The animal care and use protocol was reviewed and approved by the Purdue University Animal Care and Use Committee. A total of 144 male Pekin ducklings were obtained from a commercial hatchery (Maple Leaf Farms, Leesburg, IN), weighed, tagged, and randomly allotted to 24 cages. All ducklings were housed on raised wire floors in an environmentally controlled room and were fed from 0 to 14 d of age. Feed and water were provided ad libitum. Birds were inspected daily for health problems, and mortality was recorded as it occurred.
The corn-soybean meal based basal diets were formulated to meet or exceed the nutritional requirements of ducks from hatch to 14 days (Table 1). Four dietary treatments were arranged in a 2 × 2 factorial with 2 aflatoxin concentrations (0 and 0.2 mg/kg) and 2 crude protein concentrations (20% and 24%). The cultured aflatoxin material (50 mg/kg) was produced on ground corn using Aspergillus parasiticus (NRRL 2999) as described by Gowda et al. (2008). Four premixes, contributing 0.5% of the final diets by weight, were prepared with or without 80.3% 50 mg AFB1/kg ground corn, and then blended with the basal diet (20% CP or 24% CP) to create the 4 treatment diets, respectively. All diets were formulated to be isocaloric, and the amino acid to crude protein ratios were kept constant for all treatment diets.
Feed intake (FI) and individual BW were measured weekly. Average feed intake and BW gain were calculated and were corrected for mortality. All ducklings were euthanized by intraventricular injection of isopropyl alcohol, and two ducklings from each cage (12 ducklings per treatments) were randomly selected for blood collection. From all birds, the lower 2/3 ileal digesta were collected for dry matter, N, chromium, and energy determination. Breast muscle weight was determined from 3 birds per pen, and relative breast muscle weight was calculated as % of BW. A section of mid-jejunum was collected from 1 bird per pen and stored in 10% neutral buffered formalin for villi histology analysis. In addition, pancreas and small intestine mucosa were collected from 1 bird per pen for subsequent determination of digestive enzyme activities.
Serum Chemistry Analysis
Blood samples were centrifuged at 1,400 × g at 4◦C for 15 min for serum separation. Collected serum was preserved at −20◦C until submitted for biochemical analysis. Six replicate serum samples per treatment were analyzed at the Veterinary Medi-cal Diagnostic Laboratory of University of Missouri (Columbus, MO) for glucose, albumin, globulin, total protein, Ca, P, aspartate amino transferase (AST), γ-glutamyl transferase (GGT), and uric acid using an auto-analyzer (Kodak Ektachem Analyzer, Eastman Kodak Co., Rochester, NY).
Digestive Enzyme Activity
The pancreas and small intestine mucosa samples were immersed in PBS, respectively, to a final concentration of 0.2 g/mL. Subsequently, the mixture was homogenized, centrifuged at 1,400 × g; at 4◦C for 15 min, and the clear supernatant was separated for enzyme activity analysis. Pancreatic enzymes activities were determined using respective commercial kit. Specifically, total protein (Thermo Scientific kit 23200, Rock-ford, IL), amylase activity (Sigma kit MAK009), lipase activity (Sigma kit MAK046), and trypsin activity (BioVision kit K771) were determined according to manufacturer instructions. Mucosal activities of sucrase and maltase were determined as described by Sell et al. (1991). One unit of sucrase activity is defined as the amount of enzyme that cleaves sucrose to generate 1.0 μmol of glucose per mg of protein at 37◦C. One unit of maltase activity is defined as the amount of enzyme that cleaves maltose to generate 1.0 μmol of glucose per mg of protein at 37◦C.
Jejunal Villi Histology
Collected jejunum samples were fixed in 10% neutral buffered formalin, processed by standard paraffin sectioning, and stained with hematoxylin-eosin. The slides were then examined under a light microscope. Measurements of villus height were taken from the top of villus to the valley between villus, and measurements of crypt depth were taken from the valley to the basolateral membrane. The villus height: crypt depth ratios (VH:CD ratio) were calculated. Reported values were means of 10 villi from each bird, 6 birds per treatment.
All diet and ileal samples were analyzed for dry matter (method 934.01; AOAC International, 2006), chromium (method 990.08; AOAC International, 2000), N (method 990.03; AOAC International, 2000) using a Leco model FP 2000 N combustion analyzer (Leco Corp., St. Joseph, MI), and energy using a Parr adia-batic bomb calorimeter (Parr Instruments Co., Moline, IL). The analyzed CP concentrations for the 20% CP-and 24% CP-diets were 20.31 and 24.07%, respectively.
All data were analyzed by 2-way ANOVA using SAS (SAS 9.4, Cary, NC; SAS, 1990) for a completely randomized design. Pen was used as the experimental unit. Model included main effects of aflatoxin concentration, crude protein concentration, and their interaction. Treatment means were compared using the least significant difference procedure when significant (P ≤ 0.05) and trending (0.05 < P ≤ 0.10) treatment effects were observed. All data were tested for normality using the UNIVARIATE procedure and common variance using the GLM procedure.
Growth performance and breast muscle weight results are shown in Table 2. Based on the factorial analysis, 0.2 mg/kg AFB1 showed a significant main effect of reducing cumulative BWG, FI, and breast muscle weight by 33, 35, and 43%, respectively (P < 0.0001). Cumulative G:F ratio was not affected by dietary AFB1 (P = 0.33). Compared to birds fed 20% CP diet, dietary crude protein concentration at 24% significantly improved BWG, G:F, breast muscle weight, and relative breast muscle weight (P ≤ 0.03), with a trend to increase FI (P = 0.094). The improvement in BWG, FI, G:F, and breast muscle weight were 18, 8, 9, and 40%, respectively. There was no statistical interaction between AFB1 and dietary CP concentration for any measures (P ≥ 0.18). However, the numerical reduction of BWG from aflatoxicosis was 39% for birds fed 20% CP, but was only 27% for birds fed 24% CP. Similarly, the reduction of relative breast muscle weight from aflatoxicosis was 34% for the 20% CP group, but was only 5% for the 24% CP group.
The weekly performance followed a similar trend (Table 2). As expected, 0.2 mg/kg AFB1 started to de-press BWG (by 14%) from 0 to 7 d (P = 0.0013), but the negative effect became more substantial during 7 to 14 d (reduction was 48%; P < 0.0001). A similar trend was seen for FI (P = 0.064 and <0.001 for 0 to 7 d and 7 to 14 d, respectively), while G:F ratio was not affected by AFB1 at any time (P ≥ 0.18). Crude protein concentration at 24% significantly increased BWG from 0 to 7 d (P = 0.0013) and 7 to 14 d (P < 0.0001), and improved FI by 14% from 7 to 14 d (P < 0.0001). Gain: feed ratio was significantly increased by the higher CP diet during 0 to 7 d (by 11%; P = 0.002).
The serum biochemistry results are shown in Table 3. Aflatoxin at 0.2 mg/kg reduced serum concentrations of glucose, albumin, total protein, globulin, and calcium (P <0.002), suggesting reduced feed intake and impaired protein synthesis of the ducklings. Higher crude protein concentration did not improve serum con-tent of these 5 measures (P ≥ 0.22). Serum uric acid concentration was significantly increased by higher dietary protein (11.34 vs 7.37 mg/dL for 24% and 20% CP, respectively; P = 0.006), which may be resulted from higher protein intake and subsequent increased amino acid intake and oxidation.
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Digestive Enzyme Activity and Nutrient Digestibility
Results for digestive enzyme activity are shown in Table 4. Activity of mucosal enzymes (sucrase and mal-tase) were not affected by treatment diets (P ≥ 0.33). On the contrary, a significant effect of AFB1 was observed for pancreatic amylase and lipase (P < 0.005), with birds fed AF-contaminated diets showing higher enzyme activity. In addition, birds fed 20% CP during aflatoxicosis showed the highest lipase activity (AFB1 by CP interaction; P = 0.069). Pancreatic protease activity was not affected by treatments (P ≥ 0.22).
Consistent with the unaffected protease activity, the apparent N digestibility was not affected by dietary treatments (P ≥ 0.45) (Table 5). No changes for ap-parent DM digestibility were found between treatments (P ≥ 0.26). On the contrary, 0.2 mg/kg AFB1 showed a significant main effect of decreasing the apparent digestible energy (ADE) (P = 0.0003), which was also reduced by higher dietary CP concentration (P = 0.0003). No AFB1 by CP interaction was detected for any digestibility measures.
Jejunal Villi Histology
The jejunal villi histology results are shown in Table 6. The crypt depth (CD) from birds fed AFB1-contaminated diets were significantly increased compared to control birds (P = 0.0122). While villi height and crypt depth were not statistically affected by dietary protein concentration statistically (P ≥ 0.22), birds fed higher dietary CP showed a significantly increased VH:CD ratio (main effect P = 0.023).
With the high prevalence and continuously increasing risk of AF contamination in feedstuffs worldwide (Streit et al., 2013), better understanding and elucidation of aflatoxicosis is in particular need for ducks, the most susceptible poultry species to AF. Considering the popularity of low-protein diets in the poultry industry in the past few years, and the fact that AFB1 potently inhibit protein synthesis, the question arises as to whether interactive effects between AFB1and dietary CP exist, where lowering dietary protein supply may augment aflatoxicosis and thus lead to further production loss. In the current study, we have explored the impact of dietary protein concentration on the degree of aflatoxicosis in Pekin ducklings by evaluating growth performance, serum biochemistry panel, as well as nutrient digestion and absorption.
Using graded cultured AFB1 from 0.1 to 0.3 mg/kg, our prior study revealed that the 14 d BWG of Pekin ducklings can be reduced by 15% with every 0.1 mg/kg AFB1 increase in the diet. Consistently, in the current study, BWG reduction by 0.2 mg/kg AFB1 was approximately 33% compared to control birds (main effect of AF < 0.0001). This reduction can be attributed primarily to decreased FI (by 35%, main effect <0.0001), while G:F ratio was not affected by AFB1. However, others have documented decreased G:F ratio by 17% in ducks at 42 d when 0.04 mg/kg AFB1 contaminated diet was fed (Han et al., 2008). Shifting from FI inhibition to FCR suppression by AFB1 is possible as the ducks mature. Nevertheless, the observation herein is in agreement with our previous conclusion that inhibited FI is the most profound harm of AFB1 exposure in young ducklings (Chen et al., 2014a). On the other hand, higher CP improved the G:F ratio from 0.72 to 0.80, which may be partially attributed to the increased villi height: crypt depth ratio (P= 0.021); yet unlike the experimental hypothesis, it was not able to completely restore the dramatically inhibited FI in ducks (FI re-duction by AFB1 was 38 and 33% for birds fed 20 and 24% CP diet, respectively; main effect of CP concentration = 0.094). On the contrary, because reduced feed efficiency is a major contributor to growth depression in broiler chicks when exposed to AFB1, the high-CP diet (26%) was able to completely eliminate the BWG reduction by AF by restoring G:F ratio in broilers at 20 d (Chen et al., 2015). This discrepancy in how AF affects performance between broilers and ducks suggest that different counteractive strategies that are species-specific may warrant future consideration, although it is possible that interactive effects may become significant if ducks were fed to market age or more dietary CP concentrations were included.
The serum biochemistry panel results are in agreement with performance. As expected, serum proteins (albumin, globulin, and total protein) were all reduced significantly by AFB1. This is a consistent response that is often observed in AF-related studies in poultry (Zhao et al., 2010; Chen et al., 2014a,b; Chen et al., 2015), indicating that AFB1 impaired liver function and strongly inhibited protein synthesis. However, in contrast to our observation in broilers where serum protein concentrations were completely restored in birds fed higher CP diets (AFB by CP interaction; P < 0.001) (Chen et al., 2015), such amelioration effect by 24% CP diet compared to 20% CP diet on serum protein concentration in ducklings was not observed in the current study. Similarly, serum glucose concentration was decreased by AFB1 by approximately 41%, possibly from reduced FI. However, this reduction was not attenuated by feeding higher dietary CP. Clearly, excess supply of diet CP can minimize the effect of AF on performance by im-proving nutrient utilization only when feed efficiency, rather than feed intake, is the primary factor for impaired growth.
Digestion and absorption in the small intestine are key processes to ensure optimal nutrient utilization and consequently growth performance; these processes are very likely to be affected upon AFB1 exposure. However, there is limited literature addressing this issue as of today, especially in ducks. Han et al. (2008) reported increased digestive enzyme activities upon AFB1 exposure in 42 d Cherry Valley ducks. The authors suggest that increased proenzymes released from the injured pancreas during aflatoxicosis is a plausible reason. Similarly, in the current study, AFB1 significantly increased pancreatic amylase and lipase activity in Pekin ducklings. Another possible explanation is a compensatory effect of the birds in response to lowered FI to meet their nutrient need, but this increase in enzyme activities was not able to restore the growth impairment from AFB1. Pancreatic protease was not affected by dietary treatments, which is consistent with the unaffected apparent ileal N digestibility that was observed. Conversely, others have shown decreased N digestibility during aflatoxicosis both in broilers (Verma et al., 2002; Chen et al., 2015) and in ducks (Han et al., 2008). In the latter study, a very low concentration of AFB1 (0.04 mg/kg) was fed, thus it is likely that the AFB1 contamination was not high enough to drastically sup-press FI, but instead, exerted its effects on the nutrient digestion process. Consistent with the reduced AME in broilers fed 1 to 2 mg/kg AFB1 reported by Verma et al. (2002), a main effect of AFB1 on reducing the apparent ileal digestible energy was observed herein, which did not agree with the DM digestibility which was un-affected. In the meantime, higher CP also reduced ileal digestible energy. It is uncertain what caused this reduction, but there might be a higher endogenous loss with increased FI when birds were fed higher dietary protein. However, apparent digestible energy was increased with increasing dietary CP concentration (from 16% to 26%) in broiler chicks (Chen et al., 2015). Further studies determining the endogenous loss and metabolizable energy in ducks during aflatoxicosis are needed for verification.
Collectively, based on our results, it is clear that AFB1 as low as 0.2 mg/kg can dramatically reduce FI and thus BWG of Pekin ducklings from hatch to 14 d of age. Serum biochemistry and digestible energy were also negatively affected. There were no statistical interactive effects between aflatoxin and dietary CP concentration, but higher dietary CP had main effects of increasing G:F ratio and thus partially restored the BWG of the birds. Nevertheless, reduced FI cannot be restored by higher dietary CP supply, and therefore, feeding higher dietary CP may be a nutritional approach to attenuate aflatoxicosis in broilers, but may be less successful in Pekin ducks. Regardless, feeding a high CP diets is not always economically practical in production; rather, attention should be paid to the different mechanisms of AFs actions in these two species so that specific counteractive strategies can be developed. In ducks, future focus should be on exploring the mechanisms of how aflatoxin inhibit feed intake. In the meantime, strategies are needed to minimize AF exposure before the stage of absorption by the birds, which include regular feed sampling and analysis and use of aflatoxin adsorbents. Future directions regarding broiler chicks may include evaluating the effects of certain amino acids or other nutrient supplements that have a stimulatory effect on protein synthesis, in order to determine if they can become effective and economical approaches for ameliorating aflatoxicosis. On the other hand, in both broilers and ducks, feeding low protein diets was found to exacerbate the negative effects of AF, either significantly or numerically (which is still considerable in practice). Therefore extra caution is needed when feeding low CP diets, especially at times when the weather or storage conditions favor AF production in feed ingredients.
The authors gratefully acknowledge partial funding and research support from Maple Leaf Farms.
This article was originally published in 2016 Poultry Science 95:834–841. http://dx.doi.org/10.3382/ps/pev378. This is an Open Access article under a Creative Commons license.