On the composition of ergot and the effects of feeding two different ergot sources on piglets

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Animal Feed Science and Technology 139 (2007) 52–68

On the composition of ergot and the effects of feeding two different ergot sources on piglets Simone Mainka a , S. D¨anicke a,∗ , H. B¨ohme a , K.-H. Uebersch¨ar a , F. Liebert b a b

Institute of Animal Nutrition, Federal Agricultural Research Centre, Braunschweig (FAL), Bundesallee 50, 38116 Braunschweig, Germany Institute of Animal Physiology and Animal Nutrition, Georg-August-University G¨ottingen, Kellnerweg 6, 37077 G¨ottingen, Germany

Received 3 November 2005; received in revised form 20 November 2006; accepted 12 December 2006

Abstract It was one purpose of the study to obtain actual data on the composition of ergot (Claviceps purpurea), especially concerning the variation of the content of toxic alkaloids and the alkaloid pattern. For this reason 13 ergot batches, sorted out of naturally infected rye from different origins in Central Europe, were analysed for their crude nutrient, fatty acid, amino acid and alkaloid contents. The mean concentrations of crude protein, fat and fibre amounted to 223, 350 and 287 g/kg dry matter (DM). The concentration of the ergot specific ricinoleic acid was 14.4 g/100 g fatty acids on average. The mean total ergot alkaloid content (sum of ergometrine, ergotamine, ergocornine, ␣-ergocryptine, ergocristine, ergosine and their -inine isomers analysed by a high performance liquid chromatography (HPLC) method) was 738 mg/kg DM and showed a high variation (24–1569 mg/kg DM). The alkaloid pattern was variable as well but Ergotamine, Ergocristine and Ergosin represented the main alkaloids with average contents of 20, 24 and 15 g/100 g total alkaloids. Especially due to the high variation of the alkaloid content in ergot, the analysis of the toxic alkaloids in feed is more useful to evaluate potential risks at feeding instead of the determination of the ergot content in grain, which is current practice in the European Union up until now. Additionally, a 35-day experiment with 80 crossbred piglets from 9.2 to 24.4 kg body weight (BW), which were allocated into 5 groups, was conducted to determine the critical alkaloid contamination level in feed and to study the effect of different alkaloid patterns. Therefore, two ergot sources

∗

Corresponding author. Tel.: +49 531 5963102; fax: +49 531 5963199. E-mail address: [email protected] (S. D¨anicke).

0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.12.001

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were used, which were characterized by different alkaloid patterns, and standardised on equal total alkaloid contents in two concentrations at 5.6 and 11.2 mg/kg diet. The alkaloid pattern had no effect on growth performance and serum biochemical parameters (P>0.05). However, the extent of the dietary amount of alkaloids had an influence since the cumulative body weight gains of the high supplemented groups were significantly decreased as compared to the control (P<0.05) and showed significant linear dose–response effects (P<0.05). The serum protein content was significantly decreased at P<0.05 as well. The evaluation of the total or key alkaloid contents (sum of ergometrine, ergotamine, ergocornine, ␣-ergocryptine and ergocristine) in grain or feed seems to be practicable to evaluate the feeding hazard without special consideration of the respective alkaloid pattern. The critical contamination level in the present experiment was determined to be 3.57 mg total alkaloids/kg feed (=1.89 mg key alkaloids/kg feed) for piglets. © 2007 Elsevier B.V. All rights reserved. Keywords: Ergot composition; Alkaloid pattern; Piglet

1. Introduction To prevent ergot intoxications of animals due to contaminated feeding stuffs, a maximum amount of 1000 mg ergot/kg grain is permitted in the European Union (Council Directive 2002/32/EC of 7 May 2002). However, the content of alkaloids, which are known to be the main toxic components in ergot (Rotter et al., 1985; Wirth and Gloxhuber, 1994), is highly variable whereas only scarce recent information on the alkaloid content of ergot can be found (Young, 1981a,b; Young and Chen, 1982). Furthermore, the ergot content is no longer detectable in ground grain and the sensitivity of different animal species varies as well. For these reasons, the analysis of the toxic alkaloids in feed is more precise to evaluate the feeding hazards than a survey of the ergot contamination in grain. However, no valid critical dietary contamination levels of ergot alkaloids for the different animal species exist up until now. Additionally, the alkaloid pattern of ergot is variable (Meinicke, 1956; Young, 1981a,b). The pharmacological characteristics and the interactions of the ergot alkaloids may cause different effects on health and performance of animals when ergot containing altering alkaloid patterns is fed (Hofmann, 1964; Berde and St¨urmer, 1978; Griffith et al., 1978). For instance, Rotter et al. (1985) detected variations in the toxic effects of ergot from five sources, which were standardised to the same dietary total alkaloid content in chicks, and attributed them to the different ergot alkaloid patterns. Therefore, the following objectives of the present study were deduced: - One purpose of the present study was to obtain new data on the composition of ergot, especially alkaloids. Therefore, ergot from different sources was analysed on the alkaloid content and pattern. Moreover, the nutrient, fatty acid and amino acid contents were determined as well. - In order to investigate the critical total alkaloid content in feed for piglets on the basis of the no observed effect level, defined as “Greatest concentration or amount of a substance, found by observation or experiment, which causes no detectable effect.” (WHO, 1994),

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diets containing 5.6 mg and 11.2 mg total alkaloids/kg were prepared because a previous feeding experiment with piglets (Mainka et al., 2005a) indicated the critical level of dietary total alkaloids was in this range. - In addition, to study the effects of feeding different alkaloid patterns, two different ergot sources were selected for the piglet trial on the basis of their alkaloid composition. Hence, the diets were standardised using both ergot sources to equal total alkaloid contents in two graded levels (5.6 mg and 11.2 mg total alkaloids/kg).

2. Material and methods 2.1. Ergot screening Representative samples of ergot from different breeding areas at Lohheide (five sources), Hermannsburg (five sources), W¨urzburg (one source), Worms (one source) and from France (one source) were sorted out of the rye harvest in 2003 by a colour based technique with photoelectric cells. The contents of crude ash (CA), crude protein (CP), crude fat (CL), crude fibre (CF), starch, sugar, fatty acids, amino acids and ergot alkaloids were analysed. 2.2. Piglet feeding experiment 2.2.1. Ergot sources and experimental diets A basal uncontaminated control diet (CON) based on cereals and soybeans (Table 1) was formulated as meal (4 mm sieve) to meet the recommendations for pigs (GfE, 1987). On this basis, the experimental diets were prepared with two different ergot sources (A or B). Calculated by the alkaloid contents of Ergot Sources A and B (Table 2), the ergot incorporation levels of Diets A1 und B1, as well as A2 and B2, were standardised to contain the same total alkaloid concentrations of 5.6 and 11.2 mg/kg, respectively. 2.2.2. Animals and management Eighty crossbred piglets [Federal hybrid breeding programme (BHZP), 40 gilts and 40 barrows], weaned with 4 weeks of age, were allocated to 20 slatted floor pens (four piglets per pen, four pens per treatment). With consideration of sex and initial body weight, the animals were randomly assigned to the five treatments in order to ensure the same experimental conditions for all groups. During one pre-experimental adaptation week (5th week of live) all piglets were fed the uncontaminated basal control diet (Table 1). Feed and water were offered ad libitum. The experimental period started at an average body weight of 9.2 ± 1.1 kg. During 35 days, feed consumption (pen-based) and individual piglet body weights were registered weekly. In addition, blood samples of piglets of the control and the high supplemented groups (CON, A2 and B2) were taken from the vena jugularis externa in the morning of the last experimental day by using serum monovettes (10 ml by Sarstedt). The blood serum was analysed on the porcine growth hormone content, which distribution might be influenced by ergot alkaloids due to their partial structural homology to the neurotransmitter dopamine (Browning et al., 1997; Browning and Thompson, 2002), and different biochemical parameters which serve as indi-

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Table 1 Composition of the basal uncontaminated control diet Basal diet Components (g/kg) Barley Wheat Soybean meal extracted Maize Extruded maize Soybean oil Soybean protein concentrate (65 g CP/kg) Premixa

215.2 200.0 200.0 150.0 100.0 40.0 40.0 54.8

Calculated nutrient, amino acid and energy contents (g/kg)b Crude protein Crude fat Crude fibre N-free extract Lysine Methionine + cystine Threonine Tryptophane MEBFS c (MJ/kg)b

187.6 64.8 26.2 535.2 12.3 7.5 8.2 2.7 13.9

CP: crude protein. a Provided per kg diet: Ca, 10 g; P, 2.4 g; Na, 2.2 g; Mg, 0.4 g; Vitamin A, 16,000 IU; Vitamin D , 1600 IU; 3 Vitamin E, 48 mg; Vitamin B1 , 1.5 mg; Vitamin B2 , 4 mg; Vitamin B6 , 4 mg; Vitamin B12 , 30 mcg; Vitamin K3 , 2.1 mg; nicotinic acid, 20 mg; Ca-pantothenic acid, 13.5 mg; choline chloride, 200 mg; Fe, 160 mg; Cu, 20 mg; Mn, 107 mg; Zn, 134 mg; I, 3 mg; Se, 0.5 mg; Co, 1 mg; l-lysine HCl, 4.5 g; dl-methionine, 2.5 g; l-threonine, 2 g; l-tryptophane, 0.5 g; formic acid, 5 g; bio-feed-phytase (5000 FYT/g), 0.3 g. b Based on a dry matter content of 880 g/kg. c Metabolizable energy corrected for bacterial fermentative substance.

cators for liver damages since the alkaloids are mainly metabolized in the liver (Lorenz, 1979). The experimental design was approved by the Land Bureau for Consumer Protection and Food Safety for Lower Saxony (LAVES) in Oldenburg. 2.3. Analyses Feedstuffs and ergot were analysed for crude nutrient contents, starch, sugar and amino acids using the official German standard methods (Naumann and Bassler, 1993). The fatty acid concentrations were detected gas chromatographically after sample preparation as described by N¨urnberg et al. (1997). The esterification was conducted after the method of Schulte and Weber (1989). The alkaloid contents [ergometrine (EM); ergotamine (ET); ergocornine (ECOR); ␣ergocryptine (ECRY); ergocristine (ECRIS); ergosine (ESIN) and their -inine (-ine) isomers] of ergot and diets were analysed with a HPLC-method according to Wolff et al. (1988) as described by Mainka et al. (2005b). The detection limits were 10 ␮g/kg for EM and EM-ine and 5 ␮g/kg for the other alkaloids and their isomers at a sample weight of 5 g. The mean

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Table 2 Alkaloids and composition, as analysed, and calculated contents of ricinoleic acid and energy (according to GfE, 1987) of the diets and the two used ergot sources Ergot source/diet Ergot A

Ergot B

CON

A1

B1

A2

B2

Ergot (g/kg diet) STA (mg/kg diet)a

0.0 0.0

4.0 5.6

5.2 5.6

8.0 11.2

10.5 11.2

DM (g/kg)

907.1

903.2

905.0

905.0

904.6

0.48 0.28 0.13 0.09 0.75 0.68 0.22 0.11 0.08 0.06 0.33 0.17 3.39 1.72 71

0.40 0.45 0.44 0.19 0.59 0.68 0.09 0.20 0.21 0.12 0.19 0.19 3.75 2.06 73

0.94 0.46 0.50 0.20 1.45 1.40 0.42 0.18 0.27 0.12 0.64 0.38 6.97 3.56 71

0.63 1.93 0.41 0.28 1.01 1.24 0.17 0.94 0.16 0.16 0.41 0.31 7.66 4.26 72

(mg/kg)b

Ergot alkaloids Ergometrine Ergotamine Ergocornine ␣-Ergocryptine Ergocristine Ergosine Ergometrinine Ergotaminine Ergocorninine ␣-Ergocryptinine Ergocristinine Ergosinine Total alkaloidsc Key alkaloidsd -ine alkaloids (g/100 g total alkaloids) -inine alkaloids (g/100 g total alkaloids) Ricinoleic acid (g/kg)b Components Crude protein (g/kg)b Crude fat (g/kg)b Crude fibre (g/kg)b Crude ash (g/kg)b MEBFS e (MJ/kg)b

149 125 79 60 382 304 62 24 35 19 91 51 1381 795 80

73 278 69 62 198 233 18 44 14 12 22 34 1057 680 87

20

13

29

27

29

28

46.2

47.1

0.19

0.25

0.37

0.49

207 80 39 76 14.0

203 82 39 75 14.0

210 84 41 74 14.0

204 85 41 70 14.1

206 76 37 73 14.0

STA: standardised total alkaloid content; CON: control group; DL: detection limit; DM: dry matter. a Based on fresh matter (Ergot Source A: 88.97% DM; Ergot Source B: 88.67% DM). b Based on a dry matter content of 88%. c Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine + ergosine + ergometrinine + ergotaminine + ergocorninine + ␣-ergocryptinine + ergocristinine + ergosinine. d Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine. e Metabolizable energy corrected for bacterial fermentative substance.

recovery rate of the alkaloids in the diets was 79%. The results of the analyses were not corrected for recovery. The sum of all identified alkaloids (-ine and -inine isomers) is termed as total alkaloids. The sum of the contents of EM, ET, ECOR, ECRY and ECRIS represent the content as key alkaloids. Serum from blood was prepared by centrifugation (3000 × g at 5 ◦ C for 15 min).

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Activities of glutamate dehydrogenase (GLDH) (Schmidt, 1970), ␥-glutamyltransferase (␥-GT) (Labor + Technik, Eberhard Lehmann, Berlin, Szasz et al., 1974) aspartate aminotransferase (AST) (Labor + Technik, Eberhard Lehmann, Berlin, opt. DGKC), alanine aminotransferase (ALT) (Labor + Technik, Eberhard Lehmann, Berlin, opt. DGKC) and total bilirubin (Labor + Technik, Eberhard Lehmann, Berlin, Jendrassik and Grof, 1938) were measured in serum by photometric standard procedures. Protein content was determined using the biurette method and the albumin concentration by the bromcresolgreen standard procedure (Labor + Technik, Eberhard Lehmann, Berlin). Furthermore, porcine growth hormone (pGH) concentration in serum was determined applying the EIA-technique described by Serpek et al. (1993). 2.4. Calculations and statistics The energy contents of the diets were calculated as follows: MEBFS (MJ/kg DM) = 0.021 × digestible protein + 0.0374 × digestible fat + 0.0144 × digestible N-free extract − 0.014 × sugar − 0.068 × (BFS − 100) with sugar correction at >8 g sugar/100 g DM and BFS (bacterial fermentative substance = digestible fibre + digestible N-free extract − starch − sugar) correction at >10 g BFS/100 g DM. The ricinoleic acid contents of the diets were calculated on the basis of the ricinoleic acid concentration of the respective ergot source and the proportion of ergot in the diet. The growth performance data of the feeding experiment were first analysed by using the t-test (P<0.05) in order to determine significant differences between the experimental groups and the control group. To verify dose–response effects (linearity), the data were analysed separately for the control group and each ergot source (CON versus A1 versus A2 and CON versus B1 versus B2) as well as for the control group and the supplemented groups pooled concerning the standardised total alkaloid content (Groups A1/B1 versus A2/B2) by a one-factorial design of ANOVA. In addition, differences between ergot sources with consideration of the standardised dietary total alkaloid contents, as well as the interactions, were determined by analysing the data without the control group with a complete two by two factorial design of ANOVA. The serum parameters were analysed by a one-factorial design of ANOVA for one concerning the ergot source (A2 versus B2) and for the other concerning the standardised total alkaloid content (Group CON versus A2/B2). Significant differences between means were evaluated by the t-test or the Student–Newman–Keuls-test (P<0.05). The Mann–Whitney-U-test (P<0.05) was used for those data not normally distributed (AST and pGH). All statistics were carried out using the Statistica for WindowsTM operating system (StatSoft Inc., 1994).

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Table 3 Composition (alkaloids, composition and fatty acids) of ergot from the rye harvest 2003 Mean (n = 13)

Minimum

Maximum

Ergot alkaloids (mg/kg DM) Total alkaloidsa Key alkaloidsb -ine alkaloids (g/100 g total alkaloids) -inine alkaloids (g/100 g total alkaloids)

738 459 78 22

24 15 67 13

1569 903 87 33

Components (g/kg DM) Crude ash Crude protein Crude fat Crude fibre N-free extract Starch Sugar

32.9 223.1 349.9 286.6 107.6 46.6 9.6

29.6 205.1 319.6 199.5 16.4 34.2 2.5

44.4 247.3 385.1 371.6 198.3 59.0 21.5

Fatty acids (g/100 g fatty acids) Capric acid Lauric acid Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Eicosanic acid Eicosenic acid Eicosadienic acid Behenic acid Erucic acid Ricinoleic acid Lignoceric acid

0.05 2.05 0.55 30.41 3.19 7.49 21.09 17.66 0.75 1.30 0.22 0.07 0.39 0.16 14.38 0.24

0.03 1.30 0.38 24.74 2.53 4.85 19.11 14.97 0.19 0.92 0.17 0.06 0.33 0.11 7.21 0.18

0.09 3.30 0.66 32.95 3.55 9.06 22.64 29.48 3.47 1.45 0.42 0.08 0.43 0.33 16.50 0.29

DM: Dry matter. a Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine + ergosine + ergometrinine + ergotaminine + ergocorninine + ␣-ergocryptinine + ergocristinine + ergosinine. b Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine.

3. Results 3.1. Ergot composition The mean total alkaloid content of ergot amounted to 738 mg/kg DM with a range of 24 to 1569 mg/kg DM (Table 3). The key1 and -inine alkaloid concentrations were 78 g/100 g total alkaloids (67–87 g/100 g) and 22 g/100 g total alkaloids (13–33 g/100 g), respectively. In general, ergotamine, ergocristine and ergosine were with 20, 24 and 15 g/100 g total alkaloids the major alkaloids found (Table 4). Crude protein (223 g/kg DM), fat (350 g/kg 1

Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine.

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Table 4 Ergot alkaloid pattern (g/100 g total alkaloids) of ergot from the rye harvest 2003

Ergometrine Ergometrinine Ergotamine Ergotaminine Ergocornine Ergocorninine ␣-Ergocryptine ␣-Ergocryptinine Ergocristine Ergocristinine Ergosine Ergosinine

Mean (n = 13)

Minimum

Maximum

7 3 20 4 7 2 6 4 24 5 15 3

2 1 9 2 4 1 4 1 18 2 11 2

11 7 30 9 9 4 9 13 31 10 22 7

DM) and fibre (287 g/kg DM) were the principal compounds of ergot (Table 3). The fatty acid analysis demonstrated the highest occurrences for palmitic-, oleic-, linoleic- and ricinoleic acid with 31, 21, 18 and 14 g/100 g fatty acids (Table 3). The correlation coefficients (r) between fat and ricinoleic acid content amounted to r = 0.142, between fat and total alkaloid content to r = 0.095 and between ricinoleic acid and total alkaloid content to r = −0.249 at P>0.05. With regard to amino acids, highest concentrations were found for aspartic acid (7 g/100 g CP), threonine (8 g/100 g CP), glutamic acid (13 g/100 g CP) and lysine (7 g/100 g CP) (Table 5). The correlation coefficient between total amino acid and total alkaloid content amounted to r = 0.275 (P>0.05).

Table 5 Amino acid concentration of ergot from the rye harvest 2003

Amino acids (g/100 g crude protein) Cysteine Aspartic acid Methionine Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

Mean (n = 13)

Minimum

Maximum

1.2 7.1 1.3 8.1 4.1 13.0 4.4 3.9 4.4 5.0 3.9 4.8 2.7 2.8 2.3 6.7 4.0

1.0 6.7 1.1 7.0 3.8 11.5 3.9 3.5 3.8 4.5 3.4 4.5 2.4 2.6 1.9 5.9 3.4

1.4 7.3 1.4 9.1 4.6 16.0 5.2 4.3 4.9 5.5 4.3 5.1 4.1 3.1 2.6 7.3 4.5

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3.2. Piglet experiment 3.2.1. Nutrient and ergot alkaloid composition of the diets The nutrient and energy contents of the ergot supplemented diets differed only slightly from those of the control group (Table 2). The analysed total alkaloid contents in the diets did not exactly meet the calculated levels (5.6 and 11.2 mg/kg) on which the respective diet was standardised (Table 2). However, the duplication of the alkaloid content from A1 to A2 and B1 to B2 was achieved. The alkaloid patterns of the two ergot sources showed conspicuous differences mainly considering ergotamine, which was nearly three times higher in Ergot Source B. Otherwise the contents of ergometrine and ␣-ergocryptine were higher in Ergot Source A. The -inine isomer contents raised from 20 to13 g/100 g total alkaloids in Ergot Sources A and B, respectively, to 29 and 28 g/100 g total alkaloids, on average, in Diets A1/A2 and B1/B2 (Table 2). 3.2.2. Performance results and alkaloid intake Piglets mean body weight ranged from 9.2 ± 1.1 to 24.4 ± 4.3 kg during the trial. Cumulative feed intake and body weight gain differed significantly from the control only in the high contaminated groups (Table 6). Feed-to-gain ratios remained unaffected. The Table 6 Effects of dietary ergot supplements on performance of piglets Treatment

Ergot (g/kg diet)

STA (mg/kg diet)

Feed intake (g/day) (n = 4)

Live weight gain (g/day) (n = 16)

Feed-to-gain ratio (kg/kg) (n = 4)

CON A1 B1 A2 B2

0.00 0.40 0.52 0.80 1.05

0.0 5.6 5.6 11.2 11.2

745.2 696.1 642.6 632.1 633.7*

495.3 463.8 418.3 398.9* 410.6*

1.567 1.501 1.536 1.585 1.543

PSEM

19.07

26.88

0.012

Statistics P Ergot Source A Treatment Linear

0.221 0.091

0.040 0.015

0.060 0.128

P Ergot Source B Treatment Linear

0.104 0.057

0.072 0.036

0.819 0.293

P alkaloid content STA Linear

0.092 0.031

0.034 0.011

0.158 0.932

P alkaloid pattern × alkaloid content Ergot source STA Interaction

0.565 0.419 0.546

0.536 0.185 0.295

0.921 0.035 0.118

STA: standardised total alkaloids; CON: control group; PSEM: pooled standard error of means (CON, A1, B1, A2, B2). * Significantly different as compared to the respective control group (P<0.05).

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Table 7 Cumulative intake of alkaloids Treatment

Total alkaloidsa (mg/day)

Key alkaloidsb (mg/day)

Total alkaloidsa (mg × BW kg−1 × day−1 )

Key alkaloidsb (mg × BW kg−1 × day−1 )

CON A1 B1 A2 B2

<0.01 2.42 2.48 4.53 5.00

<0.01 1.23 1.36 2.31 2.78

<0.01 0.15 0.17 0.32 0.34

<0.01 0.08 0.09 0.16 0.19

CON: control group. a Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine + ergosine + ergometrinine + ergotaminine + ergocorninine + ␣-ergocryptinine + ergocristinine + ergosinine. b Ergometrine + ergotamine + ergocornine + ␣-ergocryptine + ergocristine.

Fig. 1. Cumulative feed intake of the contaminated groups (A1, B1, A2, B2) in relation (proportion) to the control (0).

cumulative intake of total and key alkaloids was up to 5.0 and 2.8 mg/day in Group B2 (Table 7). Relative feed intake of the contaminated groups decreased during the first 3 weeks of trial and got approached to the control in the last 2 weeks (Fig. 1). Lower live weights reflect the progress of feed intake (Fig. 2). The ANOVA showed significant linear dose–response effects for feed intake and body weight gain of the groups which were pooled concerning the standardised total alkaloid

Fig. 2. Progress of live weight of the contaminated groups (A1, B1, A2, B2) in relation (proportion) to the control (0).

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content (A1/B1 and A2/B2) (P=0.011) and for body weight gain of both separate ergot sources (CON, A1, A2 and CON, B1, B2) as well (P=0.015 and 0.036) (Table 6). Differences in performance between both ergot sources and interactions with the total alkaloid content were not found (Table 6). Also the regression equations between total body weight gain and intake of total alkaloids per box and day (n = 4) of Groups CON, A1 and A2 (y = 483.8 + (−14.66) × x; r = −0.446; P=0.146) and CON, B1 and B2 (y = 471.5 + (−13.20) × x; r = −0.501; P=0.098) differed not substantially from each other. 3.2.3. Biochemical parameters in serum Concerning the serum biochemical parameters which might indicate liver damages, the pooled effect of Groups A2 and B2, with standardised dietary total alkaloid contents of 11.2 mg/kg, decreased significantly for protein as compared to the control. The albumin content demonstrated the same tendency (Table 8). Differences between ergot sources were not found. In general, the liver associated serum parameters showed inconsistent tendencies.

4. Discussion 4.1. Composition of ergot The alkaloid contents of the ergot varied widely (Table 3) but within the range reported in literature (Lorenz, 1979; Young, 1981a,b; Young and Chen, 1982). However, methods for alkaloid analysis and considerations of the alkaloids used to estimate the total alkaloid content make correct comparisons quite difficult. The degree of isomerization of the alkaloids to the corresponding -inine form ranged from 13 to 33 g/100 g total alkaloids and is comparable with data from Young (1981a) who found an average value of 30 g/100 g total alkaloids. The extent of isomerization depends on many factors, e.g., duration of storage, the temperature and pH (Hofmann, 1964; Young et al., 1983; Mainka et al., 2005c) and may have an influence on ergot toxicity since the pharmacological efficacy of the -inine alkaloids is lower than their corresponding isomers (Hofmann, 1964). However, the process of isomerization can be reversible (Hofmann, 1964). The crude nutrient contents, as well as the fatty acid pattern, were mostly comparable to the data from literature (Guggisberg, 1954; Lorenz, 1979; Buchta and Cvak, 1999; Komarova and Tolkachev, 2001). The fatty acid pattern of ergot differs from cereals, since the triglycerides are largely esterificated with ricinoleic acid (Wolff and Richter, 1989). Furthermore, the concentrations of palmitic, palmitoleic, stearic and oleic acid are lower, and those of linoleic and linolenic acid are higher in rye fat (Bushuk, 1976; Sauvant et al., 2004). In contrast to Bharucha and Gunstone (1957), who found the ergot specific ricinoleic acid in amounts up to 34 g/100 g fatty acids, the ricinoleic acid content was lower in the present study. Our data together with those of Schulze (1953) and Kolsek et al. (1957) do not find any relationship between fat and alkaloid content of ergot. Additionally, we have not observed any relationship between ricinoleic acid and total alkaloid content, which confirms the findings of Waiblinger and Gr¨oger (1972). For this reason, the ricinoleic acid content is only useful to indicate an ergot contamination of grain or feed but does not allow any conclusion concerning toxicity.

Treatment

CON

A2

B2

A2/B2

Ergot (g/kg diet) STA (mg/kg diet) n

0.0 0.0 13

8.0 11.2 16

10.5 11.2 16

8.0/10.5 11.2 32

GLDH (U/l) ␥-GT (U/l) ASTa (U/l) ALT (U/l) Bilirubin (␮mol/l) Protein (g/l) Albumin (g/l) pGHa (ng/ml)

3.2 24.1 21.7 (13.8–27.2) 32.1 1.6 52.4a 33.3 5.8 (1.0–28.5)

3.6 24.1 23.7 (16.0–46.9) 29.8 1.11 49.3b 32.3b 2.8 (1.2–6.2)

2.7 21.8 24.0 (16.4–43.6) 33.2 1.0b 47.9b 30.1b 5.4 (1.1–18.4)

3.1 22.9 23.8 (16.0–46.9) 31.5 1.1c 48.6c,* 31.2c 4.1 (1.1–18.4)

STA: standardised total alkaloids; PSEM: pooled standard error of means (CON, A2, B2). a Evaluated applying the Mann–Whitney-U-test; mean (minimum − maximum). b n = 15. c n = 30. * Significantly different as compared to the control group (P<0.05).

PSEM

P alkaloid content (STA)

P ergot source

0.31 1.62

0.960 0.603

0.085 0.305

1.49 0.26 0.85 0.89

0.754 0.124 0.002 0.081

0.072 0.731 0.263 0.109

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Table 8 Effects of dietary ergot supplements on serum parameters of piglets at 35 day of administration

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The average contents of threonine and lysine in rye are lower and those of cysteine, glutamic acid and proline, higher as compared to ergot (AminoDat® , 2001; Table 5). However, assuming a contamination of 1000 mg ergot/kg rye, which demonstrates the maximum limit for feed grain (Council Directive 2002/32/EC of 7 May 2002), the amino acid profile of rye would not be substantially altered. Despite many amino acids serve as precursors for the biosynthesis of alkaloids in the ergot fungus (Gr¨oger and Mothes, 1956; Robertson et al., 1973; Rutschmann and Stadler, 1978), Gr¨oger and Mothes (1956) detected constant patterns of free amino acids in ergot from different sources and no close relationship between the alkaloid and amino acid contents. The data of the present study show only slight variations in the amino acid contents of the analysed ergot sources, without any relationship to the alkaloid content. All nutrients in the process of alkaloid synthesis come from the host plant (Kybal et al., 1976), which presumably explains the lack of relationship. 4.2. Influence of different alkaloid patterns in graded levels on piglets The slightly lower dietary alkaloid concentrations and higher -inine isomer contents analysed in the diets in comparison to the calculated data of the pure ergot (Table 2) are mainly probably due to alkaloid isomerization and degradation during storage (Berde and St¨urmer, 1978; Richter et al., 1990; Wolff, 1992) since representative samples of the diets, taken over the whole experimental time, were analysed 8 weeks later than the ergot sources. Ergot feeding decreased body weight gain which was predominately due to a decline in feed intake. This is in accordance to most published reports (Friend and MacIntyre, 1970; Harrold et al., 1974; Whittemore et al., 1976, 1977; Richter et al., 1989, 1990; Mainka et al., 2005a,b). The feed-to-gain ratios in the present study were not significantly different, which do not indicate a substantial ergot alkaloid induced effect on growth performance due to post-absorptive metabolic changes. In contrast, Oresanya et al. (2003) observed deleterious ergot effects on growth performance of piglets independent of feed intake. The ANOVA did not prove any significant effect of the ergot source on performance or interactions between the alkaloid pattern and the alkaloid content. Additionally, the slope of the regression lines of Ergot Sources A and B did not differ substantially from each other. For these reasons, the effect of the alkaloid pattern on performance was probably of minor importance. Otherwise, the extent of the alkaloid contamination seemed to play an important role since feeding the highest alkaloid contents resulted in the lowest growth performance, which is confirmed by the linear dose–response effects of body weight gain (Table 6). Oresanya et al. (2003) found linear effects at rising dietary ergot contents from 0.5 up to 10 g/kg on body weight gain as well. The corresponding sums of the five analysed alkaloids (ET, ECOR, ECRY, ECRIS and ESIN) amounted to 0.94–18.80 mg/kg feed. Compared with Group B2 of the present study, the alkaloid content used by Oresanya et al. (2003) was nearly four times higher. Comparing the patterns of the five mentioned alkaloids of Ergot Sources A and B (sum of ET, ECOR, ECRY, ECRIS and ESIN = 100%) with the pattern of the ergot used by Oresanya et al. (2003), ET, ECRIS and ESIN presented the major components in every ergot source (87, 85 and 85%). Oresanya et al. (2003) recommend not exceeding 1.88 mg of the sum of ET, ECOR, ECRY, ECRIS and ESIN/kg feed (fresh matter) for piglets since higher contents (4.70–18.80 mg/kg) caused severe declines in performance. The results are almost in accor-

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dance with the current study, since higher amounts than administered to Groups A1 and B1 (1.93 and 2.36 mg/kg, calculated from Table 2) depressed growth performance significantly as well. Expressed as the mean alkaloid intake of the piglets of Groups A1 and B1, the no observed effect level as the critical ingestion dose per day appeared to be 2.45 mg total alkaloids (=0.16 mg/kg BW), which corresponds to 1.30 mg key alkaloids (=0.085 mg/kg BW) and confirms the findings of Mainka et al. (2005a) who also determined the critical level of daily key alkaloid intake of 0.09 mg/kg BW. To compare all mentioned studies, it is necessary to calculate the sum of ET, ECOR, ECRY and ECRIS since these are the commonly analysed alkaloids. The results of the presented experiments indicate that the sum of ET, ECOR, ECRY and ECRIS/kg feed for piglets should not exceed 1.52 mg, which is the mean of the respective determined no observed effect levels of 1.46 mg of Diets A1 and B1 in the current study, 1.43 mg of Mainka et al. (2005a) and 1.67 mg of Oresanya et al. (2003). However, other ergot specific components than the alkaloids, e.g., ricinoleic acid or ergochromes (pigments, e.g. secalonic acids), and interactions might have an influence as well (Wolff, 1992). The serum biochemical parameters, which are used in veterinary medicine for appraisal of the liver health, were in the range of the reference values for pigs (Bickhardt, 1992; Plonait and Bickhardt, 1997; Kraft and D¨urr, 1999; Faustini et al., 2000). However, the ergot fed groups had less protein and albumin contents in serum (Table 8). Significant dose-dependent decreases in serum albumin contents were also detected by Mainka et al. (2005a) in piglets at levels of 0, 0.93 and 3.73 mg key alkaloids/kg diet over 5 weeks. The corresponding intake of key alkaloids per kg mean BW of the highest supplemented group in the experiment of Mainka et al. (2005a) amounting 0.16 mg was nearly the same as compared to Groups A2 and B2 (0.16 and 0.19 mg) of the current study. In general, orally administered ergot to piglets seems to have an influence on hepatic protein metabolism. A decrease of albumin, a protein solely synthesised by the liver, and total protein contents in serum may be due to reduced capacity of liver protein synthesis. However, distribution and elimination as well as different plasma volumes should be considered as factors influencing the serum contents as well. Since the biochemical parameters of Groups A2 and B2 did not differ noticeably from each other at comparable total alkaloid contents, an effect of the respective alkaloid pattern was probably of minor importance. As the pGH contents in serum varied in a wide range, an influence of an ergot alkaloid induced change on the pGH secretion and therefore on growth performance cannot be concluded. The unaffected feed-to-gain ratios confirm this conclusion. However, other factors, e.g. the pulsatile secretion, might have had a strong influence on the pGH content in serum as well (Hartman et al., 1993; Bluet-Pajot et al., 1998). The comparability of experiments on ergot is quite complex due to different analysed alkaloids. Nevertheless, when considering the same analysed alkaloids as a sum, the results of the different studies as discussed above were almost comparable and proved nearly the same no observed effect level for dietary ergot alkaloid contamination in piglets. For this reason the evaluation of the content of some specific ergot alkaloids (e.g., the five key alkaloids) in grain or feed seems to make sense without special consideration of the respective alkaloid pattern. With regard to the multitude of alkaloids, and consequently alkaloid patterns, a particular consideration seems not to be justified in the view that an obviously

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highly variable alkaloid pattern is of markedly less influence on the piglet performance than the total or key alkaloid concentration.

5. Conclusions Since the alkaloid content of ergot was proven to vary in a wide range, the analysis of the toxic alkaloids in feed is more useful to evaluate the feeding hazards than the consideration of ergot contamination in grain. The critical contamination level for piglets was determined to be 3.57 mg total alkaloids/kg feed (=1.89 mg key alkaloids/kg feed) in the present experiment. The evaluation of the content of some specific ergot alkaloids (e.g., the five key alkaloids) in grain or feed seems to make sense to appraise feeding hazards without special consideration of the respective alkaloid pattern. The effects of the alkaloid pattern need to be substantiated by further studies testing ergot from different sources.

Acknowledgements The authors wish to express their gratitude to the co-workers of the Institute of Animal Nutrition (FAL) in Celle and Braunschweig and the Institute of Animal Physiology and Animal Nutrition of the Georg-August-University G¨ottingen for assistance with data collection, analyses and animal care. Sincere thanks are also given to P. Aldag and F. Elsaesser of the Institute for Animal Breeding (FAL) in Mariensee for determination of serum pGH concentrations. We are grateful to the Wilhelm Schaumann Foundation for the support and the Lochow Petkus GmbH, Bergen for the provision of the ergot.

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