Efficacy of Minazel Plus ® a) In Reducing Detrimental Effects of Ochratoxin A, in Broilers
Published: January 29, 2014
By: Nedeljkovic-Trailovic Jelena1 and Petrujkic Branko2
(1 Dr Jelena Nedeljkovic Trailovic, Asis. Professor Department of Nutrition and Botany, Faculty of
Veterinary Medicine, University of Belgrade, Serbia;2 Dr Branko Petrujkic, Training instructor, Department of Nutrition and Botany, Faculty of Veterinary Medicine, University of Belgrade, Serbia)
Summary
In order to investigate the efficacy of a modified inorganic adsorbent (MIA) in the prevention of the detrimental effects of Ochratoxin A (OTA) in broilers a feeding trial was organized. Eighty one-dayold Ross broiler chickens were used in this study. The study lasted 42 days and was divided into periods of: 21, 14 and 7 days. Broilers were randomly assigned to one of the four groups and fed standard diet; diet contaminated with 2 mg/kg of OTA; diet contaminated with 2 mg/kg OTA + 0.2% of (MIA); or diet supplemented only with 0.2% (MIA).
Influence on production parameters (body weight gain, average daily gain, feed consumption and feed conversion ratio) as well as pathohistology of liver, kidneys and small intestine were monitored. Broilers fed the diet contaminated with OTA supplemented with 0.2% MIA achieved better results than the ones fed contaminated with OTA without adsorbent supplementation. Interestingly, the supplementation of MIA alone in the feed resulted in the higher body weight gain and improved feed conversion ratio than of the control group of broilers fed standard diet. This finding needs to be further investigated and clarified.
Pathohistological changes observed in liver and kidneys were most prominent in broilers fed 2 mg/kg of OTA while these changes were of much lower intensity in the group of broilers supplemented with MIA.
Introduction
Ochratoxins are highly toxic compounds commonly produced as secondary metabolites by two species of fungi: Penicillium verrucosum Dierckx and Aspergillus alutaceus (Reverberi et al., 2010). The toxin is made up of three similar compounds, of which ochratoxin A (C20H18ClNO6) is the most toxic and abundant. In recent years ochratoxin A (OTA) has drawn considerable attention because it can not only seriously affect animal performance and health, but it may also have deleterious effects on humans. Of greatest concern for the human population is its implied role in an irreversible and fatal kidney disease; Balkan endemic nephropathy (Castegnaro et al., 2006). Ochratoxin A is a natural contaminant of poultry and livestock feedstuffs.
Field out-breaks of ochratoxicosis have been documented (Danike, 2002; Hamilton et al., 1982) and experimental feeding of OTA has shown (Kubena et al., 1985; Devegowda and Aravind, 2002; Binder et al., 2007) to have detrimental effects on growing chickens, indicating that OTA is a potential hazard to poultry producers.
The diagnosis of ochratoxicosis may be difficult. In the absence of specific, well-defined clinical signs (Humphreys, 1985) the most common effects on farm animals are poor physical condition, lower performance or decreased production. Ochratoxin A exposure in poultry can cause reduced body weight gain (Kubena et al., 1984), decreased feed consumption and diminished egg production (Prior and Sisodia, 1978), listlessness, huddling, occasional diarrhea, ataxia, prostration and finally death (Prior et al., 1976) together with atrophy and degeneration of the tubules and interstitial fibrosis of the kidneys (Nedeljkovic-Trailovic et al., 2001).
In order to investigate the efficacy of a modified inorganic adsorbent in the prevention of the detrimental effects of OTA in broilers, a feeding trial was organized. Influences on production results (body weight gain, average daily gain, feed consumption and feed conversion ratio) as well as pathohistology (of liver, kidneys and small intestine) were monitored.
Material and Methods.
For the study, 80 one-day-old Ross broiler chickens (males and females) with an average body weight of 48.29±0.4 g were used. The study lasted 42 days and was divided into phases of 21, 14 and 7 days. Broilers were assigned to the one of the four groups: Control group (C), broilers fed a standard diet; Experimental group I (E-I), broilers fed diet contaminated with 2 mg/kg of Ochratoxin A (OTA); Experimental group II (E-II), broilers fed diet contaminated with 2 mg/kg OTA plus 0.2% of modified inorganic adsorbent (MIA) and Experimental group III (E-III), broilers fed a diet supplemented with 0.2% of MIA.
The OTA used for corn contamination originated from Aspergillus ochraceus Wilhelm NRRL 263.67 from the Dutch collection; while MIA originated from the zeolite tuff that was modified by the addition of an organic cation.
Broilers were kept in wire cages and had unlimited access to drinking water. Feed was provided ad libitum. Microclimate was adjusted to fit the broilers well being. All procedures were done in accordance to the guidelines of the Ethical Committee of the Faculty of Veterinary Medicine, Belgrade University as well as EU Directive 2010/63/EU for animal experiments.
Broilers were fed complete mixtures whose raw and chemical composition changed according to the age of broilers and following the NRC (National Research Council, 1994) recommendations. Three mixtures were used: a starter diet from day 1 until day 21; a grower diet from day 22 until day 35; and a finishing diet from day 36 until day 42.
The E-I and E-II groups were fed the OTA contaminated feed until the 21st day of the study and were then after fed standard diet without OTA or MIA supplementation. This was also the case in E-III group, in which MIA was fed only until the 21st day and then removed from the feed).
On the 21st day, 10 animals from each group were slaughtered and samples of liver, kidney and intestinal tissue were taken for pathohistological examination.
The production results were monitored at seven-day intervals throughout the study. The body weight of broilers was measured on an electronic scale (10-2 g) and during the entire study, feed consumption was recorded.
The samples for pathohistological examinations were fixed in a solution of 10% neutral formalin and absolute ethanol and molded by the standard paraffin technique. Tissue samples (5-8 μm) were then stained by the hematoxylin eosin technique as described by (Scheuer and Chalk, 1986).
All the results were statistically analyzed, for differences between groups, by an analysis of variance (ANOVA). The results were processed by the use of Graph Pad Prism® 5.0 software (Graph Pad Software Inc., San Diego, California, USA).
Results and discussion.
The average body weight of the broilers during the study is shown in Figure 1.
Fig. 1. The average body weight of broilers during the study (g)
The significant differences (P<0.05) in body weight were observed as early as the 7th day of the study, with the lowest body weight being recorded in the E-I group 113.2 ± 2.82 g, while the highest weight was recorded in E-III group (142.8 ± 3.68 g). The body weight of broilers fed the diet contaminated with OTA plus 0.2% MIA, (E-II) was of 118.5 ± 3.17 g and was somewhat higher than the body weight of broilers in E-I (113.5 ± 4.87 g). This trend continued during the rest of the study period.
On the 14th day of the study the body weight of broilers from E-I group (184.8 ± 3.00 g) was significantly lower (P<0.05) than the body weight of broilers in C and E-II groups (262.8 ± 8.88 and 219.6 ± 3.83 g) and highly significantly lower (P<0.01) than the weight of the broilers from E-III group (296.0 ± 9.98 g).
On the 21st day of the study the trend was similar. Broilers of E-I group had an average body weight of 293.8 ± 9.83g which was lower (P<0.05) than the body weight of E-II (358.1 ± 11.71 g) and the C group (458.3 ± 18.52 g) (P<0.05), and highly significantly lower (P<0.001) than the body weight of broilers from the E-III group (533.3 ± 12.42 g). The supplementation of both OTA and MIA ceased on the 21st day of the study. On the 42nd day of the study, the body weight of broilers from E-I (1080.0 ± 71.51 g) and E-II (1174.0 ± 31.09 g) were nearly the same and highly significantly lower (P<0.001) than the body weight of broilers from the C group (1796.0 ± 39.88 g) and E-III group (1842.0 ± 42.99 g).
These results are similar to the findings of Nedeljkovic-Trailovic et al., 2004; who fed broilers OTA contaminated mixtures (1.5 mg/kg) but for a shorter time period observing also a decrease in body weight. Also Santin et al., 2002; fed broilers mixtures containing 2 mg/kg OTA plus 0.25% of an inorganic adsorbent and recorded only a slight improvement in body weight gain.
The fact that the negative effect of OTA on body weight gain was recorded not only during OTA exposure but even in a later period could lead to the conclusion that OTA exposed broilers show a prolonged inhibition of body weight gain, which is possibly due to a cumulative effect of OTA. That needs to be elucidated in the future.
The average daily gain (ADG) of broilers during the study is shown in Figure 2.
Fig. 2. The average daily gain of broilers during the study (g)
Throughout the study, ADG was lowest for the E-I and E-II groups, and highly significantly lower (P<0.01) than that of the C group or the E-III group of broilers.
The results of our study are in the accordance with results reported by Huff et al., 1974; Ayed et al., 1991; and Nedeljkovic-Trailovic et al., 2004; who reported that the ADG is affected by the amount of OTA concentration in feed. The detrimental effect on ADG was 6.5%, when OTA was at a concentration of 0.5 mg/kg and 52% when OTA concentration was 4.0 mg/kg in the feed. Our results differ from the results of Zurovac-Kuzman, 2001; who reported that when modified clinoptilolite is supplemented (0.5%) to a feed containing 0.5 mg/kg of OTA the negative effects of OTA on body weight gain were completely prevented. This difference is probably due to the lower OTA concentration in the feed, as well as to the higher addition of the adsorbent that was used in the study.
The average feed consumption (Figure 3) showed the same trend as ADG.
Fig. 3. The average feed consumption of broilers during the study (g)
The similar results have been previously reported by Nedeljkovic-Trailovic, et al., 2004; who found that supplementation of adsorbents can partially improve the decrease in feed intake.
Overall, the feed conversion ratio (FCR), although not statistically significant, was better in the E-III group (MIA) than in the broilers fed diets supplemented with OTA (E-I group), OTA plus MIA (E-II group) or the control group of broilers fed standard diet. This is shown in Table 1.
Table 1. Feed conversion ratio (FCR) during the study.
The results of our study are in the accordance with the findings of Chang et al., 1981 who found significantly lower feed conversion ratio in broilers fed diets with 4 and 8 mg/kg of OTA.
Compared to the other production parameters FCR showed the smallest variation between the groups, probably due to the lower consumption of adsorbent supplemented feed, which is supported by the findings published by Garcia et al., 2003. Finally, we can say that OTA contamination had a detrimental effect on feed consumption since the first week of exposure.
Broilers of the control group, fed a standard diet as well as broilers fed a diet with 0.2% MIA were of good health and appetite with no deaths recorded in these groups. In broilers fed OTA contaminated feed (E-1), a 10 to 15% increase in water consumption was observed than in the other experimental groups. The cases of diarrhea in group E-I, were also observed. These symptoms disappeared after OTA was withdrawn from the feed. The increased water consumption can be attributed to the disturbance of electrolytes due to the damage of the kidney tubules by OTA, which was recorded in our study. In support of this statement are the reports of Nedeljkovic- Trailovic et al., 2004 as well as of Zurovac-Kuzman, 2001.
Deaths were observed in the group of broilers fed the diet contaminated with OTA (E-I; n=3) and in the group fed the diet contaminated with OTA plus MIA (E-II; n=1). No deaths were observed in C and E-III group.
In the broilers fed a diet contaminated by 2 mg/kg of OTA (E-I group) during a 3-week period vacuolization of hepatocytes due to diffuse accumulation of fat was observed (Figure 4). No pathohistological changes were observed in the intestines of the same group of broilers. Changes were observed in the proximal tubules of the kidneys (Figure 5). In two animals from this group multiplication of the renal canaliculi cells that resulted by the formation of adenomatous structures was observed (Figure 6).
Pathohistological Findings.
The pathohistological examination of liver in broilers fed 2 mg/kg of OTA plus 0.2% MIA (E-II group) showed focal accumulation of fat in hepatocytes (Figure 7). In the intestinal tissue of the same group of broilers, pathohistological changes were not observed. Vacuolization of kidneys cells with the reduction of tubule diameter was also recorded in this group (Figure 8).
In all experimental groups, after three weeks of OTA withdrawal, hepatocytes did undergo restitution and no changes could be observed any longer (Figure 9). This is in accordance with the findings of Garcia et al., 2003.
In the broilers fed diet contaminated with OTA (2 mg/kg) the changes of hepatocytes are similar to the findings of Harvey et al., 1987; Santin et al., 2002; and Koynarski et al., 2007. These authors also reported the similar changes in hepatocytes of broilers fed 1 and 2 mg/kg OTA contaminated feed. On the contrary, Nedeljkovic-Trailovic et al. ,2001; observed no changes in the liver of broilers exposed for 7 days to a feed containing 1.5 mg/kg OTA, probably due to a shorter exposure period. These findings support the claim of numerous authors that the intensity of pathohistological changes is dependant of the concentration of OTA in the feed and exposure length.
Conclusions
The broilers fed diet contaminated with OTA supplemented with 0.2% modified inorganic adsorbent (MIA) achieved better production performances than the broilers fed OTA contaminated diet without adsorbent supplementation. The protective effect of MIA could be seen through improved production results. Interestingly the supplementation of MIA in the feed (E-III group) resulted in the highest body weight gain and the best FCR, even when compared to the control group of broilers fed standard diet. This might be attributed to the fact that besides OTA, the broiler feed contained some other unidentified matters that affected production results and that it was successfully bounded by 0.2% MIA supplementation. This finding needs to be further investigated and clarified.
Pathohistological changes observed in liver and kidneys during this study were the most prominent in E-I (group of broilers fed 2 mg/kg of OTA) while these changes were of much lower intensity in the groups supplemented with MIA.
Acknowledgement
This work was supported by grant TR31087 Ministry of Education, Science and Technological Development of The Republic of Serbia.
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This paper was presented at the AMENA Congress, Puerto Vallarta, October 2013.
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