Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens

Abstract

In order to establish the tolerance level of ochratoxin A (OTA) in broiler chickens, day-old broiler chicks (n=160) were divided into 4 treatment groups (T1- control; T2-T1+50 ppb OTA; T3-T1+100 ppb OTA; and T4-T1+150 ppb OTA). Each diet was fed to 4 replicated groups of 10 birds each from day-old to 42 days of age. During overall growth phase (0- 6 weeks), the average body weight gain (BWG) in T3 and T4 was lower (P<0.05) than T1, whereas, the BWG of T2 was statistically similar to T1. OTA contamination at 100 ppb level in the diet resulted in reduction (P<0.05) in BWG of broiler chickens. The overall feed intake (FI) of group T2 was statistically similar to T1, however, the FI in T3 and T4 was lower (P<0.05) than T1. Inclusion of 100 ppb OTA in the diet resulted in significant reduction in feed consumption. OTA contamination at 100 ppb resulted in increased (P<0.05) FCR, thus deteriorating feed utilization efficiency of broilers. Addition of 50 ppb OTA in diet did not produce any change in relative weights of liver, kidney and bursa of Fabricius, however, OTA contamination at 100 ppb resulted in increased relative weights of liver and kidney; and reduced relative weight of bursa of Fabricius. Inclusion of 50 ppb OTA in diet (T2) did not produce any change in biochemical parameters of broiler chickens. It was concluded that broiler chickens can tolerate up to 50 ppb of dietary ochratoxin without any adverse effect on their performance, organ weight and biochemical parameters.

Keywords: Ochratoxin A, Tolerance level, Broiler chicken, Performance.

1. Introduction

Moulds (Fungi) in poultry feed are a source of significant economic losses. Molds adversely influence poultry performance by altering the nutrient composition of feed ingredients, decreasing the efficiency of nutrient utilization and by producing toxic secondary metabolites called mycotoxins (Katole et al., 2013). Ochratoxin is an isocumarin derivative linked through the carboxy group to a L-ß-phenylalanine. Ochratoxins is a general term describing a family of toxic compound consisting of three members known by the trivial name of ochratoxin A, B and C. Structurally the three toxins differ very slightly from each other; however, the differences have marked effects on their respective toxic potentials (Elaroussi et al., 2006). Ochratoxin A is the most abundant, commonly detected and frequently occurring member of this family of mycotoxins (Pathak et al., 2017). This mycotoxin was isolated as metabolites of the fungus Aspergillus ochraceus from which the toxin acquired its name. However, ochratoxin production is not unique to Aspergillus ochraceus as additional several Aspergillus species (Varga et al., 2003; Bayman et al., 2002) and several Penicillium species (Larsen et al., 2001) produce ochratoxins. Ochratoxin A is a mycotoxin that is receiving increasing attention worldwide because of the hazard it posses to animal and human health (Patil and Degloorkar, 2016a, b, 2018; Patil et al., 2017a, b). Ochratoxicosis causes a reduction in production performance viz. reduced growth rate, decreased feed consumption, poorer feed conversion (Singh et al., 2015; Singh et al., 2016b; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Singh et al., 2018) and increased mortality (Singh et al., 2015; Singh et al., 2016b). Ochratoxin contamination at 200 ppb level in broiler diet led to decreased protein, haemoglobin and creatinine, while increased uric acid, alkaline phosphatase, AST and ALT levels in blood; and impaired both cell mediated and humoral immunity (Singh et al., 2016a). Ochratoxin A has also been detected in meat, egg and milk (Jorgensen, 1998; Skaug, 1999). It inhibits the synthesis of proteins, DNA and RNA in the cell. It shows renal toxicity by inhibiting various enzyme activities in the kidney. Also, OTA induces degenerative changes and an increase in the weight of the kidney and liver (Singh et al., 2019a, b), as well as a decrease in the weights of the lymphoid organs (Stoev et al., 2000; Stoev et al., 2002; Elaroussi et al., 2006; Elaroussi et al., 2008). The objective of this study was to determine the tolerance level of ochratoxin A in the diets of broiler chickens.

2. Materials and Methods

2.1 Ochratoxin Production and Analysis

The lyophilised preparation of Aspergillus westerdijkiae NRRL 3174 was obtained from U.S. Department of Agriculture, Peoria, Illinois (USA). This lyophilised preparation was revived on potato dextrose agar medium and used for experimentation. Ochratoxin was produced as per the methodology standardized by Singh et al. (2013). Cracked maize (50 g) was taken in 250 ml conical flasks. The moisture content of substrate was adjusted to have a moisture level of 35%. Thus flasks were plugged with non-absorbent cotton and sealed with aluminium foil. The flasks were autoclaved for 20 min at 121°C and inoculated with 1- week old mycelia of Aspergillus westerdijkiae NRRL 3174. The inoculated flasks were incubated in a BOD incubator for 14 days. After removal from the incubator, the flasks were dried at 70°C and the ochratoxin assays were performed as per AOAC (1995).

2.2 Experimental Design

Experimental design was completely randomized design (CRD). There were four dietary treatments. Each dietary treatment had 4 replicates and each replicate had 10 chicks. The experiment was conducted in broiler chickens from day-old to 6 weeks of age. The various dietary treatments were prepared by mixing the required quantity of mouldy maize to get the desired concentration of OTA in basal diet. Experimental diets included T1 control (basal diet), T2 (basal diet + 50 ppb OTA), T3 (basal diet + 100 ppb OTA), and T4 (basal diet + 150 ppb OTA).

2.3 Biological Experiment and Analysis

Day-old broiler chicks (n=160) were obtained from experimental hatchery, ICAR-CARI, Izatnagar. The chicks were wing banded, weighed individually and distributed randomly into 4 groups. All birds were reared under standard management conditions from 0-6 weeks. All birds were fed with broiler starter ration from 1-21 days and broiler finisher ration from 22 to 42 days. The basal diet was prepared using maize, soybean meal and rapeseed meal. The starter diet (0-21 days of age) contained 21.5% protein, 2,890 kcal ME/kg, lysine 1.28%, methionine 0.52%, calcium 1.02% and available P 0.45%. The corresponding values in finisher diet were 19.02%, 29,991 kcal/kg, 0.93%, 0.39%, 1.09% and 0.39%. Body weight of individual birds and feed consumption of each replicate were recorded at weekly interval and FCR was calculated. The protein (AOAC, 1995) and calcium (Talapatra et al., 1940) contents were estimated, while the concentrations of lysine, methionine, available P and metabolizable energy values were calculated. At the end of the experiment, 8 birds/ dietary treatment were sacrificed randomly and their organs and blood samples were collected. The serum was separated and analysed for various biochemical parameters using commercial kits. The statistical analysis was done using SPSS 16.0 version.

3. Results and Discussion

3.1 Body Weight Gain (BWG)

The data pertaining to body weight gain (BWG) as influenced by various ochratoxin levels is given in Table 1. At first week of age, there was no significant difference in BWG among various dietary treatments. Significant (P<0.05) differences in BWG among various treatments were recorded from second week of age, wherein the BWG in groups T3 and T4 was lower (P<0.05) than that of control group (T1), however, the BWG of group T2 was statistically similar to that of control. During third to sixth weeks of age, the BWG in T2 was statistically similar to that of control, however, the BWG in T3 and T4 was lower (P<0.05) than that of control group. During overall growth phase (0-6 weeks), the average BWG in T3 and T4 was lower (P<0.05) than that of control group, whereas, the BWG of group T2 was statistically similar to that of control. In the present study, 100 ppb or higher level of OTA contamination in the diet resulted in reduction (P<0.05) in BWG in broiler chickens. Similar findings were also recorded by earlier workers who reported significant reduction in BWG at 50 to 800 ppb level of ochratoxin contamination in feed (Singh et al., 2015; Singh et al., 2016b; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Singh et al., 2018; Sakhare et al., 2007; ElBarkouky et al., 2010; Hanif et al., 2008; El-Barkouky and Abu-Taleb, 2008; Santin et al., 2006; Hatab, 2003; Elaroussii et al., 2006; Pozzo et al., 2013).

In the present investigation, dietary contamination of 50 ppb OTA did not produce any significant change in BWG of broiler chicks, indicating that broilers can tolerate up to 50 ppb OTA in their diet.

Table 1: Effect of ochratoxin on body weight gain (g/bird) of broiler chickens

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 1

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3.2 Feed Intake (FI)

During first, third and fourth weeks of age, the feed intake (FI) was statistically similar among various dietary treatments (Table 2). At second week of trial, the FI of group T2 was statistically similar to that of control (T1), however, the feed consumption of groups T3 and T4 was lower (P<0.05) than that of control group. At fifth and sixth weeks of trial, the FI of groups T2 and T3 was statistically similar to that of control (T1), however, the feed consumption of groups T4 was lower (P<0.05) than that of control. During overall growth phase (0-6 weeks), the FI of group T2 was statistically similar to that of control (T1), however, the FI of groups T3 and T4 was lower (P<0.05) than that of control group. In the present study, inclusion of 100 ppb or higher OTA in the diet of broiler chickens resulted in significant reduction in feed consumption. These results were in agreement with earlier workers who reported significant reduction in FI at 150 to 800 ppb level of dietary ochratoxin (Singh et al., 2016b; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Singh et al., 2018; El-Barkouky et al., 2010; Hanif et al., 2008; Santin et al., 2006; Hatab, 2003; Elaroussii et al., 2006). Watts et al. (2003); Singh et al. (2015); Koynarski et al. (2007); Denli et al. (2008); Sawale et al. (2009) also found that dietary inclusion of OTA adversely affected feed consumption of birds. In the present study, dietary contamination of 50 ppb OTA did not produce any significant change in feed consumption of broiler chicks, indicating that broilers can tolerate up to 50 ppb OTA in their diet.

3.3 Feed Conversion Ratio

During first week of age, the feed conversion ratio (FCR) did not vary significantly (P<0.05) among various dietary treatments (Table 3). Significant (P<0.05) differences in feed conversion ratio (FCR) among various dietary treatments were recorded from second week of age onward. During second to sixth week of age, the FCR of group T2 was statistically similar to that of control (T1), however, the FCR in groups T3 and T4 was higher (P<0.05) than that of control. With regard to overall FCR (0-6 weeks), the FCR of group T2 was statistically similar to that of control group (T1), however, the FCR of groups T3 and T4 was higher (P<0.05) than that of control. The results showed that 50 ppb contamination of ochratoxin in the diet of broilers did not affect feed utilization efficiency; however, 100 ppb or higher levels resulted in increased FCR in broiler chickens. Increased FCR due to ochratoxicosis was earlier reported in several studies (Singh et al., 2015; Singh et al., 2016b; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Singh et al., 2018). Similar results of reduced feed efficiency due to ochratoxin contamination were also reported by earlier workers (Gibson et al., 1989; El-Barkouky et al., 2010; Hanif et al., 2008). In the present study, OTA contamination at 100 ppb or higher resulted in significantly (P<0.05) increased FCR, thus deteriorating feed utilization efficiency of broilers.

3.4 Liveability

The results on week-wise liveability percentage in broilers kept on different dietary treatments are presented in Table 4. During first week of age, no mortality was recorded. During second, third and fourth weeks of age, the livability percentage did not vary significantly (P<0.05) among various treatment groups. During fifth and sixth weeks of age, the livability percentage in groups T2 and T3 was numerically lower than that of control (T1), however, the livability percentage in group T4 was significantly (P<0.05) lower than that of control. By and large, there was increase in mortality with increasing level of ochratoxin. The livability percentage was not affected in basal diet and basal diet with 50 ppb OTA. However, the liveability percentage decreased at OTA levels above 50 ppb (82.50%, and 80.00% liveability in diets with OTA level of 100 and 150 ppb, respectively). This result was in agreement with those of earlier studies (Singh et al., 2018; Singh et al., 2015; Singh et al., 2016b; El-Barkouky and Abu-Taleb, 2008; ElBarkouky et al., 2010) wherein significant decrease in survivability due to ochratoxicosis caused by 50 to 200 ppb ochratoxin was reported in broiler chickens. The results of the present study showed that broiler chickens can tolerate 50 ppb of OTA in their diet without any adverse effect on survivability.

Table 2: Effect of ochratoxin on feed consumption (g/bird) of broiler chickens

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 3

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Table 3: Effect of ochratoxin on feed conversion ratio (FCR) of broiler chickens

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 5

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Table 4: Effect of ochratoxin on livability percentage of broiler chickens

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 7

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3.5 Organ Weights

The relative weights of liver and kidney (Table 5) in group T2 was statistically similar to that of control (T1) however, the relative weights of liver and kidney in groups T3 and T4 was higher (P<0.05) than that of control (T1). The relative weight of liver and kidney between T1 and T2; and T3 and T4 did not differ significantly. In the present study, OTA contamination at 50 ppb level did not influence the relative weights of liver and kidney, however, 100 ppb or higher level resulted in increased liver and kidney weights. Significant increase in relative weight of liver and kidney due to ochratoxicosis was earlier observed in several studies (Singh et al., 2017; Singh et al., 2016b; Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Stoev et al., 2004; Gibson et al., 1989; Huff et al., 1992; Elkady, 1993). Verma et al. (2004); Hanif et al. (2008) also reported a significant increase in the relative weight of kidney when broilers were fed with OTA at a dietary levels of 0.5, 1, 2 and 4 ppm over 42 day period. El-Barkouky et al. (2010) also reported significant increase in relative weight of kidney at OTA level of 200 μg/kg feed from one-day old to 5 weeks of age in broiler chicks. The relative weights of bursa of Fabricius in group T2 was statistically similar to that of control, however, the relative weights of bursa of Fabricius in group T3 and T4 was lower (P<0.05) than that of control (T1). The relative weight of bursa between groups T3 and T4 did not vary significantly. In the present study, addition of 50 ppb OTA in diet did not produce any change in relative weights of bursa of Fabricius, however, OTA contamination at 100 ppb or higher level resulted in decreased (P<0.05) relative weight of bursa of Fabricius.

Similarly, significant decrease in relative weight of bursa due to ochratoxicosis caused by 100 to 400 ppb ochratoxin A was earlier reported in several studies (Singh et al., 2017; Singh et al., 2016b; Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b). Stoev et al. (2004) observed a significant increase in relative bursal weight in broiler chicks fed a diet contaminated with OTA at 130, 300 or 800 μg/kg feed. Kumar et al. (2004); Gupta et al. (2008); Verma et al. (2004) also recorded significant reduction in relative weight of bursa of Fabricius due to OTA levels ranging from 2 to 4 mg/kg diet in broiler chickens. The relative weight of spleen did not vary significantly (P<0.05) among different dietary treatments. In the present study, administration of OTA at any level did not produce any significant effect on relative weight of spleen in broiler chickens. This finding was in agreement with that of Singh and Mandal (2018a); Singh and Mandal (2018b); Singh et al. (2015) where ochratoxicosis caused by 100 to 400 ppb ochratoxin did not produce any effect on relative weight of spleen in broiler chickens. In the present study, addition of 50 ppb OTA did not produce any significant change in organ weights of broiler chicks, indicating that broilers can tolerate up to 50 ppb OTA in their diet. However, Pozzo et al. (2013) also reported that feeding broiler chickens, a diet contaminated with 100 ppb OTA did not produce any effect on organ weights of broiler chickens.

3.6 Biochemical Parameters

The total serum protein in group T2 was statistically similar to that of control (T1) (Table 6). The serum protein in groups T3 and T4 was lower (P<0.05) than that of control. The protein content between groups T3 and T4 did not differ significantly. The results showed that inclusion of 50 ppb OTA in the diet of broiler chicks did not influence total protein content, however, 100 ppb or higher level significantly (P<0.05) decreased the total serum protein content. The present finding was in agreement with earlier studies reported by several researchers (Singh et al., 2016a; Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Manning and Wyatt, 1984; Ramadevi et al., 2000; Stoev et al., 2000; Singh et al., 2019), who also reported decreased serum protein during induced ochratoxicosis in broilers. With regard to serum cholesterol content, it did not differ (P<0.05) among groups T1, T2 and T3, however, it was significantly (P<0.05) lower in group T4 compared to that of control. In the present study, there was a significant reduction in serum cholesterol level at 200 ppb OTA concentration in feed. In respect of serum cholesterol during ochratoxicosis, a similar trend was reported earlier by several researchers (Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Manning and Wyatt, 1984; Ramadevi et al., 2000; Stoev et al., 2000; Singh et al., 2019).

However, Singh et al. (2016a) reported no significant effect on serum cholesterol content due to ochratoxicosis caused by 200 ppb of ochratoxin in broiler chickens. The serum uric acid level in group T2 was statistically similar to that of control (T1), however, it was significantly (P<0.05) higher in groups T3 and T4 compared to that of control. In the present study, there was a significant elevation in serum uric acid level at 100 ppb or more dietary OTA concentration. The present finding was in agreement with those of earlier studies reported by several researchers (Singh et al., 2016a; Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Manning and Wyatt, 1984; Ramadevi et al., 2000; Singh et al., 2019), indicating that there was inhibition of protein accretion due to OTA contamination in feed, which might have the reason of lower body weight gains in broilers fed higher level of OTA. The activities of alkaline phosphatase in group T2 was statistically similar to that of control (T1), however, the activities of alkaline phosphatase in group T3 and T4 was significantly (P<0.05) higher than that of control (T1). The addition of 50 ppb OTA in diet did not produce any change in activities of alkaline phosphatase, however, OTA contamination at 100 ppb or higher level resulted in increased activities of alkaline phosphatase. Increased alkaline phosphatase due to ochratoxicosis in broiler chicks was also reported by several researchers (Singh et al., 2016a; Singh et al., 2015; Singh and Mandal, 2018a; Singh and Mandal, 2018b; Hanif et al., 2008; Singh et al., 2019). The results of the present study showed that broiler chickens can tolerate up to 50 ppb of OTA in their diet without any adverse effect on biochemical parameters. However, Pozzo et al. (2013) reported that 100 ppb dietary OTA did not alter biochemical parameters in broilers.

Table 5: Effect of ochratoxin on relative weights of organs (percent of live weight) of broiler chickens

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 9

Table 6: Effect of ochratoxin on serum biochemical parameters of broiler chickens fed different dietary treatments

Determination of Tolerance Level of Ochratoxin A in the Diet of Broiler Chickens - Image 10

4. Conclusion

It was concluded that broiler chickens can tolerate up to 50 ppb of dietary ochratoxin without any adverse effects on their performance, organ weight and biochemical parameters during their 0-6 weeks of growth phase.

This article was originally published in Livestock Research International, April-June, 2019. Volume 07, Issue 02, Pages 137-143.

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