Dietary mycotoxins concentrations were measured by enzyme-linked immunosorbent assay method (kits, Neogen Company, Lansing, MN, USA; Microplate Reader, Model 680, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The detection limits of the assay kits were 2 to 200 μg/kg for AFB₁, 2 to 25 μg/kg for ochratoxin, 0.025 to 0.25 μg/kg for T2 toxins, 1 to 6 μg/kg for fumonisins, 0.025 to 0.25 μg/kg for deoxynivalenol, and 0.02 to 0.5 μg/kg for zearalenone, respectively.

The Animal Welfare Committee of Southwest University of Science and Technology approved the animal care protocol used for this experiment. A total of 704 one-d-old Cherry Valley ducklings with an average initial BW of 55.9±0.2 g were weighed, tagged, and randomly allotted to 44 pens on the basis of sex and body weight. All ducklings were housed in an environmentally controlled room.

This 2-wk trial consisted of 2 treatments with 22 pens per treatment and 16 birds per pen (8 males and 8 females) in a randomized complete block design. Birds were fed from 1 to 14 d of age. All diets (Table 1) were formulated to meet or exceed the NRC [16] requirements for ducks, and the dietary treatments were control (CON) and 100% contaminated corn. Diets were fed in pellet form and feed and water were provided ad libitum.

The ducklings were weighed and feed intake was recorded on d 1, 7, and 14, and average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated. Dead birds were weighed daily, and the mortality was recorded as it occurred.

At the end of the experiment, 8 ducklings (4 males and 4 females) were randomly selected from each pen and blood samples were collected from the jugular vein into a sterile syringe and stored at 4°C. Samples were then centrifuged at 3,000×g for 15 min and serum was separated. After blood collection, the same ducks were sacrificed by cervical dislocation. The lower 2/3 ileal digesta was collected by flushing with reverse osmosis water for nitrogen (N), gross energy (GE), dry matter (DM), ether extract (EE), chromium (Cr), and amino acid (AA) determination. A section of mid jejunum was gently scraped with a glass slide, and mucosa was frozen in liquid nitrogen, and stored at −80°C for relative gene expression of Na⁺, K⁺-ATPase analysis.

All diets and ileal samples were analyzed for DM, N, EE, crude ash, calcium, and phosphorus according to AOAC [17,18]. Cr was analyzed via UV absorption spectrophotometry (Shimadzu UV-1201, Shimadzu, Kyoto, Japan) [18]. Energy was determined by measuring the heat of combustion in the samples using a Parr 6100 oxygen bomb calorimeter (Parr instrument Co., Moline, IL, USA). Amino acid contents were determined, following acid hydrolysis with 6 N HCl at 110°C for 24 h, using an AA analyzer (Biochrom 20, Pharmacia Biotech, Cambridge, England). Before acid hydrolysis, Met and Cys were oxidized with formic acid. Tryptophan was determined after NaOH hydrolysis for 22 h at 110°C.

The serum neuropeptide Y (NPY), ghrelin, leptin, cholecystokinin-8 (CCK-8), insulin, insulin-like growth factors-1 (IGF-1) levels were analyzed by radioimmunoassay method using kits (Shanghai Yuanye Biotechnology Co. Ltd., shanghai, China) according to the manufacturer’s instructions. Automatic radioimmunoassay γ counter (H-7500, Hitachi, Tokyo, Japan) was used for determination.

Total RNA extraction, reverse transcription reaction and quantitative real-time polymerase chain reaction (PCR) of Na⁺, K⁺-ATPase gene was determined as previously described [19]. Briefly, total RNA of tissues was extracted with RNAiso Reagent (TaKaRa, Kyoto, Japan) and reverse-transcribed with RT Reagents (TaKaRa, Japan) according to manufacturer’s instructions. Quantitative real-time PCR was performed using 96-well iCycler iQTM Real-Time PCR Detection System (Bio-Rad, USA). The gene-specific primers used are listed in Table 2 and purchased from TaKaRa (Japan). The PCR system consisted of 12.5 mL SYBR Green PCR Master Mix (TaKaRa, Japan), 2.0 mL of cDNA, 8.5 mL of PCR-grade water and 2.0 mL of primer pairs (100 mM forward and 100 mM reverse) for a total volume of 25 mL. All samples were assayed in triplicate. Cycling conditions were as follows: 94°C for 10 s, and 40 cycles involving a combination of 94°C for 5 s, 55.5°C for 20 s and 72°C for 15 s. Relative gene expression to the house-keeping gene (β-actin) was performed in order to correct for the variance in amounts of RNA input in the reactions. However, the relative gene expressions compared to the house-keeping gene were calculated [20]. The primer sequences of gene for Na⁺, K⁺-ATPase are presented in Table 2.

Data were analyzed by analysis of variance using the T-test procedure of SAS (SAS Inst. Inc., Cary, NC, USA) with the pen being the experimental unit. Variability in the data was expressed as the standard error of means. Probability values less than 0.05 were considered significant.

Concentrations of various mycotoxins, as well as their regulatory guidance concentration, in corn and diets are presented in Table 3. The dietary AFB₁ was 2.87 and 124.35 μg/kg for CON and AFB₁, respectively. Only AFB₁ exceeded the regulatory guidance concentration of Chinese National Standard (GB 13078-2001), while others did not exceed the regulatory limits of Chinese National Standard (GB 13078.2-2006, GB 13078.3-2007, and GB 21693-2008) and European Commission [21].

The GE and nutrient composition between normal corn and contaminated corn are presented in Table 4, which showed few differences.

From d 1 to 7, feeding AFB₁ diets decreased d 7 BW, ADG, and ADFI by 22%, 31%, and 23%, respectively (p<0.05), while FCR increased by 11% (p<0.05) (Table 5). From d 8 to 14, birds fed AFB₁ diets had lower ADG, ADFI, and FCR (p<0.05) with the reduction of 41%, 47%, and 10%, respectively. In the whole trial, the AFB₁ reduced final BW, ADG, ADFI, and FCR by 34%, 39%, 43%, and 8% (p<0.05). Mortality was increased by AFB₁ diets (p<0.01) from d 8 to 14 and d 1 to 14.

Apparent ileal digestibility of DM, N, and AA was increased (p< 0.05) by AFB₁ diets, whereas apparent ileal digestible energy digestibility was decreased (p<0.05) (Table 6). No difference was observed in apparent ileal EE digestibility between treatments (p>0.05).

Serum levels of leptin and IGF-1 was increased by AFB₁ diets (p< 0.05), while NPY, ghrelin, CCK-8 and insulin were not affected (p>0.05) (Table 7). There was no difference (p>0.05) in relative expression of Na⁺, K⁺-ATPase between treatments (Table 8).

The detrimental effects of AFB₁ on growth performance may be due to anorexia, listlessness, impaired liver function, and inhibition of protein synthesis and lipogenesis [22,23]. As expected, the ADG and ADFI was reduced by AFB₁ (124.35 μg/kg) in the current study, which was in agreement with a study that reported that the growth and feed intake decreased by naturally contaminated AFB₁ diets (120.02 μg/kg) during d 0 to 14 in Cherry valley ducklings [10]. Similar findings in ducks were observed by other studies [6–8] with purified AFB₁ (from 40 to 200 μg/kg). Consistently, a recent study indicated that purified AFB₁ (200 μg/kg) decreased the 14 d body weight gain and ADFI of Pekin ducklings by approximately 33% [11]. Interestingly, FCR was reduced by 10% and 8% during d 8 to 14 and 1 to 14, while increased by 11% during d 1 to 7 in the study herein. In contrast, others observed that FCR was improved by purified AFB₁ (40 to 200 μg/kg) in ducks [6–8,24]. However, FCR was not affected by semi-purified AFB₁ (200 μg/kg) in Pekin ducklings [11] and naturally contaminated AFB₁ (from 120.02 to 128.7 μg/kg) in Cherry valley ducklings [9,10] from d 0 to 14. This inconsistency in FCR was also observed in broilers. FCR was decreased in broilers fed naturally contaminated AFB₁ diets (44.5 μg/kg) [25], whereas FCR was improved by purified AFB₁ (1,500 μg/kg) [26]. This inconsistence may be due to AFB₁ origins (corn naturally contaminated or inoculated with purified mycotoxins), dosage and species-specificity. The lack of AFB₁ effect on FCR was attributed to the ducks maturity [11]. The decrease in the rate of excreta to pass through the digestive tract in ducks may lead to the reduction in FCR [10], which was supported by some studies that reported the fusarium toxin decreased the excreta emptying rate in growing pigs and broilers, respectively [27,28]. Therefore, the reduction in ADG may be due to the decreased ADFI, whereas the reduced ADFI may be attributed to the decrease in excreta passage rate, which was supported by the decreased FCR in this study. All the deaths caused by AFB₁ happened in the second week because of the chronic and accumulated mycotoxicosis [9]. Nevertheless, more research is needed to evaluate the influence of naturally contaminated AFB₁ diets on poultry because some reaction in enzyme activities and cell wall degradation may take place.

Several studies have revealed the AFB₁ effect on the gastro-intestinal tract [10,11]. It was reported the biotransformation of AFB₁ to the toxic AFBO was also occurred in the intestinal tract [12]. Therefore, the digestion and absorption in gastro-intestinal tract may be affected by AFB₁. However, there was limited literature about the nutrient digestibility in ducks. Reduced apparent ileal digestible energy (ADE) in birds fed AFB₁ diets was observed in this study, which was in agreement with several studies in Pekin ducklings (200 μg/kg) from d 0 to 14 [11] and in broilers (2,000 μg/kg), respectively [29]. Purified AFB₁ (200 μg/kg) did not affect apparent ileal DM and N digestibility in Pekin ducklings [11]. Reduced N digestibility upon AFB₁ exposure (200 μg/kg) in Cherry Valley ducks was observed [8]. On the contrary, apparent ileal DM, N, and AA digestibility was improved by AFB₁ in the herein study. A study indicated that improved proenzymes were released from the injured pancreas in response to AFB₁ (200 μg/kg) in Cherry Valley ducks [8]. Meanwhile, improved pancreatic amylase and lipase activity was observed in Pekin ducklings given purified AFB₁ (200 μg/kg) [11]. The authors also proposed that the compensatory effect of the birds in response to decreased ADFI to meet their nutrient need might be a possible reason, yet the improved enzyme activities was not enough to restore the damage to growth performance from AFB₁. Notwithstanding this, the reason for the increased apparent ileal DM, N, and AA digestibility requires further research to determine whether it was due to the improved enzymes or other factors.

Because of the dramatic reduction in ADFI by AFB₁, it was hypothesized that the adverse effects of AFB₁ on ducks were correlated to nutrient absorption transporter and several hormones involved in ingestion and digestion. Therefore, this was an important issue in this study. Liver is the primary target organ of AFB₁, which was demonstrated by other researchers [9,30] who observed hepatic physical change and impaired hepatic function. Leptin (a satiety hormone), is mainly produced in the adipocytes of white adipose tissue, and can regulate fat stores through depressed appetite and increased energy consumption [31]. Furthermore, fatty liver can be caused by AFB₁ [32]. Leptin was increased by AFB₁ in this study. Accordingly, it was supposed that leptin showed a compensatory increase to inhibit adipose synthesis and promote adipose lipolysis caused by fatty liver in response to AFB₁. Although IGF-1, produced mainly in the liver, was a primary mediator of the effects of growth hormone and had growth-promoting effects, it is an important cytokine of the liver inflammation and fibrosis. Dietary AFB₁ had no effect on ghrelin, NPY and CCK-8 in the current study. Ghrelin (hunger hormone) and NPY were opposed by the action of leptin, which can increase appetite. CCK-8, synthesized and released by enteroendocrine cells in the mucosal lining of the small intestine (mostly in the duodenum and jejunum), suppressed hunger and feed intake through reduced rate of gastric emptying [33].

The Na⁺, K⁺-ATPase is a solute pump that pumps sodium out of cells while pumping potassium into cells, both against their concentration gradients. The Na⁺, K⁺-ATPase helps maintain resting potential, effects transport, and regulates cellular volume [34]. Particularly, it is used to transport most nutrients in the intestinal tract, which can reflect the absorption of nutrients [35]. The AFB₁ may affect the tight junction proteins, which were the major constituent of gut barrier for the latter function, thus any damage to these proteins’ synthesis and activities may result in an increase of permeability of the selective gut barrier [11,12]. The relative expression of jejunum Na⁺, K⁺-ATPase gene was numerically increased by 22% in ducklings fed AFB₁ diets in the herein study. Although the AFB₁ effect on the relative expression of jejunum Na⁺, K⁺-ATPase gene was not significant, the large increase in number may partially mirror the gut permeability to some degree. Previous studies indicated that increased gut permeability may also facilitate the absorption of any presented mycotoxins [11,12].

It was hypothesized that the mechanism for the depressed feed intake by AFB₁ may be as follows: decreased ADFI may be due to the improved leptin, which inhibited appetite and adipose synthesis caused by fatty liver. In addition, the increase in nutrient digestibility and feed efficiency may be due to the reduced rate of gastric emptying and improved enzyme activities.