Brazilian Journal of Poultry Science Revista Brasileira de Ciência Avícola ISSN 1516-635X Oct - Dec 2006 / v.8 / n.4 / 221 - 225 221 Santin E1 Paulillo AC2 Nakagui LSO2 Alessi AC2 Maiorka A1 1 Universidade Federal do Paraná - UFPR. 2 Faculdade de Ciências Agrárias e Veterinárias – Unesp. Elizabeth Santin Departamento de Medicina Veterinária Setor de Ciências Agrárias / UFPR Rua dos Funcionários, 1540 Juveve 80.035-050. Curitiba, PR, Brazil Aflatoxin, Ochratoxin, Yeast cell wall, broilers, performance. The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo. Proc. n. 99/ 12952-7 for their financial support. Evaluation of Yeast Cell Wall on The Performance of Broiles Fed Diets With or Without Mycotoxins Mail Address Keywords Author(s) Arrived: July / 2003 Approved: November / 2006 ABSTRACT This experiment aimed at evaluating the effects of the interactions between aflatoxin (500 or 250 ppb) and ochratoxin (500 or 250 ppb), and the possible benefits of adding yeast cell wall to prevent the effects of these mycotoxins in broiler chickens. Relative organ weight gain and live performance were evaluated at 21 and 42 days of age. Results indicated that at the levels of mycotoxins included in the experimental diets, ochratoxin reduced feed intake and body weight gain, and aflatoxin only affect feed intake of 21-day-old birds. No interaction was observed between aflatoxin and ochratoxin at the levels used in experimental study. Yeast cell wall did not significantly reduced the deleterious effects of ochratoxins. No significant differences were observed in relative organ weight gain. Yeast cell wall improved feed conversion ratio when birds were fed either contaminated or non- contaminated feeds. INTRODUCTION Mycotoxins are toxic substances produced by fungi that commonly grow in cereals used in animal feeds and human food (Santin et al., 2000). Fungi infestation and the production of mycotoxins may occur any time in a cereal mass. In general, the interaction between fungi species and variations in temperature and moisture promotes a mixture of different mycotoxins in the stored grain (Ramakrishna et al., 1996). Huff & Doerr (1981) reported that aflatoxin and ochratoxin can be found as simultaneous contaminants of feed ingredients and finished feed, and the effect of the synergic action of these two mycotoxins on growth rate was far greater than the sum of the individual toxicities of these mycotoxins. According with these authors, the synergism between aflatoxin and ochratoxin A significantly decreased growth rate and numerically increased mortality, demonstrating the enhanced toxicity of contaminated feed. Recently, the most promising and practical approaches to counteract mycotoxins are the use of adsorbents. However, although several studies showed that aluminosilicates can reduce the effects of aflatoxins, due to their binding capacity (Phillips et al., 1988; Kubena et al., 1990), these adsorbents cannot alleviate the harmful effects of ochratoxin (Santin et al., 2002). This is probably due to the variation in the structure of adsorbents and of mycotoxins. A study of Huff et al. (1992) suggested that when aflatoxin is found to be the only mycotoxin present in the diet, it may be cost effective to add aluminosilicates to the diet to minimize the effects of the problem. However, if ochratoxin A is present, aluminosilicates have little effect on the toxicity of this mycotoxin, and the proportion of aflatoxin biologically available to the animal may interact with ochratoxin, resulting in a little benefit from the use of aluminosilicates. Acknowledgments Santin E, Paulillo AC, Nakagui LSO, Alessi AC, Maiorka A Evaluation of Yeast Cell Wall on The Performance of Broiles Fed Diets With or Without Mycotoxins 222 On the other hand, a live yeast, Saccharomyces cerevisiae, was found to alleviate the adverse effects of aflatoxicosis in poultry (Stanley et al., 1993). Its beneficial effects are attributed to the composition of its cell wall. The objective of the present study was to evaluate the effect of the interaction between aflatoxin and ochratoxin on relative organ weight and performance of broilers, and the effects of yeast cell wall (SCCW) to counteract these mycotoxins. MATERIAL AND METHODS Experimental birds and diets Six hundred and forty day-old Cobb male broilers were used. Birds were fed a balanced diet, based on corn and soybean meal, formulated according to the recommendations of the National Research Council (1994). The corn used was free from aflatoxin, ochratoxin, and zearalenone, as determined by thin- layer chromatography (Soares & Rodriguez-Amaya, 1989). Birds were submitted to similar management as that practiced in commercial flocks. Mycotoxin and Yeast cell wall Aflatoxin was produced by the fermentation of rice with Aspergillus parasiticus, strain NRRL 2999, according to West et al. (1973). Ochratoxin was produced by the fermentation of wheat with Aspergillus ochraceus, strain NRRL 3174, according to Manning & Wyatt (1984). After fermentation, wheat and rice were dried in an oven at 100 oC for 12 hours, and ground. Ochratoxin A and aflatoxin levels were analyzed by thin-layer chromatography, according to the technique described by Soares & Rodriguez-Amaya (1989). Saccharomyces cerevisiae var. Calsberg was cultivated in simple agar. After growth, it was replicated in liquid peptonized water with 2% of dextrose and incubated at 37 oC for 48 hours. Then, the solid phase was separated by centrifugation at 3000 x for 30 min, and lyophilized to be incorporated in feed. The inocula (500 ppb or 250 ppb of aflatoxin and ochratoxin, and 0.1% of yeast cell wall) were weekly mixed into the feed, as indicated by each treatment, from the first day until the end of the trial (1 to 42 days). Experimental design A randomized experimental design with a factorial arrangement (3 x 3 x 2) incomplete block with missing cells was used, with 640 birds distributed into eight groups of 80. Group 1 did not receive either mycotoxin or yeast cell wall in the diet, Group 2 was fed 500 ppb of aflatoxin in diet, Group 3 was fed a diet containing 500 ppb of ochratoxin, Group 4 was fed a diet containing 250 ppb of aflatoxin and 250 ppb of ochratoxin, Group 5 was fed a diet containing 0.1% yeast cell wall, Group 6 was fed a diet containing 500 ppb of aflatoxin and 0.1% yeast cell wall, Group 7 was fed a diet containing 500 ppb of ochratoxin and 0.1% yeast cell wall, and Group 8 was fed a diet containing 250 ppb aflatoxin, 250 ppb ochratoxin and 0.1% yeast cell wall. Data were submitted to analysis of variance. Necropsy and morphometric analysis of the organs On days 21 and 42 of the experimental period, eight birds from each group were slaughtered by cervical dislocation to perform macroscopic observation of the bursa of Fabricius, liver, and kidney. These organs were immediately dissected and individually weighed. Weights were expressed as a percentage of body weight, thus obtaining the relative weight of each organ. Performance analysis Birds and residual feed were weighed at the beginning and end of each experimental stage (initial stage: 1 to 21 day and final stage: 22 to 42 days). Feed conversion ratio was obtained by dividing total feed intake by weight gain of each pen added to the weight of dead birds. RESULTS AND DISCUSSION Mycotoxins may cause significant losses to poultry industry due to reduced performance and health problems in the exposed birds as was observed in the present study. The results in Table 1, 2, and 3 show that ochratoxin in the diet significantly decreased feed intake and weight gain as compared to the control group. Birds exposed to ochratoxin during initial stage (1 to 21 days), final stage (22 to 42 days), or in total experimental period (1 to 42 days) had lower average feed intake and weight gain (P <0.05). Prior et al. (1980) also showed performance reduction using the same ochratoxin levels in broilers. However, aflatoxin caused a significant (P = 0.040) decrease in feed intake at 21 days (Table 1). This mycotoxin did not influence other performance parameters at 21 or 42 days (Tables 1, 2 and 3), which is probably due to the lower levels of this mycotoxin used in the present study. It is known that aflatoxin level should be of at least 1 ppm to affect Santin E, Paulillo AC, Nakagui LSO, Alessi AC, Maiorka A Evaluation of Yeast Cell Wall on The Performance of Broiles Fed Diets With or Without Mycotoxins 223 bird performance under experimental conditions (Santin et al., 2003). In the present study, no significant (P>0.05) interaction between aflatoxin and ochratoxin was observed, contrary to the reports of Huff & Doerr (1981) and Huff et al. (1992), probably due to the lower levels of mycotoxins used in the present study as compared to those studies. The addition of SCCW to the diet containing mycotoxins did not minimize the deleterious effects of ochratoxin, but improved feed conversion ratio in birds fed diets with or without mycotoxin during the final stage (22 to 42 days) and total experimental period (1 to 42 days). A recent study from Santin et al. (2001) showed that SCCW improved intestinal mucosa parameters, and suggested that this might be the Table 1 - Feed intake, body weight gain and feed:gain of broilers at initial stage of experiment (1 to 21 day). Aflatoxin(ppb) Ochratoxin(ppb) SCCW*(%) Feed intake (g) Weight gain (g) Feed:gain(g/g) 0 0 0 1,136 694 1.642 0 500 0 1,071 667 1.606 500 0 0 1,063 699 1.520 250 250 0 1,010 639 1.579 0 0 0.1 1,136 723 1.575 0 500 0.1 1,040 660 1.578 500 0 0.1 1,078 726 1.487 250 250 0.1 1,019 640 1.596 Main effects Aflatoxin (+) 1,047 676 1.546 Aflatoxin (-) 1,096 686 1.600 Ochratoxin (+) 1,035 651 1.590 Ochratoxin (-) 1,103 710 1.556 SCCW (+) 1,069 675 1.559 SCCW (-) 1,070 687 1.587 Probabilities Aflatoxin (A) 0.040 0.417 0.195 Ochratoxin (O) 0.011 0.000 0.419 SCCW (SC) 0.948 0.313 0.501 A x O 0.629 0.251 0.235 A x SC 0.586 0.924 0.636 O x SC 0.701 0.214 0.522 A x O x SC 0,808 0,845 0,948 SCCW – Cell wall of Saccharomyces Cerevisiae. Table 2 - Feed intake, body weight gain and feed:gain of broilers at initial stage of experiment (21 to 42 day). Aflatoxin(ppb) Ochratoxin(ppb) SCCW*(%) Feed intake (g) Weight gain (g) Feed:gain(g/g) 0 0 0 3,555 1,781 1.995 0 500 0 3,329 1,658 2.008 500 0 0 3,543 1,717 2.063 250 250 0 3,405 1,665 2.045 0 0 0.1 3,437 1,813 1.903 0 500 0.1 3,459 1,700 2.034 500 0 0.1 3,452 1,781 1.941 250 250 0.1 3,261 1,673 1.949 Main effects Aflatoxin (+) 3,445 1,709 1.999 Aflatoxin (-) 3,444 1,737 1.985 Ochratoxin (+) 3,363 1,674 2.009 Ochratoxin (-) 3,496 1,773 1.976 SCCW (+) 3,402 1,741 1.957 SCCW (-) 3,457 1,705 2.028 Probabilities Aflatoxin (A) 0.636 0.240 0.665 Ochratoxin (O) 0.041 0.000 0.319 SCCW (SC) 0.375 0.140 0.042 A x O 0.620 0.436 0.255 A x SC 0.325 0.980 0.258 O x SC 0.437 0.650 0.285 A x O x SC 0.234 0.508 0.492 SCCW – Cell wall of Saccharomyces Cerevisiae. Santin E, Paulillo AC, Nakagui LSO, Alessi AC, Maiorka A Evaluation of Yeast Cell Wall on The Performance of Broiles Fed Diets With or Without Mycotoxins 224 explanation for the improvement of the performance of broilers supplemented with SCCW in that report. In fact, the yeast cell wall used in the present study was not efficient in alleviating the effects of ochratoxicosis. However, it improved feed conversion of birds fed diets with or without mycotoxins, suggesting that the effect of the SCCW used was probably not due to its role as mycotoxin adsorbent , but as a supplement that improved feed conversion ratio. Yeast cell wall usually contains mannan oligosaccharides, and some reports showed that the use of these compounds improves feed conversion ratio(Savage & Zakrzewska, 1997, Fritts & Waldroup, 2003). Although it was reported that ochratoxin and aflatoxin cause increase in relative liver and kidney weight in poultry (Huff & Doerr, 1981), the present study did not show any differences in relative organ weight among the experimental groups. A possible explanation for this finding may be the relatively higher mycotoxin levels used by Huff & Doerr (1981) as compared those used in the present study. CONCLUSIONS It may be suggested that ochratoxin causes losses in performance of broilers, and that SCCW was not efficient in reducing the losses caused by this mycotoxin. However, SCCW may be used as a supplement to improve feed conversion ratio in broilers. REFERENCES Fritts CA, Waldroup PW. Evaluation of Bio-Mos© mannan oligosaccharides as a replacement for growth promoting antibiotics in diet for turkeys. International Journal of Poultry Science 2003; 2 (1):19-22. Huff WE, Doerr J. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Science 1981; 60:550-555. Huff WE, Kubena LR, Harvey RB. Progression of ochratoxicosis in broiler chickens. Poultry Science 1992; 71:64-69. Kubena LF, Harvey RB, Phillips TD, Corrier DE, Huff WE. Diminution of aflatoxicosis in growing chickens by dietary addition of a hydrated sodium calcium aluminosilicate. Poultry Science 1990; 69:727-735. Manning RO, Wyatt RD. Toxicity of Aspergillus ochraceus contaminated wheat and different chemical forms of ochratoxin A in broiler chicks. Poultry Science 1984; 63:458-465. National Research Council, Nutrient requirements of poultry. 9th rev. ed. Washington: National Academic Press; 1994. Phillips TD, Kubena LF, Harvey RB, Taylor DR, Heidelbaurgh NH. Hydrated sodium calcium aluminosilicate: a high affinity sorbent for aflatoxin. Poultry Science 1988; 67:243-247. Prior MG, O´Neil JB, Sisodia CS. Effects of ochratoxin A on growth response and residues in broilers. Poultry Science 1980; 59:1254- 1257. Ramakrishna N, Lacey J, Smith JE. Aspergilus flavus colonization and aflatoxin B 1 formation in barley grain during interactions with other fungi. Mycopathologia 1996; 136:53-63. Table 3 - Feed intake, body weight gain and feed:gain of broilers at initial stage of experiment (1 to 42 day). Aflatoxin(ppb) Ochratoxin(ppb) SCCW*(%) Feed intake (g) Weight gain (g) Feed:gain(g/g) 0 0 0 4,691 2,475 1.896 0 500 0 4,400 2,325 1.906 500 0 0 4,600 2,417 1.905 250 250 0 4,415 2,304 1.916 0 0 0.1 4,573 2,536 1.807 0 500 0.1 4,500 2,360 1.906 500 0 0.1 4,530 2,506 1.808 250 250 0.1 4,280 2,313 1.850 Main effects Aflatoxin (+) 4,458 2,385 1.870 Aflatoxin (-) 4,541 2,424 1.876 Ochratoxin (+) 4,399 2,325 1.854 Ochratoxin (-) 4,600 2,483 1.891 SCCW (+) 4,471 2,429 1.903 SCCW (-) 4,528 2,380 1.843 Probabilities Aflatoxin (A) 0.243 0.196 0.811 Ochratoxin (O) 0.008 0.000 0.129 SCCW (SC) 0.419 0.106 0.019 A x O 0.789 0.870 0.648 A x SC 0.495 0.985 0.360 O x SC 0.577 0.375 0.169 A x O x SC 0.331 0.643 0.474 SCCW – Cell wall of Saccharomyces Cerevisiae. Santin E, Paulillo AC, Nakagui LSO, Alessi AC, Maiorka A Evaluation of Yeast Cell Wall on The Performance of Broiles Fed Diets With or Without Mycotoxins 225 Santin E, Maiorka A, Krabbe EL, Paulillo AC, Alessi AC. 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