Effect of T-2 and HT-2 toxin during the growing period on body weight, lipid peroxide and glutathione redox status of broiler chickens

s of 11th European Poultry Conferencein Bremen 140. Orosz Sz, Mézes M, Vetési M, Balogh K Investigation on bile acid concentration of blood plasma in table duck fed with barley and oat-based diet using additional bile salts. In: Füleky Gy (szerk.) Bulletin of the Szent István University. Gödöllő: Szent István Egyetemi Kiadó, 2002. pp. 83-90. Könyvfejezet/Évkönyv része/Tudományos 2001 141. Mézes M, Vetési M, Husvéth F, Kővári L A tojásminőség befolyásolása eltérő zsírkiegészítések alkalmazásával. In: Kovácsné Gaál Katalin (szerk.) Agrártermelés Életminőség: XXIX. Óvári Tudományos Napok. Mosonmagyaróvár, Magyarország, 2002.10.03-2002.10.04. NyME Mezőgazdaságés Élelmiszertudományi Kar, p. &. Konferenciacikk/Előadás vagy poszter cikke/Tudományos

Fusarium moulds are widespread in the temperate climate, producing trichothecene mycotoxins, e.g.T-2 toxin, HT-2 toxin, T-2 triol and T-2 tetraol (Scott 1990).Due to their occurrence and diverse effects, in particular impairment of the immune response (Weber et al. 2008), these mycotoxins have great importance in animal nutrition.The trichothecene mycotoxins, such as T-2 toxin and its metabolites compromise the growth rate and development of poultry (Leeson et al. 1995), and affect the antioxidant status (Atroshi et al. 2002;Rizzo et al. 1994;Weber et al. 2007), primarily due to their prooxidant effect (Mézes et al. 1998;Surai 2002).Among the Fusarium mycotoxins, the T-2 toxin and its natural metabolites are excreted relatively quickly (within about 72 h) from the organism after ingestion.Therefore, the rate of their accumulation in the different tissues is low (e.g.0.2% in muscles and 0.4% in the liver).Thus the main detrimental effects of the T-2 toxin and its metabolites are not due to their accumulation, but rather the result of other, sometimes long-term effects caused immediately after absorption and metabolism of toxins in different tissues (WHO/FAO 2000).
The objective of the present study was to evaluate the long-term effect of feeding T-2 toxin contaminated diet on the growth, rate of lipid peroxidation, and also on the glutathione redox status of broiler chickens during the growing period.

Materials and Methods
A total of 40 day-old Hubbard broiler cockerels were studied for 39 days.The birds were divided into two groups fed with control ('C' n = 20) and T-2 toxin contaminated ('T-2' n = 20) diets.The nutrient content of the diets met the requirements for broiler chickens (Table 1) according to the Hungarian standards (Hungarian Feed Code 2004a).The nutrient content of the diets was determined according to the Hungarian National Standard methods (Hungarian Feed Code 2004b).Complete feed in mash form was fed in the first phase (1-21 days) with < 0.01 mg•kg -1 T-2 toxin in the control, while the experimentally contaminated diets contained 1.04 mg•kg -1 T-2 toxin and 0.49 mg•kg -1 HT-2 toxin, respectively.The same control complete feed with < 0.01 mg•kg -1 T-2 toxin was fed in the second phase (22-39 days) with the same nutrient content as in the first phase, while the experimentally contaminated diets were loaded with 0.12 mg•kg -1 T-2 toxin and 0.02 mg•kg -1 HT-2 toxin, respectively.
However, there is no official regulation in the European Union for the maximum allowed level of T-2 toxin in the feeds, only a guideline for the limit value of 0.5 mg T-2 toxin•kg -1 is proposed (Eriksen and Pettersson 2004).T-2 toxin contamination in the first phase of the present experiment was much higher than the aforementioned maximum guideline limit value.The partially purified toxin preparation was dissolved in acetone and then sprayed onto the complete feed (100 ml/50 kg of feed).T-2 toxin was produced experimentally on maize by Fusarium sporotrichoides strain NRRL 3229 (Agricultural Research Service Culture Collection, National Centre for Agricultural Utilization Research, Peoria, IL) according to the method of Fodor et al. (2006).Extraction and purification of the toxin were carried out according to Burmeister (1971).The T-2 and HT-2 toxin concentrations in the feed were measured using the HPLC technique (Central Veterinary Institute, Budapest) according to the relevant European Union directive (EU 2005).The amounts of related trichothecene metabolites, T-2 triol and T-2 tetraol were under the detection limit.
Body weight was measured on the 7 th , 14 th , 21 st , 35 th and 39 th days of the trial.Five animals from each group were sacrificed on 21 st and 39 th days, necropsy was carried out, and blood and tissue (liver and kidney) samples were collected.Blood samples were stored at cooled place (+ 4 °C) until processing, then the plasma was separated from the blood cells by centrifugation.Red blood cells were lysed with deionized water (ratio 1 : 9) and by freezing and thawing.Liver and kidney samples were homogenized in nine-fold volume of 0.65% (w/v) sodium-chloride.The samples were stored at -20 °C until analyses.
For determination of malondialdehyde (MDA) concentration in the blood samples, the method of Placer et al. (1966) and for liver and kidney samples the method of Mihara et al. (1980) were used.The glutathione peroxidase (GSHPx) activity was measured by the method of Lawrence and Burk (1976).Protein content of the blood plasma and the red blood cell haemolysate was determined by the biuret method (Weichselbaum 1948).Total protein concentrations in the 10,000 g supernatant fraction of liver and kidney homogenates were measured according to the method of Lowry et al. (1951).The GSH concentration was analysed using the method of Sedlak and Lindsay (1968).
Statistical evaluation of the results was carried out by paired Student's t-test after calculating the means and standard deviations (S.D.) with Statistica TM 4.0 (Statsoft Inc. 1993) software.The experiment was approved by the Animal Experimental Committee of the Faculty of Agricultural and Environmental Sciences of the Szent István University (2/2005).

Pathological signs
The effects of T-2 and HT-2 toxin were found at necropsy on day 21 and also on day 39 but with different extents and rates of occurrence in the group fed mycotoxin contaminated feed.The pathological signs appeared as lesions in the oral cavity and on the tongue, also 28 haemorrhage and inflammatory symptoms especially in the proximal part of the small intestine were found (Table 2).
Body weight Body weight of the birds was significantly lower after feeding T-2 and HT-2 toxin contaminated feed at the end of the starter (high toxin concentration) period on day 21 (Table 3).During the second phase of the trial with a lower dose of mycotoxin contamination, compensatory growth occurred in the toxin-fed group.

Malondialdehyde content
Lipid peroxidation was followed by measuring the concentration of the malondialdehyde, a meta-stable end-product of peroxidation of non-saturated fatty acids.There were no significant changes in the malondialdehyde content of blood plasma and liver homogenate following the feeding of T-2 and HT-2 toxin-contaminated diet.However, significantly lower malondialdehyde content was found in the red blood cell haemolysates on day 21 and in kidney homogenates on day 39 of the birds from toxin group compared to control (Table 4).

Reduced glutathione content
Neither high nor low toxin contamination of the diet caused significant changes in the reduced glutathione content of blood plasma, red blood cell haemolysate and kidney homogenate.However, significantly lower content of reduced glutathione was found in the 10,000 g supernatant fraction of liver homogenate on day 39 of the birds from toxin group compared to control (Table 5).

Glutathione peroxidase activity
Glutathione peroxidase activity did not differ significantly in the blood plasma, red blood cell haemolysate and 10,000 g supernatant fraction of kidney homogenates as an effect of feeding T-2 and HT-2 toxin-contaminated diet (Table 6).On the contrary, the enzyme activity was significantly higher in the 10,000 g supernatant fraction of liver homogenates of the birds from the toxin group at both times of samplings, namely on days 21 and 39, compared to control (Table 6).Table 6.Glutathione peroxidase activity in blood plasma, red blood cell haemolysate, and 10,000 g supernatant fraction of liver and kidney homogenates after long-term feeding of T-2 + HT-2 toxin-contaminated feed (mean ± SD)

Table 1 .
Nutrient and mycotoxin content of the diets

Table 2 .
Clinical signs of intoxication after long term feeding of T-2 and HT-2 toxin contaminated feed

Table 5 .
Reduced glutathione content in blood plasma, red blood cell haemolysate, and 10,000 g supernatant fraction of liver and kidney homogenates after long term feeding of T-2 + HT-2 toxin-contaminated feed (mean ± SD)a,b Different superscripts in the same row mean significant difference (P < 0.05) Different superscripts in the same row mean significant difference (P < 0.05)