Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 2 (february 2022) : 175-181

​Impact of Dietary Aflatoxin-B1 on Juvenile Growth in White Pekin Ducks

S.K. Mishra1,*, C.R. Jena1, P.K. Naik1, D. Kumar1, B.K. Swain1, S.K. Panda2, J. Pamia2, G.R. Jena2
1ICAR-Directorate on Poultry Research, Regional Station, Bhubaneswar-751 003, Odisha, India.
2College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
Cite article:- Mishra S.K., Jena C.R., Naik P.K., Kumar D., Swain B.K., Panda S.K., Pamia J., Jena G.R. (2022). ​Impact of Dietary Aflatoxin-B1 on Juvenile Growth in White Pekin Ducks . Indian Journal of Animal Research. 56(2): 175-181. doi: 10.18805/IJAR.B-4556.
Background: Aflatoxicosis in ducks is reckoned as a challenge for nutritionists worldwide, for its adverse impacts on growth. A study was conducted to ascertain the precise level of Aflatoxin-tolerance in White Pekin ducklings during juvenile ages.

Methods: Day-old-ducklings (240) were randomly distributed into 3 experimental-groups viz., Control (<0.5 ppb), T1 (200 ppb), T2 (400 ppb) and reared for measuring weekly growth, feed efficiency and blood biochemical changes, with treatments terminated at 6weeks age.

Result: There was significant depression in live weights for both dietary-toxin groups from 2nd week onwards till completion. Mortality (0-6 weeks) was 85% in T2, while T1 was in between control and T2, both for mortality (45%) and morbidity. Feed-consumption and conversion for either toxin groups remained significantly (p≤0.05) poor, with T2 resulting in erratic FCRs, ranging from 1.23 to 4.95, across weeks. A typical AFB1-induced lameness in ducklings beyond 3 weeks of AFB1-exposure, emerged as a hallmarked morbidity, in varied proportions, in T1 and T2, but not in control. The study confirms that acute-depression of juvenile growth and high-morbidity are distinct outcomes from AFB1’s presence in Pekin diets @ 200 ppb level or higher, with such leg deformities emerging as  hallmarked features of Pekins reared on AFB1-spiked diets. It can be concluded that, while the AFB1 content of juvenile White Pekin ducks should be kept limited to the recommended safe levels (<10 ppb); exceeding a threshold of 200 ppb is sure to cause poor growth and FCR, with adverse blood biochemical changes, high mortality, morbidity and lameness.
Duck farming (Anas platyrhynchos) in India, is mostly concentrated in eastern-coastal states (Rath et al., 2015), which pose big challenges in the storage of poultry feed ingredients due to high relative- humidity (Madgwick et al., 2011) leading to build-up of mycotoxins (Pachauri and Reisinger, 2007), specifically, Aflatoxin-B1 (AFB1) with productivity-drops in growing and laying ducks (Pandey and Chauhan, 2007). There are conflicting reports regarding toxic threshold for naturally- occurring AFB1 in feed, beyond which morbidity could surface in the host (Fouad et al., 2019). Further to European Commission (2006 and 2011) recommendations, Mishra et al., (2016) have recommended the acceptable level of aflatoxins (AFB1) in grains as 10 ppb, for safe-usages in Poultry feeds. While AFB1 levels ranging up to 500 ppb in feed of chickens were viewed insignificant; AFB1 upto 250 ppb in diets of Turkey Poults and AFB1 levels upto 100 ppb in diets of ducklings were also viewed insignificant, to impact productivity of the respective subjects (Shabani et al., 2010; Diaz et al., 2009 and Fin et al., 2012).
Keeping the above in view, a study was conducted to ascertain the precise level of Aflatoxin-tolerance in White Pekin ducklings during juvenile ages.
The experiment was carried out at ICAR-CARI Regional Center, Bhubaneswar. White Pekin day-old ducklings (240, avg. hatch weight: 55 gms) were randomly divided into 3groups and each group having 4 replicates of 20 day old ducklings (DOD). A standard duck starter-feed (2950 Kcal/kg, 21.80% CP), with negligible (<0.5 ppb, mostly from background content in best of the sourced grains) AFB1 was treated as control diet. Two treatment diets (T1 and T2) were prepared from the control diet by spiking with lab-cultured and purified AFB1, to measure 200 and 400 ppb respectively. Randomly, the above three feeds were offered to the three groups, ad libitum, for a period of six weeks.
The culture and purification of AFB1 was carried out as per Verma et al., (2004) using Aspergillus parasiticus (strain: NRRL-299, maintained at ICAR-CARI, Izatnagar, India), which were harvested, characterized and diluted appropriately (BIS; AOAC, 1995). The feed-samples were evaluated for AFB1 content, using a standard Flurometer (VICAM®, Milford, MA, USA).
Raising of ducklings and generation of data
Juvenile growth
All the DODs were raised on floor and standard managemental practices were followed through-out the experimental period. Daily feed consumption and weekly live weight were recorded and the FCR was derived as feed consumed per unit live-weight gain.
Biochemical analysis of blood samples
Blood was collected from 5 ducklings per replicate on 21 and 42 days of age. Serum was separated out from blood and stored at -20°C till usage. Gama glutamyl transpeptidase (GGT), Serum glutamate oxalate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), Cholesterol and Triglyceride were analyzed using standard bio-chemical kits (Coral Clinical Systems®, Goa, India).
Necropsy analyses, morbidity and mortality profiling
Post mortem (P.M) was conducted routinely, immediately upon succumbing of ducklings. Necropsy was conducted for all the sacrificed ducklings (a proportion of survivors: 4 to 6 ducklings per treatment) at 6 weeks of age, upon termination. The gross pathology of the sacrificed ducklings were carried out, following histopathological analyses of vital organs (liver, Kidney and Spleen) from 5 randomly selected carcasses, as per standard procedures. All morbidities were characterized.
Ethical clearances and approval
The whole experiment was conducted, taking proper care of ducklings, under ethical clearances from Animal Ethics committee, vide letter OUAT-IAEC No.6/ Dt. 9th Mar 2018.
Statistical analysis
The data were subjected to Statistical analyses using SAS software package (version 9.2) for Analysis of variance (ANOVA) and tests of significance for various traits (Tukey’s Tests).
There was significant depression in weekly growth and feed conversion efficiency in both the Toxin-fed groups, at all ages. The Aflatoxin-B1 (AFB1) impacted the weekly growth of Pekins, where the T2 had higher quantum of damage compared to the T1 in terms of morbidity and resultant growth parameters.
Variations in Body weight of ducklings
The growth of ducklings over 2 to 6 weeks underwent significant variation (p≤0.05), where the variation remained erratic between T1 and T2 indicating differential debilitating impact from the AFB1 on different ages of Juvenile growth (Table 1). The live weight in both the Toxin groups (T1 and T2) was significantly poorer than control, at most juvenile ages of measurements, especially from 2 to 4 Weeks. The differential bodyweight between the control and both- Toxin groups, remained in 200 to 300% ranges, where in continued mortality and morbidity in both toxin groups rendered them to be with highest C.V, indicating impact of AFB1 exposure, on uniformity of ducklings. In general, the reduction in bodyweight was in a dose-dependent manner, where T2 showed higher variation than T1, where higher quantity of toxins consumed apparently caused the depression. To generalize, body weight was significantly (p<0.05) reduced for ducklings fed with diets containing AFB1 exceeding 200 ppb, resulting in wider Feed conversion ratio, similar to reports of earlier workers (Han et al., 2008) where decrease in body weight of hens fed with 2.0 to 8.0 ppm levels of AFB1, were noted across juvenile stages.

Table 1: Group-wise Means±S.E. for weekly live-weights along with C.V (%) through different weeks.

Feed consumptions
There was significant (p<0.001) decrease in feed consumption (g/day) in AFB1- treated groups, throughout 1st to 5th week age compared to control (Fig 1). Depressing effect of toxins (AFB1) was noticeable in ducklings fed AFB1 @ 400 ppb, indicating that ducklings detested AFB1’s presence, with loss of appetite. The reduced cumulative feed consumption in both treated groups over control was probably due to impaired hepatic metabolism (Verma et al., 2004).

Fig 1: Daily feed consumption patterns (per duckling), in toxin-fed groups, versus control, (till 5 weeks age).

Feed conversion efficiency
There was significantly-inferior feed efficiency (p≤0.05), in the toxin-treated ducklings (T1 and T2) during 2nd to 6th week of age (Fig 2). The poor feed conversion efficiency noted in both AFB1-groups in this study indicate that ducks are sensitive to mycotoxins with respect to feed efficiency that could be attributed to differences in source of contamination occurring in natural processes (Pandey and Chauhan, 2007). The inferior feed efficiency was observed specifically in higher-toxin diet (400 ppb) which indicated the negative effect from cumulatively-higher amount of toxin on vital organs, in supporting exuberant muscle growth and metabolism, as normally due in case of meat-type ducks (Pekins). The FCR ranged from 1.23 to 4.95 during 1 to 6 weeks. The FCR of ducklings tended to remain wider, upon increase in AFB1 level in feed: T2 (@400 ppb) as compared to T1 (@ 200 ppb) against control. Similar findings were also reported by earlier workers (Pandey and Chauhan, 2007; Chen et al., 2016).

Fig 2: Mean feed conversion ratio across different weeks of growth, for the experimental groups.

Live weight gain
The body weight gain of T1 remained lowest, during the 2nd week of rearing, where as the control group gained the highest live weight, during 4th week of age (Fig 3). The T2  remained lagging behind the other 2 groups, throughout course of study; where the growth performance remained lower in AFB1 treated groups, similar to patterns previously described by many authors (Han et al., 2008; Khlangwis et al., 2011; Murugesan et al., 2015; Mahmood et al., 2017).

Fig 3: Average weekly weight gains per duckling, through weeks of growth, for treatment groups

Serum biochemical analyses
There was significant increase (P<0.01) in levels of liver-function enzymes namely, ALT, AST and GGT for the AFB1 treated ducks, compared to control. The T1 group’s liver enzyme status remained mid-way between control and T2, showing a dose-dependent rise in these key enzyme’s levels in liver, probably as a sequalae to hepatocyte’s damage by Aflatoxin-B1 (Fernandez et al., 1994). Significant increases (P<0.01) in the level of Triglyceride and Cholesterolat 3rd week and 6th week upon impact from AFB1, was recorded (Fig 4), parallel to the findings of earlier workers (Chen et al., 2014).

Fig 4: Biochemical parameters of different AFB1 groups [ALT, AST, GGT, Triglyceride and Cholesterol], at two time points.

Gross and histopathology
Significant gross-pathological changes were observed (Fig 5) primarily across 3 vital organs, i.e. liver, kidney and heart: but characteristic lesions were also visible on spleen too, which correlated well with available literature (Madheswaran et al., 2005). There were changes in liver, signified by hepatomegaly with lesions, in ducklings fed with 400 ppb AFB1. Livers of T1-ducklings revealed congestion with adjacent pale-patches and enlarged friable, loss of architecture with cloudy swelling and necrosis of hepatocytes; whereas in case of T2 Liver, remarkable loss of architecture; necrosis with massive infiltration red blood cells (Fig 6) were prominent.

Fig 5: Necropsy profile of 42- day old White Pekin ducks: Control Vs T1 Vs T2, for selected liver samples.


Fig 6: Histopathology of 3 vital organs of Pekins (42d) receiving varying dietary AFB levels.

There was enlargement of kidney, at large, across all the specimens, with paleness evident in most samples drawn from T1 (200 ppb) and T2 (400 ppb). Nephrotic changes in kidney; including petechial hemorrhages, across whitish streaks (apparently from Uric acid deposits) were usual features of kidneys which were little-variant from those experienced across regular mortalities (Monson et al., 2014). Similarly, against the normal histological factors observed in kidney from control groups, in case of T1 kidney: slight degeneration of tubular epithelium with mild atrophy of glomerulus were noticeable and for T2 Kidneys, distinct atrophy of glomeruli with necrosis of tubular epithelium were recorded. In T1 spleen, mild congestion of red pulp with few nuclear debris and vacuoles were prominent; whereas across T2 spleens, severe congestion of red pulp with Increase in Nuclear debris and vacuoles were evident.
The disturbed histopathological profiles across major necropsy specimens covering liver, spleen and kidney of the AFB1-treated pekin’s profile, as realized in this study have also been documented by many authors (Chen et al., 2014; Peng et al., 2014), which concurs that: AFB1 at levels of 200 ppb, indeed constitutes a critical and vulnerable dose of AFB1.
Mortality and morbidity
The pattern of mortality in T2 was in distinct contrast to that of T1, where no mortality precipitated till 2nd week of age. There was 25% mortality in T2 alone in 2nd week, apparently from poor-appetite and intense toxin-impact, which were similar to reports of Resanovic and Sinovec (2006). The mortality in T1 occurred only after passage of 2nd week, where the peak-mortality became visible during 5th week of rearing. The T1 had 10% mortality in 3rd week, followed by a spurt in same (30%) during 5th week, after which the mortality rate declined.  Summarily, by 5th week of age, the T2 had suffered nearly double the mortality of T1 (cumulative mortality:  80% versus 40%, Table 2), whereas mortality in both groups came down by 6th week of age (5% in each group), which signified rising tolerance of ducklings to AFB1 intakes to some extent. The control group did not evince any mortality, throughout the 6 weeks of dietary toxicosis.

Table 2: Mortality percentages (Cumulative and [weekly]) of ducklings across experimental groups.

The control group which did not shown any morbidity and had the highest uniformity in live weight, as inferred from coefficient of variation (Table 1). The T2 remained highly morbid, with display of non-specific problems in gait (difficulty in walking), which precipitated into distinct lameness; besides poor growth; wide variation in live weight with signs of flightiness and nervousness (Khlangwis et al., 2011). After 3rd and 4th week, T1 revealed more variability compared to T2, where Poor uniformity remained a hallmark of both AFB1-fed groups (C.V ranging: 21-29%) during morbid period. The control group maintained the best live-body weight across the entire trial period, where differences in live weight between the control and both toxin-groups remained significant (p<0.001) from 3rd to 6th week period (Chen et al., 2014). The liveweight in T1 across all ages vis a vis Control and T2 indicated that morbidity in AFB1-fed groups acted in a dose dependent manner (Murugesan et al., 2015). The sensitivity of ducks to AFB1, at different levels, in the diets have been reviewed by Fouad et al., (2019), where AFB1-levels creating morbidity in ducks ranged from 20 to 1100 ppb. Our study reporting significant morbidity of ducks at 200 ppb AFB1 or above is well supported by the  analogy from earlier authors (Fouad et al., 2019), though ours varied a bit for the threshold of AFB1 @ 300 ppb reported by Fin et al., (2012).
Aflatoxin induced leg deformities
It was observed beyond 3rd week of growth that many surviving Pekins developed lameness, across the hock joints, in both T1 and T2, in varying proportion. Typically, these lame ducks continued to remain live with slower growth, but without much agility, where most deformities were irreversible, despite medical amelioration. Such lameness resulted from feeding of AFB1, could plausibly be attributed to decline in resistance of long-bones (femur and tibiotarsus) to breakage (Maurice et al., 1983) or to changes in cholecalciferol metabolism for hepato and nephrotoxic nature of AFB1 (Huff et al., 1980), which are subject to further investigations.
It can be concluded that, while the AFB1 content of juvenile White Pekin ducks should be kept limited to the recommended safe levels (<10 ppb); exceeding a threshold of 200 ppb is sure to cause poor growth and FCR, with adverse blood biochemical changes, high mortality, morbidity and lameness.
The authors are thankful to the NASF (ICAR), New Delhi and Director, ICAR-CARI for providing funding and facilities, respectively to conduct this study.
The authors declare that they have no conflict of interest.

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