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
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: Ergot sklerotia on a grain ear (Bös, 2000) Figure 1: Ergot sklerotia on a grain ear (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).
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 ring Figure 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).
AlkaloidEmpirical formula Rabbit (mg/kg i.v.)M PhenylalanineErgotamineC33H35N5O53 α-hydroxy-alaninLeucineErgosineC30H37N5O5 ValineErgovalineC29H35N5O5 PhenylalanineErgocristineC35H39N5O51.9 Prolinα-hydroxy-valinLeucineErgocryptineCHNO0.8
Components of the peptideLD50(Griffith
1: “Periodical system” of the ergot alkaloids according to Hoffmann (1964) modified by Mühle and Breuel (197 GroupFungal source (Cole and Schweikert, 2003) ouse (mg/kg oral) 3200CP Ergotamine-CP CP, ET, AC CP 324155Ergotoxine-CP ValineErgocornineC31H39N5O50.92000CP PhenylalanineErgostineC34H37N5O5CP α-hydroxy-α-amino-butyric acidLeucineErgoptineC31H39N5O5Ergoxine-CP ValineErgonineC30H37N5O5CP ErgometrineC19H23N3O23.2460CP, BE, BH, BC
et al., 1978)
7) and extended by ergometrine =Claviceps purpurea, ET =Epichloe typhina, AC =Acremonium coenophialum, BE =Balansia epichloe, BH =Balansia henningsiana, BC = Balansia claviceps
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).
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
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.
(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
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)
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
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,
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
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 ?
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
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).
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
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.
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.
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.
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
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.
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).
recovery) in 20 µl (n = 2-3) Ergo- cryptinine Ergo- cristineErgo- cristinine
Table II. Mean recovery rates [%] after an addition of 0.5 ng of the alkaloid (2.5ng for ergometrine Ergo- metrine Ergo- metrinineErgo- tamineErgo- aminineErgo- cornine Ergo- corninine Ergo- cryptine Serum 107 103 94 99 89 102 100 92 89 97 Fat 107 88 117 96 95 73 103 Muscle 80 90 70 81 83 73 72 Liver 87 75 63 68 73 64 60
52 91 65 65 75 64 52 64 55 Kidney 115 94 87 85 85 80 86 69 77 71 Bile 105 88 90 90 86 82 94 72 83 79 Urine 87 100 90 97 89 98 99 89 84 95 Feedstuff 139 103 75 81 87 58 71 56 76 45
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).
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.
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.
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.
s eart Thymus PancreasSpleen Testicles
Table IV. Effects of ergot contaminated concentrate on carcass composition and organ weights of growing bulls EBW° Dressing† LiverLung KidneyHLive weight* Abdominal fat‡Gastroin- testinaltract Ergot (g/kg concentrate)[kg][kg][%] [kg/100 kg EBW] [g /100 kg EBW] 0.00 555 492 52.0 8.454.78 1506 701 230406 105 114 218 383 0.45 555 497 52.4 8.214.60 1524 731 241 2.25 553 497 52.8 8.194.65 1513 720 226 ANOVA (probabilities) Ergot 0.7580.463 0.211 0.898 0.329 0.923 0.571 0.258 linear 0.4960.280 0.081 0.676 0.302 0.878 0.500 0.641 quadratic 0.7840.530 0.928 0.837 0.270 0.7120.409 0.119
421 101 115 231 380 421 101 105 232 398 0.674 0.915 0.3270.273 0.648 0.436 0.715 0.2290.146 0.475 0.664 0.832 0.3900.467 0.566 PSEM 2 3 0.3 0.430.09 31207 148 5 7 15 *After at least 7 h fasting ° Empty body weight: difference between live weight and the weight of the contents of the gastro-intestinal tract and of the urinary bladder † Weights of the warm carcass as percent of live weight ‡ Sum of the fat of the kidney cavity and the fat covering the gastro-intestinal tract
