Deoxynivalenol (DON), produced by the
Mycotoxins are toxic secondary metabolites produced by different types of filamentous fungi. The most relevant mycotoxins to animal health and production are produced by
Deoxynivalenol (DON), also known as vomitoxin, is a trichothecene mycotoxin produced by the
Animal feed components and finished feedstuffs normally contain this mycotoxin, it is one of the most frequently found mycotoxin in cereal grains, such as wheat, barley, and corn [
Despite the fact that rainbow trout belongs to a carnivorous species, commercial salmonoid feeds contain plant components. Due to a decrease in the availability of fish meal for the production of aquaculture feeds, alternative protein sources need to be used in fish feed production. When we use more plant source ingredients in commercial feeds for farmed fish, we increase the possibility that mycotoxins contaminate those feeds [
Deoxynivalenol causes a broad variety of toxic effects in animals, the toxicity is well recognized in mammals. The main effect at the cellular level is the inhibition of protein synthesis through it being bound to the ribosomal subunit [
The experiment was carried out on one-year-old rainbow trout (
The control fish were fed a commercial diet (BioMar, Denmark) containing rapeseed oil, blood meal, fish meal, soya cake, sunflower cake, rapeseed meal, horse beans, wheat, soya concentrate, fish oil, pea proteins, vitamins, and minerals.
The experimental diet was prepared by adding DON to commercial pellets in several separate steps. The amount of 32.50 g of Eudragit E (Basic Butylated Methacrylate Copolymer) was dissolved in 227.50 g of acetone on the electromagnetic stirrer for a period of 60 minutes (solution A). 60 mL of this solution was put aside (solution B). Into each of the three vials containing 5 mg of DON, 10 mL from the solution B was injected for the reconstitution of DON. Next, the dissolved content of these vials was added to the original solution A. The vials were then rinsed with the rest of solution B (10 mL for each vial) and solutions (A and B) were mixed together. The resultant common solution was divided into the two equal parts with the weight of 130 g that is equivalent to 7.5 mg of DON.
2470.75 g of pellets and 13.00 g of AEROSIL were added to a cubic blender and mixed for 5 minutes at 40 rpm (“Blend A”). 130 g of solution with a content of 7.5 mg of DON was uniformly and carefully poured onto the surface of mixed excipients and this moistened mixture was kneaded for 5 minutes at 40 rpm. The same procedure was performed with the “Blend B”. The final mixtures were placed in a hot air dryer and dried at 50°C for 4 hours.
The polymer forms a specific layer on the surface of the pellets, which is formulated from the solid dispersion of the active ingredient fixed in a polymer. As a result, it is assumed that there is a highly uniform content of active substance in each of the individual pellets.
The contents of deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, fumonisin B1 and B2, HT-2 toxin, T-2 toxin, nivalenol, ochratoxin A, and zearalenone in control and experimental feed were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by Metrology and Testing Laboratory (Institute of Chemical Technology, Prague). The analyzed mycotoxin contents are described in Table
Mycotoxin concentration in control and experimental feed.
Mycotoxin contamination |
Control feed | Experimental feed |
---|---|---|
Deoxynivalenol | 225 | 1964 |
3-Acetyldeoxynivalenol | ND1 | ND |
15-Acetyldeoxynivalenol | ND | ND |
Diacetoxyscirpenol | ND | ND |
Fumonisin B1 | ND | ND |
Fumonisin B2 | ND | ND |
HT-2 toxin | ND | ND |
T-2 toxin | ND | ND |
Nivalenol | ND | ND |
Ochratoxin A | ND | ND |
Zearalenone | ND | 1 |
Fourty fish were divided into control (
Blood samples were taken from each fish by puncturing the caudal vessel and stabilized with sodium heparin (50 IU per 1 mL of blood). Heparinized blood samples were used for the evaluation of haematological indicators including erythrocyte count (RBC), haemoglobin concentration (Hb), hematocrit (PCV), mean erythrocyte volume (MCV), mean erythrocyte haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and leukocyte count (WBC). Samples were determined according to Svobodova et al. [
For biochemical analysis, a part of heparinized blood, after centrifugation at 855 g for 10 min at cooled centrifuge (4°C), was used. Biochemical parameters including albumins (ALB), total proteins (TP), glucose (GLU), ammonia (NH3), triacylglycerols (TRIG), lactate (LACT), cholesterol (CHOL), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), calcium (Ca2+), and inorganic phosphate (PHOS). Plasma biochemical indicators were measured using a biochemical automatic analyzer Konelab 20i (ThermoScientific, Czech Republic) and commercial test kits (BioVendor, Czech Republic).
After blood sampling, the fish were stunned with a blow to the head and killed by spinal transection. Then, the biometrical indices were defined (total and standard length, body and liver weight) from which there were derived and calculated somatic parameters such as the Fultons condition factor and the hepatosomatic index. The Fultons condition factor (CF) was calculated using the formula CF = (body weight [g]/standard length [cm3]) × 100. The hepatosomatic index (HSI) was calculated using the following formula: HSI = liver weight/body weight × 100.
The samples of gills, skin, liver, cranial, and caudal kidney and spleen of ten fish from each group were immediately fixed in buffered 10% neutral formalin. The samples were later dehydrated, embedded in paraffin wax, sectioned on a microtome at a thickness of 4
The results of the haematological and biochemical examinations and biometric parameters were carried out with UNISTAT statistica 5.6 software. Data were first tested for normality (Shapiro-Wilk test). When necessary, logarithmic transformations were used for the analysis of variance. A one-way analysis of variance (ANOVA) and a Tukey-HSD testwere applied. If the normal distribution was not satisfied, a nonparametric Kruskal-Wallis test was applied. Significance was accepted at
The results of the analyses of the haematological indices of both control and experimental groups after 23 days from the beginning of the experiment are presented in Table
Values of haematological indices 23 days after the beginning of the experiment.
Indices | Control |
Experimental |
---|---|---|
RBC (T |
1.41 ± 0.36 | 1.58 ± 0.41 |
Hb (g |
76.94 ± 12.78 | 69.49 ± 12.92 |
PCV (L |
0.30 ± 0.06 | 0.29 ± 0.05 |
MCV (fL) | 228.14 ± 66.62 | 198.26 ± 53.05 |
MCH (pg) | 57.86 ± 16.26 | 46.93 ± 14.12* |
MCHC (g |
250.31 ± 27.58 | 235.67 ± 22.97 |
WBC (G |
12.13 ± 4.97 | 14.23 ± 3.58 |
Significant difference between test groups (
RBC: erythrocyte count, Hb: haemoglobin concentration, PCV: haematocrit, MCV: mean erythrocyte volume, MCH: mean erythrocyte haemoglobin, MCHC: mean corpuscular haemoglobin concentration, WBC: leukocyte count.
The results of plasma biochemical indicators are presented in Table
Values of biochemical parameters 23 days after the beginning of the experiment.
Indices | Control |
Experimental |
---|---|---|
ALB (g |
15.69 ± 2.90 | 15.25 ± 2.74 |
TP (g |
37.45 ± 5.77 | 36.38 ± 3.98 |
GLU (mmol |
4.84 ± 0.79 | 4.36 ± 0.48* |
NH3 ( |
398.14 ± 75.85 | 280.79 ± 57.99** |
TRIG (mmol |
1.90 ± 0.40 | 1.63 ± 0.68 |
LACT (mmol |
2.62 ± 0.98 | 2.10 ± 0.69 |
CHOL (mmol |
6.50 ± 1.43 | 5.54 ± 1.12* |
ALP ( |
1.64 ± 0.78 | 1.40 ± 0.69 |
ALT ( |
0.33 ± 0.12 | 0.42 ± 0.28 |
AST ( |
7.66 ± 2.35 | 7.71 ± 1.85 |
LDH-L ( |
16.57 ± 6.50 | 16.77 ± 6.81 |
Ca2+ (mmol |
2.32 ± 0.15 | 2.32 ± 0.16 |
PHOS (mmol |
3.49 ± 0.41 | 3.67 ± 0.42 |
Significant difference between test groups (
ALB: albumins, TP: total proteins, GLU: glucose concentration, NH3: ammonia, TRIG: triacylglycerols, LACT: lactate, CHOL: cholesterol, ALP: alkaline phosphatase, ALT: alanine aminotransferase, AST: aspartate aminotransferase, LDH: lactate dehydrogenase, Ca2+: calcium, PHOS: inorganic phosphate.
The mean values of fish total and standard body length, body and liver weight, Fultons condition factor, and hepatosomatic index did not show significant differences (Table
Biometrical indices 23 days after the beginning of the experiment.
Indice | Control |
Experimental |
---|---|---|
Total length | 26.10 ± 1.37 | 25.89 ± 0.85 |
Standard length | 23.44 ± 1.25 | 23.67 ± 0.85 |
Body weight | 199.33 ± 33.22 | 185.48 ± 29.95 |
Liver weight | 2.98 ± 0.69 | 2.92 ± 0.66 |
Fultons condition factor | 1.54 ± 0.17 | 1.39 ± 0.17 |
Hepatosomatic index | 1.49 ± 0.26 | 1.58 ± 0.33 |
The histopathological examination revealed severe hyaline droplet degeneration in the tubular epithelial cells (tubulonephrosis) of the caudal kidney in 9 out of 10 fish fed the diet containing DON (Figure
Effect of deoxynivalenol exposure on caudal kidney histology. Hyaline droplet degeneration of tubular epithelial cells (arrows). HE, 400x.
The study showed posttreatment changes in the haematological and biochemical profiles and histopathological changes in rainbow trout fed a commercial feed with the addition of the mycotoxin deoxynivalenol. No fish mortality was observed in the control or experimental groups during the test.
The main haematological response of rainbow trout after 23 days exposure to DON was a statistically significant decrease in MCH (
Experimental rainbow trout after exposure of DON-haemorrhages in the liver and gastrointestinal tract.
The main biochemical response of rainbow trout to the effect of DON showed a decrease in glucose, cholesterol (
We observed severe hyaline droplet degeneration in the tubular epithelial cells of the renal tubules of the caudal kidney in the experimental group. The kidney is a target organ of certain toxicants; it is a major route for the excretion of foreign chemicals. On the other hand, Hooft et al. reported that deoxynivalenol caused considerable morphological changes in the liver, including subcapsular haemorrhages, subcapsular edema, altered hepatocytes, and fatty infiltration [
In conclusion, the results of the present study indicate that exposure of deoxynivalenol in a dose of 2 mg/kg feed induces significant changes in the haematological and biochemical parameters and in the histopathological examination of rainbow trout. The alterations of these parameters may provide a better understanding of the toxicological effect of mycotoxin deoxynivalenol on aquaculture fish, such as rainbow trout.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research was supported by the Project IGA 34/2013/FVHE and the Project KUS QJ1210013. The authors would like to thank Charles du Parc for proofreading this paper.