Toxicological evaluation of low dietary aflatoxin B1 concentrations on performance and residues in laying ducks

N Tansakul, J Phakam, S Choochuay and N Paochoosak

Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
natthasitt@yahoo.com

Abstract

The adverse effects of aflatoxin B1 (AFB1) concentrations on duck laying performance regarding egg productivity, serum-hematological profiles and residue were investigated. Experiments were performed under simulated conditions at low-level aflatoxin contamination commonly found in natural feedstuff with sub-chronic ingestion. The samples were 100 Khaki Campbell Ducks at 22 weeks (the initial age for laying). The toxin was introduced into the mixed feed as rations containing AFB1 at 0.025, 0.050 and 0.100 mg/kg for an experimental period of 14 days. Results showed that feeding rations containing AFB1 at a low-level did not significantly affect performance and residue in laying ducks but influenced liver and spleen weight. Serum-hematological profiles showed alteration patterns in which only total plasma protein (TP) and alkaline phosphatase (ALP) were significantly altered. At the end of the experimental 14 day period, the serum patterns of aspartate aminotransferase (AST): alanine transaminase (ALT) ratio ranged from 1.2 to 2.6.

Key word: egg production, growth performance, mortality, serum-hematological profiles, toxicity

Introduction

Animal feeds are frequently contaminated with mycotoxins, with aflatoxin the most common mycotoxin found in tropical climates. Poultry feed is prone to contamination with mycotoxigenic fungi and mycotoxins, particularly aflatoxin (Kavisarasai 2010; Monson et al 2015). Aflatoxin is a carcinogenic and mutagenic metabolite which is produced by the fungal strains of Aspergillus spp. There are four major aflatoxin analogues including aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) of which AFB1 is the most toxic and detected worldwide (Monson et al 2015).

In poultry production, aflatoxicosis as the poisoning that results from ingesting aflatoxins has a significant impact on health by increasing the mortality rate, reducing the absorption of nutrients, feed intake and growth rate, impairing the immune function and decreasing performance. Aflatoxicosis is also associated with serum-hematological changes involving decreased total protein, albumin, cholesterol and blood coagulation patterns (Méndez-Albores et al 2007, Rawal et al 2010, Siloto et al 2011). Aflatoxin has a marked effect on liver and serum enzyme activities. Activity elevation of aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine transaminase (ALT) by aflatoxin exposure was reported by Méndez-Albores et al (2007) and Chen et al (2014). High concentrations and long-term exposure to aflatoxin manifest as histopathological alteration through an enlarged and fatty liver, hepatocellular necrosis, bile duct proliferation and hepatocellular carcinoma (Bintvihok 2001, Rawal et al 2010, Siloto et al 2011). Extensive studies of aflatoxicosis in chickens have been carried out while ducks are also very sensitive to the disease; however, reports on ducks regarding the effect of low-dose aflatoxicosis, particularly at the age for laying are limited. Contaminated feed may cause aflatoxin residues in food of animal origin such as meat and eggs. Residues of AFB1 and the carry-over rate of aflatoxin into tissues and eggs were reported by Oliveira et al (2000) and Bintvihok et al (2002).

In Thailand, low-level contamination of aflatoxin in poultry feed is frequently found (Kavisarasai 2010), possibly due to Thai animal feed regulations which limit the toxin concentration to below 0.03-0.1 mg/kg. In addition, results from a local questionnaire determined that most poultry farmers only store animal feed for 1 to 2 weeks. However, farmers always believe that aflatoxicosis is a major root of cause whenever the duck is ill or performing poorly. Consequently, many farmers added mycotoxin adsorbent to the feed to prevent intestinal absorption of toxins and economic loss due to aflatoxicosis. Little is known regarding the effects of aflatoxicosis in laying ducks, particularly from short-term exposure at low dosages. To shed light on this issue, an investigation was conducted into the adverse effects of AFB1 on duck performance, egg productivity, serum-hematological profiles and residue under simulated low-level conditions frequently found in natural feedstuff following sub-chronic ingestion.

Materials and methods

One hundred Khaki Campbell Ducks at 22 weeks (the initial age for laying) were used to investigate the effect of AFB1 on growth performance, serum-hematological profiles and residue in tissue. All ducks were healthy and free from pathogen infections. The ducks were acclimatised for seven days before starting the experiment. A basal diet was formulated containing 18.7% crude protein, 2,750 kcal/kg of energy following standard nutrient composition for laying ducks by the Department of Animal Husbandry. Water and feed were offered ad libitum. Experimental procedures were in accordance with the ‘Ethical Principles for the Use of Animals for Scientific Purposes’ issued by the National Research Council of Thailand (NRCT). Groups of 25 ducks were randomly allotted to four cages with Ctl as the control. The remaining three groups AF-L, AF-M and AF-H were fed with rations containing AFB1 at 0.025, 0.050 and 0.100 mg/kg feed respectively for 14 days. Conditions of aflatoxin levels were simulated relative to ranges previously reported in Thailand by farmers keeping stock feed for only 1 to 2 weeks. AFB1 standard (Fermentex^®) was used. Prior to contamination, the feed was tested for the presence of major mycotoxins including AFB1, deoxynivalenol and ochratoxin A by ELISA which can detect levels below 0.01 and 0.035 mg/kg feed. No mycotoxins were recorded.

Data were collected of body weight gain, feed intake, egg productivity, appearance and aflatoxin residue in the egg, liver and ovary tissue. Serum and plasma samples were taken on days 1, 5, 10 and 14. Hematological parameters including red blood cell count (RBC), white blood cell count (WBC), haemoglobin (Hb), haematocrit or packed cell value (PCV) and serum biochemical parameters as aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), total plasma protein (TP) and albumin (Alb) were observed. At the end of the experiment, the ducks were sacrificed to observe lesions and to measure the weight of the internal organs including liver, gizzard, ovary, proventriculus, spleen and kidney. Hematological and serum biochemical measurements determined erythrocyte and white blood cell numbers using an automatic cell counter. Differential blood counts e.g. lymphocyte (Lymp) were performed on blood smears stained by a standardised method. Concentrations of total serum proteins and albumin were determined using the Biuret and Bromocresol methods respectively. Aflatoxin residue in the eggs and tissues was detected using the HPLC system following the standard method of the Association of Official Analytical Chemistry (AOAC 2005). The limit for quantification of tissue spiking validation was 0.005 mg/kg.

Statistical analysis

Data were presented as means with variability expressed as the standard error of the mean (SEM) for each group. Parametric data were statistically analysed by the GLM option using one-way ANOVA. Probability values less than 0.05 (p < 0.05) were considered as significant.

Results and Discussion

Influence of AFB1 on hematological parameters

Data alteration of RBC, WBC and Hb by aflatoxicosis showed no significance between the groups or for different time periods (Table 1). Total WBC of the AF-M group was higher than the Ctl and AF-L groups at day five post-treatment. PCV values were significantly different between groups; however, pattern variations were too inconsistent to draw any conclusions. Results indicated that a low-level of AFB1 had no effect on the hematopoietic processes in laying ducks. In contrast, numerous avian studies showed that PCV, RBC and WBC depletion occurred at high levels of aflatoxin contamination (Huff et al 1986, Agag 2004, Safameher and Shivazad 2008). Recently, ducklings fed diets contained AFB1 showed decreased Hb, PCV (Khajarern et al 2003, He et al 2013, Rattanasinthuphong et al 2017) and RBC (He et al 2013).

Regarding the differential white cell count, the lymphocyte count at day 14 in groups AF-M and AF-H reduced, which differed from the Ctl and AF-L groups, whereas other differential leukocyte counts did not change (data not shown). Data variation among previous studies concerning total and differential leucocyte counts with concurrent lymphopenia and heterophilia were reported by Agag (2004) and Monson et al (2015). However, dose-related aflatoxicosis caused lymphocytopenia and impaired innate immune dynamics in poultry (Jewers 1990, Safamezer and Shivazad 2008, Chen et al 2014).

Influence of AFB1 on serum parameters

Our results showed variations in the pattern of observed serum chemistry levels (Table 2). Blood serum levels of AST, ALT and Alb did not show significant alteration patterns related to aflatoxicosis exposure time, while TP and ALP values showed significant differences between groups. This finding concurred with Li et al (2012) who found that AFB1 led to elevated ALP levels in Cherry Valley ducks. Although Alb levels reduced over time in group AF-H, they were not significantly different from the other groups. This was consistent with Chen et al (2014) who demonstrated that a linear reduction of Alb and TP levels correlated with increasing AFB1 concentration. An increased level of liver enzymes including AST, ALT and ALP were linked to liver damage since elevation of AST levels in avian species damages the liver, skeleton, heart and renal tubules. High activity of AST and ALT serums in ducks following dietary aflatoxin contamination has been reported (Cheng et al 2001, Bintvihok and Davitiyananda 2002, Han et al 2008, He et al 2013).

Our results demonstrated that ingestion of a diet containing up to 0.1 mg/kg AFB1 elevated the biochemical enzymes AST and ALP; however, significant difference (p < 0.05) was only seen for ALP compared to other groups. This finding agreed with a recent report that AST and ALP serum levels increased when ducklings received an AFB1 intoxicated diet equivalent to 0.1 mg/kg (Chen et al 2014). Similarly, other researchers reported that a feeding trial of aflatoxin at 0.1 mg/kg on ducklings led to increased AST and ALT enzymes and AST:ALT ratio in the blood circulation (Cheng et al 2001, Méndez-Albores et al 2007) as well as increased ALT and ALP (Bintvihok 2001). Conversely, Cheng et al (2001) reported that AFB1 did not affect the ALP level when fed to one-day-old Mule ducklings at 0.2 mg/kg for three weeks.

In contrast, we found that activity of duck serum ALT was not affected by feeding a low-dose of AFB1 which agreed with Chen et al (2014), whereas other reports recorded elevation in this enzyme (Bintvihok 2001, Méndez-Albores et al 2007). The AST:ALT ratio is used as an indicator for liver function. Our results showed that the AST:ALT ratio at day 14 ranged from 1.2 to 2.6. This result agreed with Chen et al (2014) who demonstrated that ducklings fed with 0.21 mg/kg AFB1 recorded increased AST:ALT ratios of up to 2.5 and this was associated with liver fibrosis in Pekin ducks. Additionally, low concentrations of AFB1 led to impaired liver function and gene expression in ducklings (Chen et al 2014).

Effect of AFB1 on the performance of laying ducks and tissue lesions

Ducks are approximately 200 times more susceptible to aflatoxicosis than chicken broilers and layers. In Thailand, the Department of Livestock Development (DLD) has set the limit of aflatoxin in general animal feed to 0.030-0.100 mg/kg depending on the type of feed and animal species, with feed for ducks limited to 0.030 mg/kg. In our experiments, no abnormal appearances were recorded for egg quality including pigment, eggshell thickening and blood spots. Mean values of individual egg production for all groups showed no significant difference (data not shown). Average body weight gain, feed intake and organ weights of ducks fed with different levels of AFB1 are presented in Table 3. Results indicated no appreciable change in the mean values of the parameters for all groups.

Chen et al (2014) reported that feed intake was reduced by up to 55% in ducklings that received AFB1 at 0.14 mg/kg, whereas the feed intake reduction of chickens was only 17.4% when fed with 2 mg/kg of AFB1. Similarly, AFB1 caused reduced feed intake and body weight gain in ducklings (He et al 2013, Wan et al 2013, Rattanasinthuphong et al 2017). In contrast to these previous studies, our results revealed that low levels of AFB1 did not affect either feed intake or body weight gain in laying ducks. This could be due to the breed, age differences of duck and AFB1 concentrations used in the experiment which are all factors for toxin susceptibility. Aflatoxin has a marked effect on the liver by causing fat accumulation and bile duct proliferation. The precursors for both yolk and albumen are synthesised in the liver; hence, liver damage may affect egg production.

We observed morphological changes as microscopic lesions in the kidneys, heart, spleen, thymus and intestine while subcutaneous tissues did not show any variation in appearance, size, weight and consistency both within and between groups. Liver lesions of one duck in each of the AF-M and AF-H groups showed slight swelling as a pale colour with the fatty liver moderately degenerated.

However, as shown in Figures 1 and 2, ingestion of AFB1 at low levels led to a dose-dependent phenomenon which correlated with liver and spleen weight. This finding indicated that liver weight (He et al 2013, Rattanasinthuphong et al 2017) and spleen weight (Khajarern 2003, He et al 2013) could be elevated by AFB1 contaminated feed. However, conversely, chronic exposure of ducklings to AFB1 caused liver weight depletion (Wan et al 2013).

No lesions were found on the ovaries and oviducts for all groups. On the contrary, Hafez et al (1982) found that aflatoxin caused an increase in ovarian follicular atresia in poultry. Our results showed that low-level exposure to AFB1 had no effect on the egg productive organs in ducks. Moreover, the animals appeared clinically normal with no mortality throughout the experiments.

Figure 1. Effect of AFB1 on liver weight

Figure 2. Effect of AFB1 on spleen weight

AFB1 residue in tissues and eggs of laying ducks

Normally, aflatoxin is rapidly absorbed and slowly excreted in poultry. Risk potential of aflatoxin contamination in feed should be considered for the transmission of toxins into foodstuffs of animal origin such as meat, milk and eggs. Extensive studies of AFB1 residue in hen eggs have been conducted but little information from duck eggs is documented. Our study did not detect residual value of aflatoxin in duck eggs in any sample for both yolk and albumen. In addition, no aflatoxin residual was found in ovary tissue. Thus, low-concentrations of aflatoxin may marginally accumulate in egg productive tissue but it does not penetrate the yolk sac in the ovary nor remain in the egg. Aflatoxin was not detected in the liver, in agreement with Anong and team (Bintvihok et al 2002) who detected a trace amount of AFB1 in the liver when the ducks ingested aflatoxin at high concentrations of up to 3 mg/kg for seven days. They also stated that the ratio of feed level to aflatoxin residual level was 4,615 and 3,816 in the yolk and albumen of hens, respectively; a higher value than recorded in duck and quail. Numerous studies have indicated wide variation in the calculated transfer rate of aflatoxin into eggs ranging from 2,200:1 up to 66,200:1 (Oliveira et al 2000; Bintvihok et al 2002).

Conclusion

• Consumption of AFB1 at low levels had no adverse effect on growth performance, mortality and residue in ducks at the beginning of laying age which showed relative tolerance to aflatoxicosis.
  
  
• Certain serums and hematological profiles including ALP, PCV and TP were significantly affected by diets containing AFB1-contaminated feed. Moreover, a dose-dependence phenomenon was found for liver and spleen weight depending on AFB1 concentrations.
  
  
• Variation of the toxin transfer rate into eggs and its effect on animal health depends on species, breed, age, toxin concentration and duration of exposure. Generally, the feed is a cocktail of mycotoxin contamination and this may have a mycotoxicosis synergist effect.
  
  
• Although aflatoxin was not found in any duck egg or tissue examined in this study, a rigorous monitoring programme to ensure the effectiveness of toxin control should not be ignored.

Acknowledgements

The authors gratefully acknowledge support of competitive grant from the Kasetsart University Research and Development Institute (KURDI).

References

Agag BI 2004 Mycotoxins in foods and feeds-aflatoxins. Assiut University bulletin for environmental researches.7:173-206.

AOAC 2005 In House Method TE-CH-025 based on AOAC 2005. Code #991.31, 994.08.

Bintvihok A 2001 Controlling aflatoxin danger to ducks and duck meat. World Poultry Science Association. 17:18-19.

Bintvihok A, Thiengnin S, Doi K and Kumagai S 2002 Residues of aflatoxins in the liver, muscle and eggs of domestic fowls. Journal of Veterinary Medicine Science. 64:1037-1039.

Bintvihok A and Davitiyananda D 2002 Aflatoxins and their metabolite residues in chicken tissues from 5parts (10 provinces) of Thailand. Journal of Health Research.16:37-50.

Chen X, Horn N, Cotter P F, and Applegate T J 2014 Growth, serum biochemistry, complement activity, and liver gene expression responses of Pekin ducklings to graded levels of cultured aflatoxin B1. Poultry Science. 93:2028-2036.

Cheng Y H, Shen T F, Pang V F and Chen B J 2001 Effects of aflatoxin and carotenoids on growth performance and immune response in mule duckling. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 128:19-26.

Hafez A H, Megalla S E, Abdel-Fattah H M and Kamel Y Y 1982 Aflatoxin and aflatoxicosis II. Effects of aflatoxin on ovaries and testicles in mature domestic fowls. Mycopathologia. 77:137-139.

Han X Y, Huang Q C, Li W F, Jiang J F and Xu Z R 2008 Changes in growth performance, digestive enzyme activities and nutrient digestibility of cherry valley ducks in response to aflatoxin B1 level. Livestock Science. 119:216-220.

He J, Zhang K Y, Chen D W, Ding X M, Feng G D and Ao X 2013 Effects of maize naturally contaminated with aflatoxin B1 on growth performance, blood profiles and hepatic histopathology in ducks. Livestock Science 152:192-199.

Huff W E, Kubena L F, Harvey R B, Corrier D E and Mollenhaur D D 1986 Progression of aflatoxicosis in broiler chickens. Poultry Science. 65:1891-1899.

Jewers K 1990 Mycotoxins and their effect on poultry production. Options Méditerranéennes, Sér. A. 7:195-202.

Kavisarasai P 2010 Study on aflatoxin B1 and ochratoxinA in animal feed for chicken layer and egg. M.Sc. Thesis, University of Thammasart, Thailand.

Khajarern J M, Khajarern S, Moon T H and Lee J H 2003 Effects of Dietary Supplementation of Fermented Chitinchitosan (FERMKIT) on Toxicity of Mycotoxin in Ducks. Asian-Australasian Journal of Animal Sciences. 16:706-713.

Khan W A, Khan M Z, Khan A, Hassan Z U, Rafique S, Saleemi M K and Ahad A 2014 Dietary vitamin E in White Leghorn layer breeder hens: a strategy to combat aflatoxin B1-induced damage. Avian Pathology. 43:89-95.

Li Y, Liu Y H, Yang Z B, Wan X L and Chi F 2012 The efficacy of clay enterosorbent to ameliorate the toxicity of aflatoxin B1 from contaminated maize on hematology, serum biochemistry, and oxidative stress in ducklings. Journal of Applied Poultry Research 21:806–815.

Méndez-Albores A, Del Rio-Garcia J C and Moreno-Martinez E 2007 Decontamination of aflatoxin duckling feed with aqueous citric acid treatment. Animal Feed Science and Technology. 135:249-262.

Monson M S, Coulombe R A and Reed K M 2015 Aflatoxicosis: lessons from toxicity and responses to aflatoxin B1 in poultry. Agriculture. 5:742-777.

Oliveira C A, Kobashigawa F E, Reis T A, Mestieri L, Albuquerque R and Correa B 2000 Aflatoxin B1 residues in eggs of laying hens fed a diet containing different levels of the mycotoxin. Food Additives and Contaminants. 17:459-462.

Rawal S, Kim J E and Coulombe R 2010 Aflatoxin B1 in poultry: toxicology, metabolism and prevention. Research in Veterinary Science. 89:325-331.

Rattanasinthuphong K, Tengjaroenkul B, Tengjaroenkul U and Pakdee P 2017 Efficacy of mycosorbents to ameliorate the adverse effects of natural aflatoxin contamination in the diets of Cherry Valley ducks. Livestock Research for Rural Development. Volume 29, Article #48. Retrieved September 11, 2017, from http://www.lrrd.org/lrrd29/3/teng29048.html

Salwa A A and Anwer W 2009 Effect of naturally contaminated feed with aflatoxins on performance of laying hens and the carryover of aflatoxin B1 residues in table eggs. Pakistan Journal of Nutrition. 8:181-186.

Safameher A and Shivazad M 2008 The effects of Saccharomyces Cerevisiae on performance and haematological characters in broiler chicks during aflatoxicosis. Proceedings of the The 15^th Congress of FAVA, FAVA - OIE Joint Symposium on Emerging Diseases. 27-30 October, Bangkok, Thailand, pp. 155-156.

Sheth S G, Flamm S L, Gordon F D and Chopra S 1998 AST/ALT ratio predicts cirrhosis in patients with chronic hepatitis C virus infection. American Journal of Gastroenterology. 9344-48.

Siloto E V, Sartori D R S, Oliveira E F A, Sartori J R, Fascina V B and Berto D A 2011 Performance and eggquality of laying hens fed diets containing aflatoxin, fumonisin and adsorbent. Brazilian Journal of Poultry Science.13:21-28.

Verma J, Johri T S, Swain B K and Aeena S 2004 Effect of graded levels of aflatoxin, ochratoxin and their combinations on the performance and immune response of broilers. British Poultry Science. 45(4):512-518.

Wan X L, Yang Z B, Yang W R, Jiang S Z, Zhang G G, Johnston S L and Chi F 2013 Toxicity of increasing aflatoxin B1 concentrations from contaminated maize with or without clay adsorbent supplementation in ducklings. Poultry Science. 92:1244-1253.

Received 25 July 2017; Accepted 7 October 2017; Published 2 November 2017

Go to top