲) of the present study. [Numbers over the symbols denote individual cows]
This increase of the catalytic enzyme activities with a higher OMI might be caused by the
higher nutrient flow, which in turn was possibly associated with a higher feed related flow of
toxic substances affecting the liver. Thus, serum activities seem to be influenced by other
factors than experimental toxin administration and are probably not always a useful
instrument to control ergot-related effects on the liver.
The body temperature increased by 0.07 °C in cows after a maximum alkaloid exposure of
16.3 µg/d*kg LW (Paper III). The fact that ergot may influence the body temperature
conforms to the reports of many authors analysing the effects of ergot consumption (Ross et
Organic matter intake (kg/d)
γ-G
T (U
/l)
1 7
11
2
3
86
9
5 4
71 1
10
4
12
8
9
3
6
5
2
20
40
60
80
100
120
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
γ-G
T (U
/l)
1 7
11
2
3
86
9
5 4
71 1
10
4
12
8
9
3
6
5
2
20
40
60
80
100
120
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
AST
(U/l)
1
7 11
2 3
8
6
9
54
71
1
10 4
12
8
9
3
65
2
60
100
140
180
220
260
300
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
AST
(U/l)
1
7 11
2 3
8
6
9
54
71
1
10 4
12
8
9
3
65
2
60
100
140
180
220
260
300
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
GLD
H (U
/l)
1 711
2 38
6
9
5
4711
104
12
8
9
3
65
2
0
20
40
60
80
100
120
140
160
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
GLD
H (U
/l)
1 711
2 38
6
9
5
4711
104
12
8
9
3
65
2
0
20
40
60
80
100
120
140
160
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
12
34
5
6
7
8
9
10
1112
1314
12
3
45
6
7
8
9
10
11 12
13
14
0
20
30
40
50
60
70
80
6 8 10 12 14 16 18 20Organic matter intake (kg/d)
1
2
3 45
67
8
9
10
11
12 13
14
12
3 4 5 6 78
9
10
11
12
13
14
0
20
40
60
80
100
120
6 8 10 12 14 16 18
Organic matter intake (kg/d)
12
34
5
6
7
8
9
10
1112
1314
12
3
45
6
7
8
9
10
11 12
13
14
0
20
30
40
50
60
70
80
6 8 10 12 14 16 18 20Organic matter intake (kg/d)
1
2
3 45
67
8
9
10
11
12 13
14
12
3 4 5 6 78
9
10
11
12
13
14
0
20
40
60
80
100
120
6 8 10 12 14 16 18
Organic matter intake (kg/d)
1
2 3
4
5
6
7
8
9
10
1112
13
14
12
3
4 5 6
7
8
9
10
11
12
1314
0
20
30
40
50
60
70
80
90
6 8 10 12 14 16 18 20
Organic matter intake (kg/d)
1
2 3
4
5
6
7
8
9
10
1112
13
14
12
3
4 5 6
7
8
9
10
11
12
1314
0
20
30
40
50
60
70
80
90
6 8 10 12 14 16 18 20
AST GLDH γ-GT
101
General discussion
al., 1989), alkaloid injection (Clark and Clark, 1980; Mcleay et al., 2002) or the consumption
of endophyte infected grasses on animals (Zanzalari et al., 1989; Schmidt and Osborne, 1993;
Cross, 2003). Endophyte concerning data might here serve for comparison, especially because
insufficient data for Claviceps purpurea related effects are available. These endophytes
(Acremonium) share a common phylogeny with Claviceptaecae and their alkaloids cause
some similar toxicosis symptoms. However, in the case of hyperthermia, effects on the body
temperature were reported to be less distinct after ergotamine than after ergovaline
administration (Mcleay et al., 2002). Thus, it might be assumed that also the consumption of
ergot sclerotia causes slighter effects than the same amount of Acremonium sclerotia, for
example.
Al-Tamimi et al. (2003) described a dose of 10 µg total ergopeptine alkaloids/d*kg LW as a
level for minimal induction of the ergot toxicity response since body temperatures in cattle
consuming this amount over 10 days increased 0.14°C more compared to those of the control
group during summer heat stress. In the current study, maximum doses of 15.2 µg
ergopeptines/d*kg LW were fed. Hence, the present results confirm the thesis of Al-Tamimi
et al. (2003).
Metabolism and carry over of ergot alkaloids
Another aim of this study was to examine a potential carry over of toxic alkaloids into edible
tissue. In this context, metabolic and kinetic fates of alkaloids in the ruminant are also of
interest. Westendorf et al. (1993) used abomasally cannulated sheep to analyse abomasal and
faecal recoveries after an administration of 945 mg and 2346 mg ergovaline and its isomer per
day. They detected 50 – 60 % of the alkaloid intake in the abomasal content and only 5 % in
the faeces. In spite of its main fungal source, which is Acremonium coenophialum and not
Claviceps purpurea, ergovaline is very closely related to ergotamine (Cole and Schweikert,
2003). This circumstance justifies a direct comparison of these two alkaloids. In the current
study, ergotamine was the main alkaloid in the ergot. In the duodenal digesta 45.2 ± 14 % of
the ergotamine intake was recovered and an excretion rate via the faeces of approximately
27.0 ± 8 % of intake was determined for this alkaloid. By contrast its isomer ergotaminine
revealed a flow at the duodenum of 133.3 ± 18 % and an excretion with the faeces of 31.1 ± 9
% of the intake of this alkaloid. Figure 5 demonstrates the relationship between the intake and
the flow to the duodenum of these two alkaloids.
102
General discussion
Ergotamine intake (µg/d)
Duo
dena
lerg
otam
ine
(% o
f int
ake)
1
23
6
4
5
9
810
7
1
12
25
35
45
55
65
75
1000 1400 1800 2200 2600 3000
Ergotamine intake (µg/d)
Duo
dena
lerg
otam
ine
(% o
f int
ake)
1
23
6
4
5
9
810
7
1
12
25
35
45
55
65
75
1000 1400 1800 2200 2600 3000
Ergotaminine intake (µg/d)D
uode
nale
rgot
amin
ine
(% o
f int
ake)
1
23
6
4
5
9
8
10
7
1
12
115
125
135
145
155
165
175
400 600 800 1000 1200 1400 1600
Ergotaminine intake (µg/d)D
uode
nale
rgot
amin
ine
(% o
f int
ake)
1
23
6
4
5
9
8
10
7
1
12
115
125
135
145
155
165
175
400 600 800 1000 1200 1400 1600
Figure 5: Relationship between the intake and the flow to the duodenum of ergotamine
(y=65.4 - 0.011*x; r² = 0.252) and its isomer.
Apparently, the forestomach system seemed to have some absorption capacity, which was
already postulated by different authors (Stuedemann et al., 1998; Hill et al., 2001) (Paper
III). But besides this potential absorption, there must also be a high conversion rate to the –
inine isomers of the alkaloids prior to the duodenum. These isomers are, due to a decreased
pharmacological activity, less toxic and occur mainly after a prolonged storage or at lower
pH-values (Wolff et al., 1988; Wolff, 1992; Gloxhuber et al., 1994; Buchta and Cvak, 1999).
As the pH of the abomasum is acidic, this could explain the higher percentage of
ergotaminine compared to ergotamine at the proximal duodenum. With a higher OMI, the
percentage of ergotamine slightly decreases (Figure 6). Since higher amounts of feed
additionally decrease the pH in the rumen, an increased isomerisation rate might be caused.
In the duodenum the pH rises again and stays slightly basic to the rectum. Thus, since the
conversion to the –inine isomers is reversible (Wolff, 1992), the ergotaminine fraction again
decreases to the faeces (Paper III). Similar conversion rates apply for ergocristine and its
isomer. In the duodenal chyme 43.2 ± 13 % of the ergocristine intake and 136.8 ± 64 % of the
ergocristinine intake was detected whereas 10.7 ± 2 % and 16.5 ± 3 % of it respectively were
analysed in the faecal samples, which may confirm the above compiled thesis.
Figure 6 demonstrates the relationship between the intake of ergotamine and ergocristine
inclusive their isomers and the flow of the single alkaloids at the duodenum.
103
General discussion
Intake of ergotamine + ergotaminine (µg/d)
1
2
3
64
59
8
10
7
1
12
1
23
64
5
9
8
1071
12
600
1000
1400
1800
2200
2600
1600 2200 2800 3400 4000Intake of ergotamine + ergotaminine (µg/d)
1
2
3
64
59
8
10
7
1
12
1
23
64
5
9
8
1071
12
600
1000
1400
1800
2200
2600
1600 2200 2800 3400 4000Intake of ergocristine + ergocristinine (µg/d)
1
2
36
4
5
9
8
107 1
12
1
2
36
4
5
9
8
107 1
12
300
500
700
900
1100
1300
1000 1800 2600 3400Intake of ergocristine + ergocristinine (µg/d)
1
2
36
4
5
9
8
107 1
12
1
2
36
4
5
9
8
107 1
12
300
500
700
900
1100
1300
1000 1800 2600 3400
Figure 6: Relationship between the intake of alkaloids (ergotamine and ergocristine) inclusive its isomers and the flow of the alkaloid itself (full) and its isomer (Δ) at the duodenum (µg/d)
Figure 7 demonstrates the relationship between the intake and the flow at the duodenum and
to the faeces respectively of different alkaloids.
a b
1
2
36
4
5
9
8
107
1
12
2500
4500
6500
8500
5000 7000 9000 11000
Total alkaloid intake (µg/d)
Alk
aloi
d flo
wat
the
duod
enum
(µg/
d)
1
2
36
4
5
9
8
107
1
12
2500
4500
6500
8500
5000 7000 9000 11000
Total alkaloid intake (µg/d)
Alk
aloi
d flo
wat
the
duod
enum
(µg/
d)
1
23
64
5
98
10
7
1 12
800
1600
2400
3200
2500 4500 6500 8500Alkaloid flow at the duodenum (µg/d)
Faec
alal
kalo
idex
cret
ion
(µg/
d)
1
23
64
5
98
10
7
1 12
800
1600
2400
3200
2500 4500 6500 8500Alkaloid flow at the duodenum (µg/d)
Faec
alal
kalo
idex
cret
ion
(µg/
d)
c d
1
2
3
64
5
9
8
107
1
12
500
700
900
1100
1300
1000 1400 1800 2200 2600 3000
Ergotamine intake (µg/d)
Ergo
tam
ine
flow
at th
edu
oden
um(µ
g/d)
1
2
3
64
5
9
8
107
1
12
500
700
900
1100
1300
1000 1400 1800 2200 2600 3000
Ergotamine intake (µg/d)
1
2
3
64
5
9
8
107
1
12
500
700
900
1100
1300
1000 1400 1800 2200 2600 3000
Ergotamine intake (µg/d)
Ergo
tam
ine
flow
at th
edu
oden
um(µ
g/d)
1
2
3
64
5
9
8
10
7
1
12
0
200
400
600
800
1000
500 700 900 1100 1300Ergotamine flow at the duodenum (µg/d)
Faec
aler
gota
min
eex
cret
ion
(µg/
d)
1
2
3
64
5
9
8
10
7
1
12
0
200
400
600
800
1000
500 700 900 1100 1300Ergotamine flow at the duodenum (µg/d)
Faec
aler
gota
min
eex
cret
ion
(µg/
d)
104
General discussion
e f
1
23
64
5
9
810
7
1
12
150
250
350
450
550
800 1600 2400Ergotaminine flow at the duodenum (µg/d)
Faec
aler
gota
min
ine
excr
etio
n(µ
/d)
1
23
64
5
9
810
7
1
12
150
250
350
450
550
800 1600 2400Ergotaminine flow at the duodenum (µg/d)
Faec
aler
gota
min
ine
excr
etio
n(µ
/d)
Ergotaminine intake (µg/d)
1
23
6 4
59
8
107
1
12
800
1200
1600
2000
2400
2800
400 800 1200 1600
Ergo
tam
inin
eflo
wat
the
duod
enum
(µg/
d)
Ergotaminine intake (µg/d)
1
23
6 4
59
8
107
1
12
800
1200
1600
2000
2400
2800
400 800 1200 1600
Ergo
tam
inin
eflo
wat
the
duod
enum
(µg/
d)
1
23
6 4
59
8
107
1
12
800
1200
1600
2000
2400
2800
400 800 1200 1600
Ergo
tam
inin
eflo
wat
the
duod
enum
(µg/
d)
g h
1
2
36
4
5
9
8
1071
12
200
400
600
800
1000
800 1600 2400Ergocristine intake (µg/d)Er
gocr
istin
eflo
wat
the
duod
enum
(µg/
d)
1
2
36
4
5
9
8
1071
12
200
400
600
800
1000
800 1600 2400Ergocristine intake (µg/d)Er
gocr
istin
eflo
wat
the
duod
enum
(µg/
d)
1
2
3
6
4
59
8
10
7 1
12
60
100
140
180
220
260
200 400 600 800 1000Ergocristine flow at the duodenum (µg/d)
Faec
aler
gocr
istin
eex
cret
ion
(µg/
d)
1
2
3
6
4
59
8
10
7 1
12
60
100
140
180
220
260
200 400 600 800 1000Ergocristine flow at the duodenum (µg/d)
Faec
aler
gocr
istin
eex
cret
ion
(µg/
d)
Figure 7: Relationship between the alkaloid intake and the alkaloid flow at the duodenum and to the faeces respectively for ergotamine, ergotaminine, ergocristine and the sum of total alkaloids a : y = - 212 + 0.701 * x; r² = 0.69 b : y = 74.2 + 0.346 * x; r² = 0.44 c : y = 299 + 0.262 * x ; r² = 0.32 d : y = 90.1 + 0.506 * x ; r² = 0.44 e : y = - 113 + 1.46 * x ; r² = 0.84 f : y = 106 + 0.144 * x ; r² = 0.30 g : y = 181 + 0.266 * x ; r² = 0.56 h : y = 25.6 + 0.209 * x; r² = 0.54 The percentages redetected at the duodenum differed between the alkaloids (Figure 7), which
might be caused by the differing chemical properties again. However, in evaluating the slopes
in Figure 7, it has to be considered that most of the r²-values are too low to get exact data.
Approximately 70 % of the ingested total alkaloids and even 146 % of the ingested
ergotaminine were found in the duodenal digesta whereas the duodenal flow of ergotamine
and ergocristine was much lower. From the alkaloids flowing at the duodenum, excretion
rates via the faeces lay between 14 and 51 %.
105
General discussion
A loss of detectable alkaloids on the way through the animal may have many reasons. Besides
absorption and isomerisation, a potential degradation by microbes or due to chemical changes
(for example the pH) must be considered. Young and Marquardt (1982) examined in growing
chicken that about 5 % of the alkaloid fed was excreted unchanged and 15 – 20 % of the
alkaloid intake was detected as a complex mixture of 16 possible metabolites. Metabolites and
degraded alkaloids might be undetectable with the method used, but may still have effects on
the animal, which should be clarified in following studies.
Once absorbed and passed into the blood stream, the alkaloids might be spread over the whole
organism and thus might also be carried over into milk or other edible tissues which is a very
important question from a consumer protection point of view.
In this context in the present study blood and organ samples were analysed for alkaloids
(Paper I, II and III).
A preliminary study revealed that the time between the collection of the blood sample and
deep-freezing of the prepared serum sample was without influence on the alkaloid
concentration within the tested period of six hours when the blood sample was initially spiked
with alkaloids. Thus the failure of alkaloid detection in blood samples collected during the
experiments with calves, bulls and cows was not related to the period between blood
collection and deep-freezing of the serum sample.
No carry over of ergot alkaloids into blood, milk or organ samples could be detected.
Since the alkaloid concentration in the blood is reported to climax 2 hours after the
administration (Kalberer, 1970), the time of sampling is of great importance. The feeding time
in the bull experiment, as well as with the calves, was not terminable, because automatic self
feeders were used. Thus it may not be excluded that in both experiments the moment of
sampling was too long after feeding to find detectable amounts of alkaloids in the blood
(Paper I and II).
In the third experiment with dairy cows the time of sampling was exactly two hours after the
beginning of the morning feeding (Paper III), even so the alkaloid content was also lower
than the indicated detection limits. Moubarak et al. (1996) administered 14 µg ergotamine/kg
LW to steers by intravenous injection. As expected, the alkaloid concentration in the blood
peaked immediately after the injection, but within 10 min approximately 70 % of it were
metabolised or excreted. Same results were found for ergosine and ergine, which indicates a
very small time slot for analysis.
106
General discussion
Some new data on a 209-day exposure to endophyte-infected tall fescue demonstrate that
there is potential carry over into beef tissue (Realini et al., 2006). Unfortunately the alkaloid
content of the tall fescue is not detailed and nothing can be said about the intake. Thus a
comparison to the current study is quite difficult.
The present results demonstrate that an alkaloid exposure not exceeding 9.1 µg/d*kg LW did
not result in a detectable carry over into edible tissue of bulls (Paper I).
Several other scientists examined a potential carry over of ergot alkaloids into milk.
Cunningham et al. (1944a) fed dairy cows with doses up to 24 g total alkaloids per day but, in
spite of clinical symptoms, no alkaloids could be detected in milk, meat or viscera by analysis
or animal feeding experiment in which rats were fed the organs and tissues of the sickened
cows. Due to the outdated colorimetric procedure of analysis used in this study, which was
featured for a detection limit of 500 ng/g, the results have to be judged carefully.
Wolff et al. (1995), who analysed milk samples as negative after a mean alkaloid exposure of
3 µg/d*kg LW over a period of five weeks assumed that the rate of alkaloid excretion is less
than ten per cent, if at all and that an accumulation of the alkaloids in the milk might be nearly
excluded.
In the present experiments no carry over into milk could be detected. But considering the
detection limit for each alkaloid of 6 ng/g DM (except for ergometrine and its isomer, where it
was 11 ng/g DM), a potential carry over of 82 ng total alkaloids/g DM, may not be excluded
(worst-case-scenario).
This worst case calculation would climax in a potential carry over rate of 1.7 % calculated
with a mean milk yield of 19.5 kg/day and an alkaloid intake of 10.6 mg/day.
107
Conclusions
Conclusions Since the alkaloid content of different ergot samples varies in wide ranges, information on the
alkaloid exposure is necessary in studies dealing with the effects of ergot on animals.
A long-term exposure of bulls to alkaloid doses not exceeding 9.1 µg/kg LW and day seemed
to be tolerated quite well. No negative effects on performance or carcass composition could
be proven and a carry over into edible tissue could not be detected.
In the present experiment, after an exposure of up to 30.4 µg ergot alkaloids/kg LW and day,
calves in the first third of the study reacted with a decreased DMI. This dose was indeed twice
as high as in the cow experiment, where no effects on DMI were detected. But calves in the
second third of the experiment were even exposed to higher alkaloid doses and their DMI
returned to the level of the control group. Since the rumen of the calves at the beginning of the
study was not yet completely developed, a possible relation to the rumen development might
be drawn. The rumen microbes might present a first line of defence against toxic substances
and thus might help the calves to tolerate the ergot intake. Another reason for the
normalisation of DMI after the first six weeks of the study might be an adaptation to the
potentially adverse taste of the ergot supplemented feed. And it has to be considered in
interpreting the results, that the concentrate intake of the calves was restricted, and that this
restriction might have masked the overall difference in DMI between the groups.
Maximum doses of 16.3 µg/kg LW and day in dairy cows were shown to eventually cause a
shift in the microbial population of the rumen followed by changes in the protein and NDF
digestibility. Further research should comprise the direct analysis of the microbial
composition in the rumen.
Furthermore, at doses of 16.3 µg/kg LW and day in dairy cows, the body temperature was
significantly elevated due to ergot feeding which indicates a physiological response by the
animal.
A carry over into the milk of dairy cows could not be detected at this exposure.
In the present experiment due to experimental conditions prolactine levels in the blood have
not been analysed, but in following studies this additional parameter might provide some
useful information.
108
Conclusions
In the current study, approximately 67 ± 13 % of the alkaloid intake were recovered in the
duodenal digesta, and approximately 24 ± 8 % were excreted with the faeces. These
percentages were not influenced by the feed intake level or the passage rate. Since the mean
percentage of –in isomers of total alkaloids was 73 ± 7 % in the concentrate, 48 ± 3 % in the
duodenal chyme and 68 ± 8 % in the faeces, a reversible isomerisation in the abomasum due
to the low pH or transforming microbes might be assumed. This relationship was also
revealed for single alkaloids as for example for ergotamine.
Analysing specific serum enzyme activities to get information about the liver status may not
always be a useful method, since these activities in the present study seemed to be influenced
by other factors, such as for example the amount of feed intake. In the following studies, a
histological analysis of the liver might probably provide some useful information.
The given weight-based upper limit of 1000 mg ergot/kg unground cereal is not justifiable.
Considering the extremely varying alkaloid content, the upper limit should be based on the
alkaloid concentration in the diet. This would also be more practical as it allows the
assessment of pelleted feed stuffs and not only unground cereals.
The alkaloid doses used for the calves (up to 35.5 µg/d*kg LW) and the fattening bulls (up to
9.1 µg/d*kg LW) were too low to titrate a NOEL, since no significant differences between the
feeding groups could be detected in either experiment. Thus further research with wide-
graded levels of alkaloid intake comprising the doses used in the present experiment, and also
exceeding them, is needed for NOEL-titration.
In dairy cows, even at the low alkaloid dose used (4.1 – 16.3 µg/d*kg LW), significant
physiological effects have been observed, which necessitate dose-response experiments
including ergot administrations lower than that, to clarify the effects on dairy cows.
Additional parameters as for example the age, the race, the outside temperature and the rate of
passage through the rumen, should be considered in following studies, since these factors
seemed to influence the ergot-induced effects.
With regard to consumer protection it might be concluded that carry over rates into edible
tissue at doses not exceeding 9.1 µg/d*kg LW (meat) and 16.3 µg/d*kg LW (milk)
respectively are negligible.
109
Summary
Summary
Barbara Schumann
Effects of ergot on health and performance of ruminants and carry over of the ergot
alkaloids into edible tissue
The sclerotia of Claviceps purpurea, referred to as ergot, may cause toxic effects in farm
animals.
During the past few years, ergotism re-gained in importance. This might be due to the wet
weather conditions, which facilitated the spreading of the fungus. But it might also be caused
by the use of high yielding, but susceptible rye hybrids and the abolition of the rye
intervention, which was followed by an increased rye-feeding.
Until now, only a weight-based upper limit of 1000 mg ergot/kg unground cereal applies for
farm animals. But the alkaloid content of the ergot varies in wide ranges, and literature
concerning the effects of ergot feeding on ruminants is scarce. Thus, it was the aim of this
study to reveal potential relations between a defined alkaloid exposure and different
performance parameters, the physiological response of cattle and carry over into edible tissues
and milk by conducting experiments with animals of different ages and production stages.
In a first long-term experiment with 38 Holstein-Friesian bulls over a period of approximately
230 days, doses of 0, 0.45 and 2.25 g ergot sclerotia/kg concentrate were fed, which
corresponded to an alkaloid exposure of 1.2 – 9.1 µg/kg BW and day. At a mean live weight
of 550 kg, bulls were slaughtered and samples of blood, urine, kidneys, liver, muscle and fat
tissue were taken for alkaloid analysis.
Statistically no significant differences could be detected between the three feeding groups.
With a mean DMI of 7.35 kg/d, bulls gained 1.41 kg live weight per day. Liver enzyme
activities measured in the blood and carcass composition were not significantly influenced by
ergot feeding. The alkaloid content of all samples analysed (edible tissue, blood and urine)
was lower than the indicated detection limits.
A second 84-day experiment with 35 calves of the same race was conducted. The ergot dose
was raised to 0, 1 and 5 g/kg concentrate (maximum exposure of 35.5 µg ergot alkaloids/kg
LW and day). DMI and LWG were not significantly influenced by increasing ergot
proportions when the whole experimental period was considered, although there was a trend
for an ergot-related decrease in concentrate intake during the first 6 weeks of the experiment.
110
Summary
This temporary decrease might be caused by a taste aversion, but might also be due to the
proceeding rumen development. Neither altered liver enzyme activities nor an alkaloid carry
over could be detected in blood samples, taken at the end of the experiment.
In the present digestibility study with 12 double fistulated dairy cows (one cannula in the
rumen and the other in the proximal duodenum), ergot doses of 0 and 2.25 g/kg concentrate
were used, which corresponded to 4.1 – 16.3 µg alkaloids/kg LW and day. The aim of this
experiment was to reveal potential relations between an ergot-contamination of the
concentrate at differing levels of feed intake and the ruminal fermentation and physiological
response of the cows. Each cow was fed with the control diet and with the ergot diet at a ratio
of 40 % concentrate and 60 % maize silage in two different periods. The amount of feed
intake was adjusted to the production stage, which resulted in a DMI between 6 and 18.5 kg/d.
Each period consisted of two weeks of adaptation, one week of digesta sampling and one
week for collecting ruminal fluid, faeces, urine, milk and blood samples.
Isovalerate, propionate and NH3-N concentrations in the rumen fluid were significantly
influenced by ergot feeding, and the amount of ruminally undegraded protein, as well as the
fermentation of NDF, tended to be increased with the ergot supplementation at higher levels
of feed intake, which might indicate a shift in the microbial population and/or activity.
Approximately 67 % of the alkaloids fed were recovered in the duodenal digesta and
approximately 24 % of the intake were excreted with the faeces. The anally measured body
temperature of the cows significantly increased after ergot administration (0.07°C). No
alkaloid residues could be detected in the blood, urine or milk samples and the activities of
liver enzymes in the serum were independent of ergot feeding.
It may be concluded that in terms of consumer protection, carry over rates of ergot alkaloids
into edible tissue at doses not exceeding 9.1 µg alkaloids/kg LW and day (meat) and 16.3 µg
alkaloids/kg LW and day (milk), respectively, are negligible.
However, even these low doses seemed to influence the rumen population and, as the body
temperature in dairy cows increased, to induce a physiological reaction by the animal.
At an exposure of calves up to 35.5 µg ergot alkaloids/kg LW and day, slight clinical effects
seem to be caused (temporary decrease in DMI of the calves).
To derive a NOEL, further experiments with defined and more widely graded alkaloid
exposures and cattle of different ages and production stages are necessary.
111
Summary
Based on such NOELs, upper limits of alkaloid exposure per kg LW and day could be
established which then would be applicable also for pelleted or mixed feedstuffs.
112
Zusammenfassung
Zusammenfassung
Barbara Schumann
Einfluss von Mutterkorn auf Gesundheit und Leistung von Wiederkäuern und Carry
Over der Ergotalkaloide in essbare Gewebe
Das als Mutterkorn bezeichnete Sklerotium des Feldpilzes Claviceps purpurea kann, wenn es
in den Futtertrog von Rindern gelangt, Vergiftungssymptome auslösen.
Durch die feuchte Witterung, die eine Ausbreitung des Pilzes begünstigt, und die
Abschaffung der Roggenintervention, die zu vermehrter Verfütterung der neuen für
Mutterkorn anfälligen Roggenlinien führte, gewann Mutterkorn wieder an Bedeutung in den
letzten Jahren.
Bisher gilt für Nutztiere nur ein Höchstwert von 1000 mg Mutterkorn/kg unzerkleinertes
Getreide. Da aber der Alkaloidgehalt verschiedener Mutterkörner sehr stark variiert und es
bisher kaum Untersuchungen zu diesem Thema gibt, war es Ziel dieser Arbeit anhand erster
orientierender Dosis-Wirkungsversuche die Zusammenhänge zwischen einer definierten
Alkaloidaufnahme und physiologischen Werten, verschiedenen Leistungsparametern, sowie
dem Carry-Over in essbare Gewebe und Milch bei Tieren unterschiedlichen Alters und
verschiedener Produktionsstufen zu bestimmen.
In einem ersten Langzeitversuch an 38 Mastbullen (Deutsche Holstein) wurde über einen
Zeitraum von ungefähr 230 Tagen 0, 0,45, bzw. 2,25 g Mutterkorn/kg Kraftfutter verfüttert,
was einer Alkaloidexposition von 1,2 – 9,1 µg/kg Lebendmasse (LM) und Tag entsprach. Mit
einem Gewicht von ca. 550 kg wurden die Tiere geschlachtet und Blut, Harn, Nieren-,
Muskel-, Leber- und Fettgewebe für die Mutterkornanalyse gewonnen.
Statistisch konnten keine Unterschiede zwischen den drei Fütterungsgruppen nachgewiesen
werden. Bei einer mittleren Trockensubstanzaufnahme (TSA) von 7,35 kg/Tag wurde im
Mittel über alle Gruppen eine Lebendmassezunahme (LMZ) von 1,41 kg/Tag ermittelt.
Sowohl die Aktivitäten der im Blut gemessen Leberenzyme, als auch die
Schlachtkörperzusammensetzung unterschieden sich statistisch nicht. Die Alkaloidgehalte
sämtlicher untersuchter Proben war niedriger als die jeweilige Nachweisgrenze.
Im Anschluss wurde ein 84-Tage-Versuch mit 35 Kälbern derselben Rasse durchgeführt. Der
Mutterkorngehalt des Kraftfutters wurde auf 0, 1, bzw. 5 g/kg angehoben (maximale
Alkaloidexposition von 35,5 µg/kg LM und Tag). TSA und LMZ waren, über den gesamten
113
Zusammenfassung
Zeitraum betrachtet, durch ansteigende Mutterkornkonzentrationen im Kraftfutter nicht
signifikant beeinflusst, dennoch gab es in den ersten sechs Wochen einen Trend zu einer
Mutterkornbedingten Abnahme der TSA bezogen auf das Kraftfutter. Gründe hierfür können
sowohl eine anfängliche Geschmacksaversion als auch die fortschreitende Pansenentwicklung
sein. In einer am Versuchsende gewonnenen Blutprobe konnten weder erhöhte Leberwerte,
noch übergetretene Alkaloide nachgewiesen werden.
In einem parallel dazu durchgeführten Verdauungsversuch mit 12 doppelt fistulierten
Milchkühen (Pansen und proximales Duodenum) wurden Mutterkornkonzentration von 0,
bzw. 2,25 g/kg Kraftfutter verfüttert, was einer Alkaloidexposition von 4,1 – 16,3 µg/kg LM
und Tag entsprach. Ziel dieses Versuches war es eventuelle Zusammenhänge zwischen einer
Mutterkornkontamination des Futters bei unterschiedlichem Futteraufnahmeniveau und der
Pansenfermentation sowie physiologischen Parametern der Kühe zu untersuchen. Jede Kuh
wurde sowohl einmal mit der Kontrollration als auch einmal mit der kontaminierten Ration im
Verhältnis 40 % Kraftfutter zu 60 % Maissilage gefüttert, wobei die jeweilige Menge dem
Laktationsstadium angepasst wurde. Die einzelnen Perioden bestanden aus 2 Wochen
Anfütterung, einer Woche Darmsaftsammlung und einer Woche, in der Pansensaft, Kot, Harn,
Milch und Blut gewonnen wurde.
Isovalerat-, Propionat- und NH3-N-konzentrationen im Pansensaft waren durch die
Mutterkornzulage signifikant beeinflusst. Auch die Menge an im Pansen nicht abgebautem
Futterprotein und die NDF-Fermentation nahmen mit der Mutterkornfütterung zu, was auf
einen Verschiebung in der Mikrobenpopulation und/oder ihrer Aktivität hindeuten könnte.
Von der verfütterten Menge an Alkaloiden wurden ungefähr 67 % im Darmsaft wieder
gefunden und ca. 24 % in den Kotproben. Die anal-gemessene Körpertemperatur der Tiere
stieg mutterkorn-bedingt signifikant (um ca. 0.07 °C) an. Es konnten weder
Alkaloidübergänge in Blut, Urin oder Milch noch erhöhte Leberwerte im Serum nach
Mutterkornzulage festgestellt werden.
Zusammenfassend ist zu sagen, dass den Verbraucherschutz betreffend bei
Alkaloidexpositionen bis zu 9.1 µg/kg LM und Tag (Fleisch), bzw. 16.3 µg/kg LM und Tag
(Milch) kein bedrohlicher Übergang der Toxine in essbare Gewebe zu erwarten ist. Trotzdem
scheinen auch diese niedrigen Konzentrationen schon die Pansenpopulation zu beeinflussen
und, wie durch die erhöhte Körpertemperatur der Milchkühe verdeutlicht, eine Reaktion im
Körper der Tiere auszulösen. Bei Expositionen bis zu 35.5 µg/kg LM und Tag scheinen
114
Zusammenfassung
bereits erste klinische Effekte deutlich zu werden (vorübergehend verringerte Futteraufnahme
der Kälber).
Um ein NOEL bestimmen zu können sind weitere Versuche mit definierter und noch weiter
gefächerter Alkaloidexposition an Tieren unterschiedlicher Produktionsstufen nötig.
Daraus ließen sich dann neue Richtwerte, bezogen auf die Alkaloidaufnahme pro kg LM und
Tag ableiten, die sich dann auch auf Mischfutter anwenden ließen.
115
References
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Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, dass ich die vorliegende Dissertation „Effects of ergot on
health and performance of ruminants and carry over of the ergot alkaloids into edible tissue”
selbständig verfasst habe. Bei der Anfertigung wurden folgende Hilfen Dritter in Anspruch
genommen:
Die Mykotoxinanalysen wurden durch Herrn Dr. Ueberschär und Mitarbeiter an der FAL
Celle durchgeführt. Die Bestimmung der Serumenzyme und des Bilirubins wurde von der
Klinik für Rinder der Tierärztlichen Hochschule Hannover durchgeführt.
Das statistische Modell in Paper III wurde mit Hilfe von Herrn Prof. Dr. Spilke der
Landwirtschaftlichen Fakultät der Martin-Luther-Universität in Halle-Wittenberg entwickelt.
Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten
(Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir
unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die im
Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.
Ich habe die Dissertation am Institut für Tierernährung der Bundesforschungsanstalt für
Landwirtschaft (FAL) in Braunschweig angefertigt.
Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen ähnlichen
Zweck zur Beurteilung eingereicht.
Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig und der
Wahrheit entsprechend gemacht habe.
Hannover, den
Barbara Schumann
Danksagung Ich danke Herrn Prof. G. Flachowsky und Herrn Dr. S. Dänicke für die Überlassung des
Themas und die Möglichkeit diese Arbeit am Institut für Tierernährung der
Bundesforschungsanstalt für Landwirtschaft (FAL) in Braunschweig durchführen zu können.
Herrn Dr. S. Dänicke gilt mein besonderer Dank ebenfalls für die hervorragende Betreuung,
die Unterstützung bei der Planung und Durchführung der Versuche, sowie die nicht endende
Geduld bei der Beantwortung von Fragen meinerseits.
Ferner möchte ich mich bei Herrn Prof. G. Breves für die gute Zusammenarbeit und
Betreuung dieser Arbeit an der Tierärztlichen Hochschule Hannover bedanken.
Bei sämtlichen Labormitarbeitern möchte ich mich für die gute Laune, die stets freundliche
Arbeitsatmosphäre im Labor und die große Hilfsbereitschaft beim Analysieren der vielen
Proben bedanken. Mein ganz besonderer Dank gilt hierbei Herrn Axel Jagow für die große
Unterstützung und die geduldigen Erklärungen bei den Chromanalysen.
Ich danke den Mitarbeitern der Versuchsstation und des Tierhauses für die Betreuung der
Tiere, sowie Sabine Hartinger für die spontane Hilfe bei jeglichen „Computerfragen“.
Ein großes Dankeschön geht außerdem an „Kalle“ Kiemann für die große Hilfsbereitschaft
und die Durchführung der Ergotanalysen in Celle.
Schließlich danke ich meinen Mitdoktoranden/-innen und –diplomanden/-innen für den Spaß,
den wir inner- und außerhalb der FAL miteinander hatten und allen Darmsaftsammlern für die
große Unterstützung während der Sammelwochen.
Mein besonderer Dank gilt Christina und Ginie, die es auch in anstrengenden Phasen so tapfer
mit mir zusammen in einem Büro, bzw. einer Wohnung ausgehalten haben, und meiner
Familie für Ihre nicht endende Unterstützung und ihr Vertrauen. Ihnen sei diese Arbeit
gewidmet.