Evaluation of the detoxifying effect of yeast glucomannan on aflatoxicosis in broilers as assessed by gross examination and histopathology

This paper was published in "British Poultry Science Volume 46, Number 3 (June 2005), pp. 394–400"

INTRODUCTION

Aflatoxins are a major concern in poultry production because of serious economic losses. The toxicity of aflatoxins in broiler chickens has been widely investigated by determining their teratogenic, carcinogenic, mutagenic and growth inhibitory effects (Oguz and Kurtoglu, 2000). The biochemical—haematological (Oguz et al., 2000), immunological (Qureshi et al., 1998), pathological (Kiran et al., 1998; Ortatatli and Oguz, 2001) and toxic effects of aflatoxins have also been well described. Aflatoxins also cause important gross and microscopic changes in the liver, kidney and spleen (Espada et al., 1992; Ledoux et al., 1999), unfavourable reproductive changes (Ortatatli et al., 2002), impair the humoral and cellular immune responses and increase susceptibility to some environmental and infectious agents (Gabal and Azzam, 1998).

Preventing mould growth and aflatoxin contamination in feed and feedstuffs is very important but when contamination cannot be prevented, decontamination of aflatoxin is needed before using these materials. Practical and cost-effective methods of detoxifying aflatoxin-containing feed are in great demand. Since the early 1990s, studies with adsorbents have proven successful, but high inclusion rates and potential interactions with feed nutrients are causes for concern (Rosa et al., 2001). Also, previous studies suggested that the best approach for decontamination would be biological degradation which could allow removal of aflatoxin under mild conditions, without using harmful chemicals or causing appreciable losses in nutritive value and palatability (Bata and Lasztity, 1999). As a result, researchers have directed efforts toward finding effective means of biological degradation of aflatoxin

A live yeast, Saccharomyces cerevisiae, initially used as a performance promoter in the early 1990s, was found to have beneficial effects on weight gain and immune response in broilers exposed to aflatoxin (Stanley et al., 1993). The beneficial effects of Saccharomyces cerevisiae have been attributed to mannan in the yeast cell wall. Mannan is extracted from the glucan-rich inner cell wall of this yeast. Yeast glucomannan showed markedly high binding ability in vitro (75 to 90%) and in vivo with aflatoxin (Murthy and Devegowda, 2004), and it has been preferred for detoxification of aflatoxin in poultry species. The studies performed using yeast glucomannan (0.5 and 1 g/kg) with different concentrations of aflatoxin (0.2 to 2 mg/kg feed) in broilers (Raju and Devegowda, 2000; Aravind et al., 2003; Santin et al., 2003; Girish and Devegowda, 2004) showed that yeast glucomannan reversed the adverse effect of aflatoxin on performance, biochemistry—haematology and immune responses of birds. Yeast glucomannan was also used for detoxification of other mycotoxins (Devegowda and Aravind, 2004).

The purpose of the present study was to examine the toxic effects of aflatoxin (2 mg/kg) on gross and microscopic changes; to evaluate efficacy of two dietary levels of yeast glucomannan (0.5 and 1 g/kg) in reducing or preventing the pathological changes induced by aflatoxin; and to compare the efficacy of these different dietary concentrations of yeast glucomannan in broilers.

MATERIALS AND METHODS

Birds and diet

Two hundred and forty 1-d-old male unvaccinated broiler chicks (Ross-308) were obtained from a commercial hatchery. Individually weighed chicks were divided at random into groups. The chicks were housed in floor pens with continuous lighting and were fed ad libitum with commercial feed. The basal diet was formulated to fulfil the requirements of NRC (1994) for all nutrients, and did not contain antibiotics, coccidiostats or growth promoters (maize and soybean meal-based diet providing 231.5 g protein, 12.97 MJ ME/kg). The basal diet was also tested for possible residual aflatoxin before feeding (Howel and Taylor, 1981) and there were no detectable levels present (detection limit 1g/kg feed, recovery of the extraction method 95%).

Experimental design

The experimental design consisted of 6 dietary treatment groups. (1) Control: basal diet; (2) AF: basal diet plus 2 mg aflatoxin/kg diet; (3) YG (0.5 g/kg): basal diet plus 0.5 g yeast glucomannan/kg diet; (4) AF þ YG (0.5 g/kg): basal diet plus 2 mg aflatoxin plus 0.5 g yeast glucomannan/kg diet; (5) YG (1 g/kg): basal diet plus 1 g yeast glucomannan/kg diet; (6) AF þ YG (1 g/kg): basal diet plus 2 mg aflatoxin plus 1 g yeast glucomannan/kg diet. Yeast glucomannan (Mycosorb) was provided by Alltech Inc. (Kentucky, USA).

Aflatoxin

The aflatoxin was produced from Aspergillus parasiticus NRRL 2999 culture (USDA, Agricultural Research Service, Peoria, IL, USA) via fermentation of rice by the method of Shotwell et al. (1966). Successfully fermented rice was then steamed to kill the fungus, dried and ground to a fine powder. The aflatoxin content in rice powder was analysed by the method of Shotwell et al. (1966) and measured on a thin layer chromatography (TLC)-fluorometric densitometer (Camag-II, Basel, Switzerland) on a TLC spot (plates from Merck, Germany, other equipment from Desega, Germany). The aflatoxin within the rice powder consisted of 82.72% Aflatoxin B1, 5.50% Aflatoxin B2, 10.20% Aflatoxin G1 and 1.58% Aflatoxin G2 (detection limit 1g aflatoxin/kg rice powder, recovery of the extraction method 92%). The rice powder was incorporated into the basal diet to provide the required 2 mg aflatoxin/kg feed. Aflatoxin was incorporated into the basal diet before yeast glucomannan was added.

Pathological examination

When the chicks reached 3 weeks of age, the feeding trial was terminated and 10 broilers from each group were selected at random and killed for pathological examination. Other chicks in the groups were used to measure performance and biochemical—haematological variables and the results have been published elsewhere (Basmacioglu et al., 2004). A detailed necropsy was conducted. Tissue samples from liver, bursa of Fabricius, thymus, spleen and kidney were collected in 10% neutral buffered formalin. After fixation, samples were dehydrated in alcohol, cleared in xylene and embedded in paraffin blocks. Five-micron sections were cut and stained with haematoxylin and eosin and Sudan black staining methods. Statistical analysis was by chisquare test.

RESULTS

The gross and histopathological lesions and the number of affected animals are summarised in the Table. In the aflatoxin group, the livers and kidneys of chicks were mostly swollen and pale yellow—red, as shown for the livers (Figure 1). The spleens were enlarged and congested whereas the thymuses were atrophied (Figure 2). The bursa of Fabricius had no visible morphological changes. Both in the aflatoxin plus 0.5 g/kg yeast glucomannan and aflatoxin plus 1 g/kg yeast glucomannan groups, the severity of macroscopic lesions was decreased. The number of affected livers in the aflatoxin group was 7 whereas it was only three and one in the aflatoxin plus 0.5 g/kg group and the aflatoxin plus 1 g/kg group, respectively (Table). The difference between the aflatoxin and aflatoxin plus 1 g/kg yeast glucomannan groups was significant (P < 0.05).

Evaluation of the detoxifying effect of yeast glucomannan on aflatoxicosis in broilers as assessed by gross examination and histopathology - Image 1

Microscopically, the livers of chicks fed the aflatoxin-containing diet revealed moderate to severe hydropic degeneration and fatty changes in hepatocytes (Figure 3), bile duct proliferation and periportal fibrosis in the portal areas (Figure 4). Occasionally, nodular lymphoid cell accumulations were seen within the hepatic lobules. While the number of chicks displaying histopathological changes in the liver was only slightly reduced in the aflatoxin plus yeast glucomannan (0.5 g/kg) group, this number significantly decreased in birds given aflatoxin plus yeast glucomannan (1 g/kg; P < 0.05), compared to the aflatoxin group.

The bursa of Fabricius (Figure 5) and spleen in the aflatoxin-treated group had mild to moderate lymphocytic depletion in their follicles. Medullary extension and cortical atrophy in the thymus and hydropic degeneration in the tubular epithelium of kidneys were noted in this group. The addition of yeast glucomannan (0.5 g/kg) to the aflatoxin-containing diet had little effect on the prevention of the degenerative changes in the bursa of Fabricius, thymus, spleen and kidneys. When 1 g/kg yeast glucomannan was added to the aflatoxin-contaminated diet, some decreases were seen in the number and severity of lesions in these organs, although differences were not significant (P > 0.05; Table). Neither the 0.5 g/kg nor the 1 g/kg doses caused any significant pathological changes in these organs, compared to the control.

DISCUSSION

This study was conducted to determine whether inclusion of yeast glucomannan (0.5 or 1.0 g/kg diet) in broiler feed was effective in alleviating lesions caused by exposure to aflatoxin. Gross and histopathological changes are useful tools for evaluating toxic effects of aflatoxin in target organs and for examining the efficacy of the detoxifying agents in broilers (Ledoux et al., 1999; Ortatatli and Oguz, 2001; Rosa et al., 2001). The liver is a main target organ for aflatoxin (Dafalla et al., 1987; Ortatatli et al., 2005), so the livers of chicks were examined for pathological changes in our study. Hepatomegaly, hydropic and fatty changes and acinar arrangements in hepatocytes, periportal fibrosis and bile duct proliferation were observed and paralleled previous findings described for aflatoxin (approximately 2 mg/kg feed; Dafalla et al., 1987; Kiran et al., 1998).

The lesions described for other organs in the aflatoxin group were diminished by both 0.5 and 1 g/kg yeast glucomannan but the improvement was greater for 1 g/kg (P < 0.05) and was not significant for the 0.5 g/kg group (Table). The improvements in the livers of the aflatoxin plus 1 g/kg group paralleled the amelioration of the haematological and biochemical values in the study by Basmacioglu et al. (2005). Overall, our results suggest that adding 1 g/kg yeast glucomannan to feeds contaminated with aflatoxin can reduce the pathological (structural) and haematological—biochemical toxicity of aflatoxin in the whole body of broilers (Figure 1).

Figure 1. Comparative appearance of livers. (A) Control group. Normal liver. (B) AF plus YG (1 g/kg) group. The liver is slightly affected when compared to C. (C) AF-treated group. Severely affected liver is enlarged and pale yellow—red.

The toxic effects of aflatoxin on haematopoietic and immune system organs in broilers are well known (Gabal and Azzam, 1998; Oguz et al., 2000; Ortatatli and Oguz, 2001). Similar pathological changes were observed in the bursa of Fabricius, thymus and spleen in our study in chicks receiving 2 mg/kg aflatoxin, and were ameliorated by 1 g/kg yeast glucomannan, though the effects were statistically not significant (Figures 2 and 5, Table).

Yeast glucomannan (1 g/kg) significantly ameliorated the effects of aflatoxin in broilers in experiments mainly performed by Devegowda and co-workers (Raju and Devegowda, 2000; Aravind et al., 2003; Santin et al., 2003; Girish and Devegowda, 2004) with regard to performance, biochemistry—haematology and immune responses. The beneficial effects of dietary yeast glucomannan have been attributed to its ability to bind selectively to aflatoxin molecules (Murthy and Devegowda, 2004), to alter the intestinal microbial environment and to modulate the immune response (Newman, 1994). Adding yeast glucomannan (both 0.5 and 1 g/kg) to an aflatoxinfree diet did not produce any significant changes compared with the controls (P > 0.05) in this study.

In conclusion, yeast glucomannan was inert and harmless for broilers and its inclusion in diets at low rates could be an advantage over other adsorbents. Addition of yeast glucomannan (1 g/kg) to the aflatoxin-containing (2 mg/kg) diet significantly and/or moderately decreased the number and severity of pathological lesions. The improvements noted in this study may contribute to establishing a safe and practical decontamination procedure and progress toward a solution to the aflatoxin problem in broiler chickens.

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Evaluation of the detoxifying effect of yeast glucomannan on aflatoxicosis in broilers as assessed by gross examination and histopathology

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