2002 Poultry Science Association, Inc. EFFECT OF HYDRATED SODIUM CALCIUM ALUMINOSILICATE ON THE PREVENTION OF THE TOXIC EFFECTS OF OCHRATOXIN E. SANTIN, 1 A. MAIORKA, E. L. KRABBE, A. C. PAULILLO, and A. C. ALESSI Faculdade de Ciências Agrárias e Veterinárias Campus de Jaboticabal-Unesp, Cep: 14870-000 Jaboticabal SP Brazil Phone: 55 16 323 2500 ext. 117 FAX: 55 16 322 4275 e-mail: besantin@fcav.unesp.br Primary Audience: Nutritionists, Researchers, Broiler Production Managers SUMMARY We studied the efficacy of hydrated sodium calcium aluminosilicate (HSCAS) as an inhibitor of the toxic effects of ochratoxin in broilers from 1 to 42 d of age. A total of 288 broilers was distributed into four treatments with four replicates of 18 birds each: T1, control; T2, 0.25% HSCAS; T3, 2 ppm of ochratoxin; T4, 0.25% HSCAS + 2 ppm ochratoxin. The parameters evaluated were feed intake; weight gain; feed conversion; relative weights of the liver, kidneys, and bursa; and serum levels of Ca, P, total protein (TP), aspartate aminotransferase (AST) and γ-glutamiltransferase (GGT). Ochratoxin in the diet negatively affected (P < 0.05) all performance parameters evaluated when the birds were 21 and 42 d of age. However, HSCAS did not affect performance, and there was no interaction between HSCAS and dietary ochratoxin. The liver and the kidneys of birds fed ochratoxin with or without HSCAS were relatively heavier (P < 0.05) than those of the control birds, demonstrating the influence of ochratoxin, but not of HSCAS, on the relative weight of these organs. Although the bursa of birds exposed to ochratoxin with or without HSCAS had a lower relative weight as compared to control birds, the difference was not significant. Ca, P, and TP serum levels were lower (P < 0.05) in birds exposed to ochratoxin, whereas AST and GGT levels were higher (P < 0.05) in these birds. These results reflect that ochratoxin in the diet impaired the productivity indexes and that HSCAS did not improve these parameters. Key words: broiler, performance, hydrated sodium calcium aluminosilicate, ochratoxin 2002 J. Appl. Poult. Res. 11:22 28 DESCRIPTION OF PROBLEM Ochratoxins are mycotoxins isolated from Aspergillus ochraceus but can also be produced by a series of Aspergillus and Penicillium species. Of this group of isocumarins, only ochratoxin A has been naturally isolated from cere- als, and it is the most toxic mycotoxin for birds [1]. These mycotoxins occur in several parts of the world, contaminating cereals that are traditionally used in poultry feeds. Ochratoxin A is classified as the second most important mycotoxin in terms of economic losses worldwide, aflatoxins being the first [2]. Intoxication of birds by ochratoxin results in reduced weight 1 To whom correspondence should be addressed.
SANTIN ET AL.: BROILER PERFORMANCE 23 gain, impaired feed efficiency, reduced egg production and quality [3], and also induced immunosuppression in broilers [4]. In the last few years, several studies have suggested that aluminosilicates can reduce the effects of mycotoxins in several animal species, due to their ability to bind to or to adsorb mycotoxins [5]. However, the actions have been explored mostly in relation to aflatoxicosis [5]. Despite that some studies have shown these minerals do not adsorb ochratoxin in vitro [6] or in vivo [7], the poultry industry has sometimes used aluminosilicates to control ochratoxicosis because they believe that the altered chemical structure of some aluminosilicates might be able to adsorb other mycotoxins than aflatoxin. The present experiment was carried out to evaluate the efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) CaO Na 2 O - K 2 O MgO A 12 O 3 4SiO 2 (OH) 2 2H 2 O in preventing performance losses in broilers exposed to ochratoxin. MATERIALS AND METHODS EXPERIMENTAL BIRDS AND MANAGEMENT A total of 288 sexed (male) Cobb 1-d-old broilers was housed in 16 cages in an experimental house and received feed and water ad libitum. Birds were distributed into the following four treatments: T1, control (0.0 HSCAS + 0 ochratoxin); T2, 0.25% HSCAS; T3, 2 ppm ochratoxin; and T4, 0.25% HSCAS + 2 ppm ochratoxin. The diet was based on corn and soybean meal, containing or exceeding the nutritional requirements recommended by the NRC [8]. HSCAS and ochratoxin were included in the feed, according to each treatment, for the duration of the experiment. During the experimental period, diets were analyzed for zearalenone and aflatoxin with the technique described by Soares and Rodriguez-Amaya [9]. These mycotoxins were not detected in the feeds. Birds and residual feed were weighed at the beginning and end of each experimental stage (initial stage: 1 to 21 d and final stage: 22 to 42 d). Feed conversion was obtained by dividing total feed intake by the weight gain of each pen added to the weights of dead birds. STATISTICAL ANALYSIS The statistical analysis used a completely random experimental design in a factorial (2 2) arrangement. Data were subjected to analysis of variance, and when significant differences were obtained, Tukey s test was used. OCHRATOXIN PRODUCTION AND ALUMINOSILICATE Ochratoxin was produced in wheat by Aspergillus ochraceus, Strain NRRL 3174, at 46% humidity [10]. After 11 d, this wheat was dried in an oven at 100 C for 12 h and ground, and its ochratoxin level was analyzed by thin-layer chromatography [9]. This ochratoxin was added weekly to the feed at 2 ppm with the aid of a Y mixer level, according to the experimental treatment. Basal diets containing ochratoxin were analyzed by thin-layer chromatography [9] to confirm the desired level of ochratoxin. The aluminosilicate used was CaO Na 2 O K 2 O MgO A 12 O 3 4SiO 2 (OH) 2 2H 2 O. BIOCHEMICAL ANALYSIS At 18 d of age, 16 birds per treatment (four birds from four repetitions) were bled by brachial vein puncture to collect blood samples for total protein (TP), Ca, P, aspartate aminotransferase (AST), and γ-glutamiltransferase (GGT) analyses. The biochemical analyses were performed by colorimetric method by using commercial kits [11]. MACROSCOPIC AND MORPHOMETRIC ANALYSIS OF THE ORGANS At 21 and 42 d of age, eight birds from each treatment were slaughtered by cervical dislocation to perform macroscopic observations of the bursa, liver, and kidneys. These organs were immediately dissected and individually weighed. Weights were expressed as a percentage of body weight, thus obtaining the relative weight of organs. RESULTS AND DISCUSSION Ochratoxins may cause significant losses to the poultry industry due to reduced performance and health problems in the exposed
24 JAPR: Research Report TABLE 1. Feed intake, body weight gain, and feed:gain of broilers at 1 to 21 d of age HSCAS A OCHRATOXIN FEED INTAKE BODY WEIGHT FEED:GAIN TREATMENT (%) (ppm) (g) GAIN (g) (g/g) T1 0.00 0 933 650 1.434 T2 0.25 0 940 651 1.447 T3 0.00 2 520 301 1.726 T4 0.25 2 449 259 1.735 CV (%) 39 42 10.12 Ochratoxin (+) 484 280 1.732 Ochratoxin ( ) 937 651 1.443 HSCAS (+) 695 475 1.599 HSCAS ( ) 727 455 1.582 Ochratoxin (O) 0.001 0.001 0.001 HSCAS (A) 0.253 0.178 0.745 A O 0.168 0.164 0.953 birds. The use of aluminosilicates as absorbent materials has been extensively studied in the past [12] due to the ability of these substances to bind to mycotoxins, especially to aflatoxins [5]. The present study showed that 1-to-42-dold broilers fed 2 ppm of ochratoxin in their diets had a significant decrease in feed intake and weight gain as compared to the control group. Birds exposed to ochratoxin, at 21 or 42 d of age, had lower average feed intake and weight gain (P < 0.01), as well as poorer feed conversion (P < 0.05), as compared to control birds. Addition of HSCAS to the diet containing the mycotoxin did not minimize these effects and did not influence these parameters when included in the control diet with no mycotoxin (Tables 1, 2, and 3). Despite other studies that have shown the efficacy of these minerals in cases of aflatoxicosis [5, 12, 13], recent research has demonstrated that aluminosilicates are not effective for all mycotoxins [7, 14, 15]. Bailey et al. [14] and Kubena et al. [16] have found that different aluminosilicates did not protect birds against detrimental effects of T- 2 toxin. This divergence in results on the ad- TABLE 2. Feed intake, body weight gain, and feed:gain of broilers at 22 to 42 d of age HSCAS A OCHRATOXIN FEED INTAKE BODY WEIGHT FEED:GAIN TREATMENT (%) (ppm) (g) GAIN (g) (g/g) T1 0.00 0 2,960 1,678 1.764 T2 0.25 0 2,890 1,717 1.688 T3 0.00 2 2,231 1,182 1.888 T4 0.25 2 2,092 1,117 1.882 CV (%) 16.81 20.64 8.87 Ochratoxin (+) 2,161 1,150 1.885 Ochratoxin ( ) 2,925 1,697 1.726 HSCAS (+) 2,491 1,417 1.785 HSCAS ( ) 2,595 1,430 1.826 Ochratoxin (O) 0.001 0.001 0.056 HSCAS (A) 0.245 0.749 0.599 A O 0.699 0.235 0.652
SANTIN ET AL.: BROILER PERFORMANCE 25 TABLE 3. Feed intake, body weight gain, and feed:gain of broilers at 1 to 42 d of age HSCAS A OCHRATOXIN FEED INTAKE BODY WEIGHT FEED:GAIN TREATMENT (%) (ppm) (g) GAIN (g) (g/g) T1 0.00 0 3,893 2,328 1.672 T2 0.25 0 3,830 2,368 1.621 T3 0.00 2 2,751 1,483 1.856 T4 0.25 2 2,540 1,376 1.854 CV (%) 20.18 25.67 8.79 Ochratoxin (+) 2,646 1,429 1.855 Ochratoxin ( ) 3,862 2,348 1.646 HSCAS (+) 3,185 1,872 1.737 HSCAS ( ) 3,322 1,905 1.764 Ochratoxin (O) 0.001 0.001 0.005 HSCAS (A) 0.189 0.514 0.671 A O 0.468 0.174 0.689 sorption of mycotoxins is probably due to variation in the structure of adsorbents and mycotoxins, as adsorption is a function of ion and/ or molecule exchange between these substances. It should be noted that even for aflatoxins there are differences in adsorption related to variation in ph, density, volume and pore diameter, and surface area of the binding material [17]. Although some studies demonstrated that dietary aluminosilicates might have negative influence on performance, Morris et al. [18], similarly to the present study, did not observe any change in productivity indexes of birds fed only aluminosilicate in the diet. Total protein serum levels were significantly reduced (P < 0.01) at 18 d of age in broilers exposed to ochratoxin (Table 4). This finding is probably related to protein synthesis inhibition or reduction promoted by this mycotoxin [19]. Although the mechanism of protein synthesis inhibition is still not fully understood, ochratoxin probably interferes in the synthesis of enzymes by competitively inhibiting phenylalanine-trna [20]. Ochratoxin also disturbs calcium homeostasis, inhibiting ATP produc- TABLE 4. Serum biochemical values of total protein (TP), Ca, and P and enzyme activities of aspartate amino transferase (AST) and γ-glutamyl transferase (GGT) in broilers at 18 d of age HSCAS A OCHRATOXIN TP Ca P AST GGT TREATMENT (%) (ppm) (g/dl) (mg/dl) (mg/dl) (U/mL) (U/mL) T1 0.00 0 3.73 ± 0.33 7.15 ± 0.68 4.67 ± 0.80 99.75 ± 7.7 10.30 ± 3.1 T2 0.25 0 3.42 ± 0.60 5.17 ± 0.97 5.08 ± 1.10 89.62 ± 4.7 13.47 ± 3.8 T3 0.00 2 3.00 ± 0.41 6.64 ± 0.73 3.85 ± 0.54 115.50 ± 14 17.42 ± 2.7 T4 0.25 2 2.36 ± 0.38 5.15 ± 0.68 3.77 ± 0.80 107.88 ± 2.7 19.00 ± 2.1 Ochratoxin (+) 2.68 5.89 3.81 111.69 18.61 Ochratoxin ( ) 3.58 6.89 4.88 94.68 11.88 HSCAS (+) 2.89 5.16 4.43 107.63 16.63 HSCAS ( ) 3.37 6.89 4.26 98.75 13.86 Ochratoxin (O) 0.001 0.001 0.002 0.009 0.001 HSCAS (A) 0.008 0.372 0.596 0.156 0.081 A O 0.337 0.407 0.445 0.839 0.723
26 JAPR: Research Report TABLE 5. Relative weights of liver, kidney, and bursa of Fabricius in broilers at 21 d of age HSCAS A OCHRATOXIN LIVER KIDNEY BURSA OF FABRICIUS TREATMENT (%) (ppm) (g/100 g BW) (g/100 g BW) (g/100 g BW) T1 0.00 0 3.64 1.15 0.25 T2 0.25 0 4.19 0.99 0.24 T3 0.00 2 5.67 1.92 0.23 T4 0.25 2 5.36 1.66 0.21 CV (%) 21.88 35.43 26.21 Ochratoxin (+) 5.52 1.79 0.23 Ochratoxin ( ) 3.91 1.07 0.25 HSCAS (+) 4.77 1.33 0.23 HSCAS ( ) 4.65 1.57 0.24 Ochratoxin (O) 0.001 0.002 0.543 HSCAS (A) 0.715 0.285 0.622 A O 0.201 0.782 0.895 tion in the mitochondria, suggesting that protein synthesis inhibition is also a consequence of a constraint in the synthesis of aminoacyltrna due to a lower availability of ATP [21]. In this experiment, there was a reduction (P < 0.01) in the serum levels of Ca and P in 18-d-old birds exposed to dietary ochratoxin (Table 4). According to Kubena et al. [15], this finding may be due to lower feed intake, but it must also be taken into account that the nutrient absorption capacity of the gastrointestinal tract is reduced in these birds. Serum levels of AST and GGT were statistically high (P < 0.01) in birds fed the diet with ochratoxin (Table 4). High GGT levels are related to liver lesions observed in birds exposed to ochratoxin [21]. AST enzyme is associated to the process of glyconeogenesis, which is increased in cases of ochratoxicosis [22]. Glyconeogenesis is described as a special form of glucose synthesis under low availability of exogenous nutrients or of reduced use of nutrients in the diet. In cases of ochratoxicosis, glyconeogenesis is probably due to lower avail- TABLE 6. Relative weights of liver, kidney, and bursa of Fabricius in broilers at 42 d of age HSCAS A OCHRATOXIN LIVER KIDNEY BURSA OF FABRICIUS TREATMENT (%) (ppm) (g/100 g BW) (g/100 g BW) (g/100 g BW) T1 0.00 0 2.17 0.70 0.26 T2 0.25 0 2.51 0.71 0.24 T3 0.00 2 4.43 1.24 0.21 T4 0.25 2 4.17 1.51 0.17 CV (%) 33.46 40.51 38.52 Ochratoxin (+) 4.30 1.37 0.19 Ochratoxin ( ) 2.34 0.70 0.25 HSCAS (+) 3.47 1.10 0.21 HSCAS ( ) 3.16 0.97 0.24 Ochratoxin (O) 0.001 0.001 0.225 HSCAS (A) 0.230 0.291 0.471 A O 0.878 0.323 0.859
SANTIN ET AL.: BROILER PERFORMANCE 27 ability of enzymes and to metabolic alkalosis, which results from ion and glucose losses due to kidney lesions. The interference of HSCAS in the TP serum levels is not reported in the literature. Some aluminosilicates may decrease Ca and Cl serum levels in broilers [23]. According to Shryock et al. [24], certain aluminosilicates may chemically interact with many diet components, promoting their adsorption. Therefore, it is possible that the studied aluminosilicate might have adsorbed some dietary micronutrients involved in the molecular cycle of protein synthesis, reducing their absorption by the birds and conse- quently interfering in their biochemical levels in the birds sera. Macroscopic and relative weight changes in the livers and kidneys of birds exposed to ochratoxin in the present experiment are comparable to those described in the literature on ochratoxicosis [25, 26]. Liver had higher relative weights, a yellowish color, and was friable at palpation, and the kidneys were pale and also had higher relative weights (Table 5 and 6). The bursa of the Fabricius did not show significant changes in relative weight (P > 0.05) in birds exposed to ochratoxin in the feed. The inclusion of HSCAS in the feed did not affect the relative weight of these organs. CONCLUSIONS AND APPLICATIONS 1. Ochratoxin negatively influenced feed intake, weight gain, and feed conversion in broilers. 2. Ochratoxin reduced serum levels of TP, Ca, and P and increased serum levels of AST and GGT in broilers. 3. Ochratoxin increased the relative weights of livers and kidneys of broilers. 4. The HSCAS used in this study did not reduce the harmful effects of ochratoxin. REFERENCES AND NOTES 1. Gibson, R.M., C.A. Bailey, L.F. Kubena, W.E. Huff, and R.B. Harvey, 1990. 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