Abstract
To evaluate the impact of aflatoxin B1 (AFB1), ochratoxin A (OTA) and their interactions on blood biochemicals and organ histopathology, dayold broiler chicks (n=288) were divided into 9 treatment groups (T1: control; T2: T1+150 ppb AFB1; T3: T1+300 ppb AFB1; T4: T1+150 ppb OTA; T5: T1+250 ppb OTA; T6: T1+150 ppb AFB1+150 ppb OTA; T7: T1+150 ppb AFB1+250 ppb OTA; T8: T1+300 ppb AFB1+150 ppb OTA; T9: T1+300 ppb AFB1+250 ppb OTA). Each diet was fed to 4 replicated groups of 8 birds each for 42 days. The results showed that individual feeding of mycotoxins in T2, T3, T4 and T5 resulted in decreased levels of serum protein, cholesterol and uric acid. Simultaneous feeding of mycotoxins in T6, T7, T8 and T9 caused additive effect on protein concentration and synergistic effect on cholesterol and uric acid concentration. Individual feeding of mycotoxins in groups T2, T3, T4 and T5 resulted in increased activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Simultaneous feeding of mycotoxins in groups T6, T7, T8 and T9 caused synergistic effect on ALP; and additive effect on AST and ALT activities. Simultaneous contamination of feed at various levels i.e., 150 ppb AFB1+150 ppb OTA (T6); 150 ppb AFB1+250 ppb OTA (T7) and 300 ppb AFB1+150 ppb OTA (T8) resulted in additive adverse effect, however, 300 ppb AFB1+250 ppb OTA (T9) resulted in synergistic adverse effect on histopathological lesions in liver, intestine and kidneys of birds. It was concluded that aflatoxicosis and ochratoxicosis individually resulted in decreased concentration of serum protein, cholesterol and uric acid; and increased activity of ALP, AST and ALT. However, simultaneous exposure caused additive or synergistic effect on serum biochemistry as well as on histopathology of organs in broiler chickens.
Keywords: Aflatoxin B1, Ochratoxin A, Serum biochemicals, Histopathology, Broiler chicken.
1. Introduction
Mycotoxins are structurally diverse and produced by various moulds belonging chiefly to species Aspergillus, Penicillium and Fusarium and Alternaria, invading crops in the field or may grow on feeds during handling and storage under favourable conditions of temperature and humidity. As per estimate of FAO (Food and Agriculture Organization) (2004), 25% of the world's crops are affected with mycotoxins each year. The toxic effects of mycotoxins i.e. mycoxicosis, is characterized by its nephrotoxic, immunosuppressive, hepatotoxic, carcinogenic, mutagenic and teratogenic effects in animals and poultry (Patil et al., 2005, 2006, 2014, 2017a, b; Katole et al., 2013; Patial et al., 2013; Patel et al., 2015; Patil and Degloorkar, 2016a, b, 2018; Singh, 2019a-g; Singh et al., 2019a, b). Singh et al. (2010) also reported that aflatoxins are of great concern in warm and humid climatic conditions like India. Further, Singh and Shrivastav (2011) reported that 90% of the maize samples were positive for AFB1 and the values ranged from non-detectable to 0.80 ppm, with an average of 0.14 ppm of AFB1. Aflatoxin B1 toxicity in poultry causes lowered performance in terms of reduced body weight gain, feed intake and feed efficiency (Silambarasan et al., 2013; Singh et al., 2015a; Singh et al., 2016c), reduced nutrient utilisation (Silambarasan et al., 2013), increased mortality (Khatke et al., 2012b; Sharma et al., 2014), anemia (Singh et al., 2015a; Singh et al., 2016c), hepatotoxicosis and haemorrhage (Churchil et al., 2014; Singh et al., 2015a; Singh et al., 2016c; Pathak et al., 2017), altered biochemistry (Singh and Mandal, 2013; Singh et al., 2013a). Moreover, it impairs humoral and cellular immune responses in poultry and increases susceptibility to environmental and infectious agents (Khatke et al., 2012c; Singh, 2019a, b, e, f) leading to severe economic losses. Ochratoxin is also one of the most commonly occurring mycotoxins and is produced by several species of Aspergillus (Aspergillus ochraceus) and Penicillium (Penicillium verrucosum). It causes a reduction in production performance viz. reduced growth rate, decreased feed consumption, poorer feed conversion and increased mortality (Patil et al., 2005, 2014, 2017a, b; Patial et al., 2013; Singh et al., 2015b; Singh et al., 2016b; Patil and Degloorkar, 2016a, b, 2018; Singh et al., 2019a, b). 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. Moreover, ochratoxin impaired both cell mediated and humoral immunity (Singh et al., 2016a). Several mycotoxins produced by a single or several fungal species may be occurring simultaneously on various agriculture commodities, thereby adding more potential risk of mixed mycotoxicosis. Aflatoxin B1 and ochratoxin A are the two most commonly occurring mycotoxins in animal feed and have detrimental effects on both animal and poultry (IARC, 1993; Katole et al., 2013; Patel et al., 2015). The objective of the present study was to evaluate the interactive effect of aflatoxin and ochratoxin on blood biochemicals and organs histopathology in broiler chickens.
2. Materials and Methods
2.1 Production and Analysis of Mycotoxins
Aflatoxin was produced by growing Aspergillus flavus NRRL 6513 on maize. This fungus was obtained from US Department of Agriculture, Illinois, USA. Fermentations were carried out in batches as per the method described by Shotwell et al. (1966). The extraction and estimation of aflatoxin was done as per the procedure of Pons et al. (1966). Aflatoxin contents were finally quantified using a UV spectrophotometer. Ochratoxin was produced as per the method described by Singh et al. (2013b) using Aspergillus westerdijkiae NRRL 3174. This fungus was also obtained from US Department of Agriculture, Illinois, USA. The extraction and estimation of ochratoxin was done as per the procedure of AOAC (1995).
2.2 Experimental Design
Experimental design was completely randomized design. There were nine dietary treatments. Each dietary treatment had 4 replicates and each replicate had 8 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 mycotoxins to get their desired concentration in basal diet (Table 1).
Table 1: Experimental groups and treatments
2.3 Biological Experiment and Analysis
Day-old broiler chicks were obtained from experimental hatchery, ICAR-Central Avian Research Institute, Izatnagar. The chicks were wing banded, weighed individually and distributed randomly into nine treatment groups on equal weight basis. All birds were reared under standard management conditions from 0-6 weeks of age. All birds were fed with broiler starter ration for 1-21 days and broiler finisher ration from 22 to 42 days. The composition of broiler starter and finisher ration was as below: The starter diet with maize 53.93, soybean meal 42.70, limestone 0.9, dicalcium phosphate 1.70, common salt 1.30, DLmethionine 0.16, TM premix 0.10, vitamin premix 0.15, B complex 0.015, choline chloride 0.05; and finisher diet with maize 63.63, soybean meal 33.40, limestone 0.95, dicalcium phosphate 1.40, common salt 0.20, DL-methionine 0.11, TM premix 0.10, vitamin premix 0.15, B complex 0.015, choline chloride 0.05 were formulated. TM premix supplied Mg, 300; Mn, 55; I, 0.4; Fe, 56; Zn, 30; Cu, 4 mg/kg diet. Vitamin premix supplied Vit A, 8250 IU; Vit. D3, 1200 ICU; Vit. K, 1 mg; vitamin E 40 IU per kg diet. B complex supplied Vit. B1, 2 mg; Vit. B2, 4 mg; Vit. B12, 10 mcg; niacin, 60 mg; pantothenic acid, 10 mg; choline, 500 mg per kg diet. The starter diet contained 22.09% crude protein, 2,848 Kcal ME/kg, calcium 0.99%,available P 0.45%, lysine 1.27%, methionine 0.53% and threonine 1.01%. The corresponding values in finisher diet were 19.04%, 2,946 Kcal/kg, 0.90%, 0.38%, 1.05%, 0.44% and 0.87%. The protein as per AOAC (1995) and calcium contents as per Talapatra et al. (1940) were estimated, while the concentrations of lysine, methionine, threonine, available P and metabolizable energy values were calculated. At 14th , 28th and 42nd days post-hatch the blood samples from each treatment group were collected. The serum was separated and stored at -200C and analyzed for various biochemical parameters. At the end of experiment, liver, kidneys and intestine samples were collected and fixed in 10% formal saline. The formal saline fixed samples were cut into pieces of 2-3 mm thickness and washed thoroughly in tap water overnight before dehydrating the tissues in ascending grades of alcohol (50%, 60%, 70%, 80%, 90% absolute alcohol I and II). The dehydrated tissues were cleared in benzene and embedded in paraffin blocks. Serial sections of 5- micron thickness were cut and stained with hematoxyline and eosin (Culling, 1968) and examined for various histopathological changes.
2.4 Statistical Analysis
All data were statistically analyzed using SPSS software package version 20.0 following one way analysis. All the observations were recorded at 95% (P≤0.05) level of significance.
3. Results and Discussion
3.1 Serum Biochemicals
The data pertaining to various serum biochemicals (Serum protein, cholesterol, uric acid, ALP, AST and ALT) was statistically analysed and the mean values are presented in Table 2.
3.1.1 Total Serum Protein
The serum protein value in groups T3, T5, T6, T7, T8 and T9 was lower (P<0.05) than that of control (T1). In groups T2, T3, T4 and T5 when AFB1 and OTA were administered individually, a decrease in serum protein content by 9.77, 14.77, 9.77 and 20.00%, respectively was recorded. However, in groups T6, T7, T8 and T9 during combine feeding of mycotoxins, a decrease in serum protein content by 18.63, 22.72, 20.45 and 30.90%, respectively was observed. In the present study, dietary administration of aflatoxin at 300 ppb level caused reduction (P<0.05) in total serum protein content. A significant decrease in serum protein due to feeding AFB1 contaminated diet has also been reported by earlier workers (Singh, 2019g; Singh, 2019c; Singh and Mandal, 2013; Khatke et al., 2012a; Singh et al., 2013a; Singh et al., 2013c; Silambarasan et al., 2016).
The decrease in total serum protein by AFB1 feeding has been reported due to reduced content of albumin and β-globulin (Pier, 1992). Other researchers reported that decrease in serum protein by aflatoxin feeding was attributed to failure in digestion and absorption of protein in gastro-intestinal tract (Voight et al., 1980) and inhibition of protein synthesis due to AFB1 contamination in diet (Sarasin and Moule, 1973). Groopman et al. (1996) also reported that the decline in serum protein may be due to decline in protein synthesis by forming adduct with DNA, RNA and protein and inhibit RNA synthesis and DNA-dependent RNA polymerase activity as well as causing degranulation of endoplasmic reticulum. The results of this study further indicated that ochratoxin contamination of feed at 250 ppb level caused significant (P<0.05) reduction in total serum protein content. Significant reduction in serum protein content due to ochratoxicosis was also reported by several researchers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b). The results of the present study revealed that combine feeding of aflatoxin and ochratoxin at all levels of contamination resulted in additive interaction on serum protein content of broiler chickens.
3.1.2 Serum Cholesterol
The serum cholesterol content in groups T2, T3, T4, T5, T6, T7, and T8 was almost similar to that of control (T1). The cholesterol content of group T9 was significantly (P<0.05) lower than that of T1. In groups T2, T3, T4 and T5 where dietary AFB1 and OTA were given separately, a decrease in serum cholesterol content by 1.13, 4.22, 2.39 and 5.61%, respectively was observed. However, in groups T6, T7, T8 and T9, wherein both the mycotoxins were given simultaneously, a decrease in cholesterol content by 8.82, 8.91, 8.92 and 13.06%, respectively was recorded. In the present study, feeding of aflatoxin at 150 and 300 ppb levels resulted in numerical reduction in serum cholesterol content. This result was more or less in agreement with those of earlier researchers (Singh and Mandal, 2013; Khatke et al., 2012a; Singh et al., 2013a; Singh et al., 2013c; Silambarasan et al., 2016; Singh, 2019c, g). The study further revealed that ochratoxin contamination of feed at 150 and 250 ppb levels also resulted in numerical reduction in serum cholesterol content. Significant reduction in serum cholesterol content due to ochratoxicosis was also reported by earlier researchers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b). The results of the present investigation revealed that simultaneous feeding of aflatoxin and ochratoxin at all levels of contamination (T6 to T9) resulted in synergistic interaction on serum cholesterol content of broiler chickens.
Table 2: Certain serum biochemical constituents of broilers fed different treatments
3.1.3 Uric Acid
The serum uric acid value in groups T2 to T8 was almost similar to that of control (T1). The uric acid value of group T9 was significantly (P<0.05) lower than that of T1. In groups T2, T3, T4 and T5 wherein AFB1 and OTA were administered individually, a decrease in serum uric acid content by 3.96, 7.35, 4.15 and 8.30%, respectively was reported. In the case of simultaneous feeding of both the mycotoxins in groups T6, T7, T8 and T9, a decrease in uric acid content by 10.56, 9.81, 13.01 and 31.50%, respectively was observed. In the present study, dietary aflatoxin at 150 and 300 ppb levels resulted in numerical reduction in uric acid content. This result was in agreement with those of earlier researchers (Singh and Mandal, 2013; Khatke et al., 2012a; Singh et al., 2013c; Silambarasan et al., 2016; Singh, 2019c, g) who reported reduced uric acid content due to aflatoxicosis in broiler chickens. The study further revealed that ochratoxin contamination of feed at 150 and 250 ppb levels also resulted in numerical reduction in serum uric acid content. Significant reduction in serum cholesterol content due to ochratoxicosis was earlier reported by several researchers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b). The results of the present investigation revealed that simultaneous feeding of aflatoxin and ochratoxin at all levels of contamination (T6 to T9) resulted in synergistic interaction on serum uric acid content of broiler chickens.
3.1.4 Alkaline Phosphatase
The ALP value in groups T3 to T9 was higher (P<0.05) than that of control (T1). In groups T2, T3, T4 and T5 wherein AFB1 and OTA were administered individually, an increase in ALP value by 18.32, 29.00, 87.29 and 93.27%, respectively was reported. In the case of simultaneous feeding of mycotoxins in groups T6, T7, T8 and T9, an increase in ALP value by 122.09, 118.96, 115.10 and 140.23%, respectively was recorded. In the present study, dietary inclusion of aflatoxin at 300 ppb level caused significant (P<0.05) increase in ALP value. Contrary to this, Bailey et al. (1998) reported a decrease in the serum ALP values when feed contaminated with 5 ppm AFB1 was fed to broilers. The study further revealed that feed contaminated with 150 and 250 ppb OTA resulted in significant (P<0.05) increase in ALP values in broiler chickens. Significant increase in serum ALP due to feeding ochratoxin contaminated diet has also been reported by earlier workers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b).
3.1.5 Aspartate Aminotransferase
The AST activity value in T2 to T9 was higher (P<0.05) than that of T1. In groups T2, T3, T4 and T5 wherein AFB1 and OTA were administered individually, an increase in AST value by 6.36, 8.86, 7.55 and 9.05%, respectively was recorded. However, with simultaneous feeding of mycotoxins in groups T6, T7, T8 and T9, an increase in AST value by 10.02, 12.81, 11.62 and 15.00%, respectively was observed. In the present study, dietary inclusion of AFB1 at 150 and 300 ppb levels caused significant (P<0.05) increase in AST value. This result was in agreement with those of earlier researchers (Singh and Mandal, 2013; Khatke et al., 2012a; Singh et al., 2013c; Silambarasan et al., 2016; Singh, 2019c, g) who reported increased AST activity value due to aflatoxicosis in broiler chickens. The study further revealed that feed contaminated with 150 and 250 ppb OTA resulted in significant (P<0.05) increase in AST activity value. This result was in agreement with those studies earlier reported by several researchers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b). The results of the present investigation further revealed that simultaneous feeding of aflatoxin and ochratoxin at all levels of contamination (T6 to T9) resulted in additive adverse effects on AST activity in broiler chickens.
3.1.6 Alanine Aminotransferase
The ALT activity value in groups T2 to T9 was higher (P<0.05) than that of control (T1). In the case of individual feeding of aflatoxin and ochratoxin in groups T2, T3, T4 and T5, an increase in ALT activity value due to mycotoxin feeding by 3.65, 8.05, 9.38 and 9.40%, respectively was observed. In the case of simultaneous feeding of both the mycotoxins in groups T6, T7, T8 and T9, an increase in ALT activity value by 10.12, 9.96, 15.59 and 15.74%, respectively was reported. In the present study, dietary inclusion of aflatoxin at 150 and 300 ppb levels resulted in significant (P<0.05) increase in ALT activity value. This result was in agreement with those of earlier researchers (Singh and Mandal, 2013; Khatke et al., 2012a; Singh et al., 2013c; Silambarasan et al., 2016; Singh, 2019c, g) who also reported increased ALT activity value due to feeding of AFB1 in broiler chickens. Moreover, the study further revealed that feed contaminated with 150 and 250 ppb OTA caused significant (P<0.05) increase in ALT activity. This result was in agreement with those earlier reported by several researchers (Singh et al., 2016a; Singh et al., 2015b; Singh and Mandal, 2018a; Singh and Mandal, 2018b). The results of the present study further revealed that simultaneous feeding of aflatoxin and ochratoxin at all levels of contamination (T6 to T9) resulted in additive adverse effects on ALT activity of broiler chickens.
3.2 Histopathological Studies
3.2.1 Liver
Liver is the primary organ for the detoxification of toxins therefore, the major alterations were observed in the liver parenchyma. The architectural and cellular organization of liver parenchyma was normal in group T1. Groups T2, T3, T4 and T5 showed sinusoidal dilatation and infiltration of inflammatory cells mainly MNCs but condition was more severe in group T3. Micrograph of liver from T6 to T9 showed progressive degenerative and necrotic changes. However, group T9 exhibited more severe degeneration in hepatic parenchyma. The liver samples collected from group T7 showed proliferation of bile duct along with periportal infiltration of inflammatory cells. The histopathological section of liver in T8 and T9 exhibited focal area lymphoid aggregation. However, more and large section of lymphoid aggregates was found in group T9. In the present study, diet containing 300 ppb of AFB1 resulted in major alterations in the liver tissue like sinusoidal dilatation and infiltration of inflammatory cells. Lesions were progressively increased in severity from combination of AFB1 and OTA. Most prominent damage to liver parenchyma was seen in 300 ppb AFB1+250 ppb OTA fed group (T9). Similar histopathological changes were observed by Karaman et al. (2010); Khatke et al. (2012c); Silambarasan et al. (2016) in broiler chicken fed 300 ppb AFB1. Sharma (2013) also found similar histopathological lesions on liver when feed contaminated with 250 ppb AFB1 was fed to broiler chickens. Singh (2019a, b) also reported similar histopathological lesions in liver at 400 and 600 ppb levels of AFB1 in the feed of Japanese quails. In the present study, it was further observed that 300 ppb AFB1+250 ppb OTA group (T9) resulted in synergistic effect on histopathology of liver in broiler chickens. With regard to OTA toxicity, similar histopathological changes as seen due to OTA contamination in diet were also observed by Sakhare et al. (2007) who subjected one-day old broiler chicks to OTA-contaminated diet (200 ppb) for 6 weeks of age. They showed degenerative changes in liver parenchyma and most of the hepatocytes were swollen, congested and vacuolated along with scattered infilteration of lymphocyte and heterophils in necrotic area. Also, Santin et al. (2002); Kumar et al. (2004) reported hepatocyte vacuolation, megalocytosis with accompanying hyperplasia of the biliary epithelium, degenerative changes and mononuclear cell infiltrations in the liver of 2 ppm OTA-treated chicks. These histopathological lesions were also in agreement with those of Singh et al. (2019a, b) when feed contaminated with 200 ppb OTA was fed to broiler chickens. Aflatoxicosis increased the liver lipid, which was absent when ochratoxin A was present Huff et al. (1988). This type of result was also seen in our findings, where vacuolation due to fat accumulation was observed in AFB1 contaminated fed group, however this type of lesion was absent when AFB1 and OTA was given simultaneously.
3.2.2 Intestine
In case of histopathology of intestine, cellular organization of intestine was normal in control group (T1). At low level of AFB1 (150 ppb) (T2) and OTA (150 ppb) (T4) only slight degenerative change in villus epithelium and increased goblet cells number was seen in villus. However, in T3 and T5 degeneration was more severe. Micrograph of intestine from T6 to T9 exhibited many pathological lesions which included mild to severe degeneration and necrosis in both villi and crypt areas surrounding infiltration of inflammatory cells, detachatment of villus, increased number of goblet cells and increased number of crypt cells. All these lesions were progressive towards group T9. The results showed that mycotoxins interaction resulted in additive effect on histopathology of intestine in groups T6, T7, T8 and synergistic effect in group T9. Result of the present study revealed that low level of both AFB1 (150 ppb) (T2) and OTA (150 ppb) (T4) was unable to initiate the severe damage to intestinal epithelium. However, slight degenerative change in villus epithelium and increased goblet cells number was seen in villus. Intestine is the first and continuous organ to be exposed to any toxin present in feed, so that the chance of physical damage to intestine is common during mycotoxicosis. Therefore, AFB1 caused damage to the gastric and intestinal mucosal barrier. Similar result was found by Kurt et al. (2009) who demonstrated that aflatoxin B1 causes a significant reduction in mucus and phospholipids levels of gastric mucosal barrier. AFB1 administration caused colon damage characterized by aberrant crypt foci, which authenticated with the increase in mucous production due to increased in goblet cell number. Sharma (2013) also found similar result like degenerative changes, sloughing and focal area showed sever necrosis along with infiltration of inflammatory cells were seen in 250 ppb aflatoxin fed group. In the present study, 300 ppb AFB1+250 ppb OTA combination (T9) caused most severe damage which, included mild to severe degeneration and necrosis in both villi and crypt areas surrounding infiltration of inflammatory cells, detachatment of villus, increased number of goblet cells and increased number of crypt cells which is designated as synergism between aflatoxin and ochratoxin.
3.2.3 Kidneys
Histological examination of kidneys revealed normal renal parenchyma and did not show any alteration or histopathological changes in control birds (T1). Feeding both levels of AFB1, 150 ppb (T2) and 300 ppb (T3), exhibited widened bowmen with slight increased in cellularity and inflammatory changes. Similar histopathological changes were observed by Khatke et al. (2012c); Silambarasan et al. (2016) in broiler chicken fed 300 ppb aflatoxin. Ochratoxin had target organ as kidneys so major pathological damage such as hypercellular glomeruli with mild degeneration in group T4 and in group T5 severe degenerative changes in renal parenchyma with dilated lumen and multifocal aggregates of mononuclear cells (MNCs) also noted. Kidneys of groups T6, T7, T8 and T9 showed moderate to severe degeneration widened bowman capsule, in addition, T9 exhibited severe necrosis with denuded tubular epithelium. The present study revealed that feeding both levels of AFB1 (150) ppb (T2) and 300 ppb (T3) contaminated diet exhibited widen bowmen with slight increased in cellularity and inflammatory changes. OTA contamination resulted in hypercellular glomeruli with mild degenerated kidneys in groups T4 and T5. This result was in agreement with those of Singh et al. (2019c, g) wherein feed contaminated with 200 ppb OTA was fed to broiler chickens. In the present study, feeding AFB1 and OTA simultaneously (T6 to T9) caused severe lesions like severe degenerative changes in renal parenchyma with dilated lumen and multifocal aggregates of MNCs, which indicated the additive effect of mycotoxins on histopathology of kidney. Moreover, 300 ppb AFB1+250 ppb OTA (T9) also resulted in severe necrosis with denuded tubular epithelium.
4. Conclusion
It was concluded that aflatoxicosis and ochratoxicosis individually resulted in decreased concentration of serum protein, cholesterol and uric acid; and increased activity of ALP, AST and ALT. However, simultaneous exposure caused additive effect on serum protein, AST and ALT; and synergistic effect on cholesterol, uric acid and ALP. Further feeding of 150 ppb AFB1+150 ppb OTA; 150 ppb AFB1+250 ppb OTA and 300 ppb AFB1+150 ppb OTA resulted in additive adverse effect and 300 ppb AFB1+250 ppb OTA resulted in synergistic effect on histopathology of internal organs in broiler chickens.
This article was originally published in Livestock Research International, April-June 2019, Volume 07, Issue 02, Pages 98-106.
