Among the factors that negatively affect animal production, mycotoxins play an important role because these natural contaminants are ubiquitous contaminants in feeds and feedstuffs [1
]. Besides this, cereal byproducts that are rejected for human consumption during processing for mycotoxin removal, as well as raw materials such as dried distillers’ grains with solubles (DDGS), can enter the animal feed chain, increasing the exposure risk [2
]. In most cases, contamination levels are low enough to ensure compliance with feed safety recommendations. However, mycotoxin contamination might still exert adverse effects on animals, and, at an economic level, the major mycotoxins risks are linked to suboptimal production and not to disease.
There is evidence that even at levels below authorities’ feed safety recommendations, deoxynivalenol (DON) may decrease resistance to infectious disease in broilers [3
]. A previous study showed that broilers fed a diet containing 3000–4000 ppb DON, i.e., at levels below the European maximum guidance (5000 ppb in the complete diet) [4
], are predisposed to develop necrotic enteritis. Also, broilers fed 1500 ppb DON combined with 20,000 ppb fumonisins are more susceptible to coccidiosis [5
]. This poses a concern for the broiler industry, and there is an ongoing discussion about the reduction of antibiotics and the potential impact on the use of anticoccidials, particularly those from the class of ionophores. This class of anticoccidial fits the classical definition of an antibiotic because they have some antibacterial activity. This means that the importance of mycotoxins in the poultry industry may increase in a situation where ionophore anticoccidials are banned from feed. Besides this, the subclinical and indirect effects of mycotoxins are often underestimated because no typical mycotoxicosis symptoms are observed. In general, mycotoxins may be involved in numerous subclinical symptoms, and will potentiate the negative effect of diseases or simply lead to impaired performance.
To mimic on-farm conditions, it is important to expose broilers to feed naturally contaminated with mycotoxins as opposed to experimental contamination with synthetic mycotoxins. Furthermore, realistic contamination levels have to be considered when applying naturally contaminated diets. For instance, it is not common to find feedstuffs highly contaminated with fumonisins. Therefore, the probability of producing a diet with a final concentration between 15,000 ppb and 20,000 ppb (20,000 ppb is the European threshold level in complete feed) [4
] is extremely low. The maximum acceptable level of DON in cereals and cereal products used for feed production is 8000 ppb, while for maize byproduct feed materials, it is 12,000 ppb (EC 576/2006) [4
]. Considering that northwestern European broiler diets contain approximately 30% wheat, it will be difficult to reach 4000 ppb in the final diet. Based on this, we can expect only low to moderate levels of DON in broiler diets, e.g., 1000 ppb to 3000 ppb.
The type of feedstuff added to the diet also interferes with intestinal health and broiler performance. Wheat and rye evoke increased intestinal viscosity in broilers due to high levels of soluble non-starch polysaccharides (NSP), and this results in impaired nutrient digestibility and predisposes to infections [6
]. This effect is usually prevented by the inclusion of enzymes that break down soluble NSP. For this trial, we decided to keep the inclusion level of wheat close to practice, whereas a small amount of rye was included to induce a mild intestinal challenge.
For this study, we exposed broilers during the starter and grower phase to two different levels of DON (900 ppb or 2300 ppb). In the finisher period, they received a diet with a negligible level of DON (57.3 ppb) to evaluate carryover effects. As a positive control, activated charcoal was tested at each DON level. Furthermore, feed was not supplemented with coccidiostats or NSP enzymes, with the aim to evaluate animal performance and intestinal integrity.
In the present study, we demonstrate that even at moderate levels (2300 ppb), DON can impair the performance of broiler chickens. Also, this negative effect is not mitigated when the chickens are subsequently fed a diet with negligible levels (57.3 ppb) of DON for 7 days. Anticipating the potentially increasing impact of mycotoxins when ionophore anticoccidials are banned from feed, we decided not to supplement the experimental diets with anticoccidials. In a previous study, broiler chickens fed diets containing 1600 ppb DON and simultaneously challenged with Eimeria
] had an impaired performance, and one of the factors involved was the ability of DON to modulate the host immune response to coccidial infections [8
]. In the present study, instead of challenging the birds with Eimeria
spp. oocysts, we excluded anticoccidials from the diet formulation. Coccidiosis not only leads to clinical signs but can also result in poor performance [9
]. Another factor that can challenge gut function is an increase in digesta viscosity, because it can decrease nutrient digestibility [7
] and increases the retention time of the diet in the intestine, inducing competition with gut microbiota for digestible nutrients. Subsequently, this increases the risks of infection [10
]. Therefore, in the present study, we used a wheat-based diet without NSP enzymes together with rye at an inclusion level of 5% in the starter (D0–14) and grower (D14–28) diets, thus increasing viscosity.
Although the diets were contaminated with a variety of mycotoxins, the negative impact observed in the present study was basically caused by DON. Besides DON, the other mycotoxins present in the diets were deoxynivalenol-3-glucoside (DON-3-G), enniatins B and B1 (ENNB+B1), zearalenone (ZEN), ochratoxin A (OTA), alternariol (AOH), and alternariol methyl ether (AME). The levels of ZEN, OTA, AOH, and AME were negligible. When DON is conjugated with glucose, resulting in the modified form of DON-3-G, it becomes unable to bind to the ribosome peptidyl transferase center, the main target of intestinal toxicity [11
]. In pigs but not in broiler chickens, DON-3-G can be hydrolyzed to DON [12
]. Therefore, the observed dietary levels between 10.7 ppb and 1670 ppb in the present study should not be a reason for concern. Enniatin B has a low intestinal toxicity in broiler chickens [13
]. Furthermore, it was remarkable that ENNB+B1 levels were higher in MD than in LD diets.
Increasing the DON level from 900 ppb to 2300 ppb in the starter and grower diets resulted in a decreased BWG. This negative effect on BWG was partly counteracted by activated charcoal only in the MD diet. The same pattern was observed with FCR, which was negatively affected in broilers fed the MD diet without activated charcoal. One must bear in mind that the goal of this study was not to promote activated charcoal as a DON adsorbent, since it may also absorb nutrients [14
], but to use it as an extra control [15
]. Instead, it was shown that even when supplementing a diet with a compound that can bind DON, performance losses are not completely avoided. When the diet was replaced by a marginally contaminated feed in the finisher phase (D28–35), no differences in performance were observed in this period. However, the final body weight of the birds fed the MD diet during the starter and grower period was impaired, regardless of the supplementation with activated charcoal. Moreover, considering the complete production period (D0–35), BWG and FCR were impaired in broilers fed the MD diet compared with those fed the LD diet, whereas FI was not affected by the experimental diets. Based on a previous study, DON has a low oral availability (~19%) and a high plasma clearance (~0.12 L/min kg) in broilers [16
]. Therefore, the impaired performance was not a result of mycotoxin accumulation or anorexia caused by DON. Previously, Lucke et al. [17
] did not observe any difference in broiler performance after 28 days of dietary exposure to 5000 ppb DON, and only observed differences after 35 days when BWG was significantly decreased. Besides the dietary differences, in this later study, broiler chickens were fed diets artificially contaminated with DON. Importantly, to achieve homogeneity, this mycotoxin was mixed with inulin at a rate of 0.03% to 0.20% depending on the desired DON dietary level. Inulin is a fructo-oligosaccharide (FOS) able to increase the production of cecal butyric acid, which provides energy for the growth of the intestinal epithelium [18
]. In another study, broiler performance was even improved, and intestinal viscosity decreased when the diet was contaminated with 1500 ppb DON [19
]. The former authors suggested that physicochemical alterations in the wheat, caused by mycotoxins, resulted in better nutrient digestibility and in a lower viscosity in the jejunum and ileum. However, we did not observe a similar decrease in viscosity, which was measured in the duodenum. Instead, we observed an increase when birds were fed the MD diet during the starter phase. The prolonged exposure time to DON did not affect duodenal viscosity at D28 (8–9 cP), although it was still considerably higher than that observed in birds fed a marginally contaminated diet in the finisher period (2.2 cP). Also, the finisher diet contained NSP enzymes, which can explain the reduced viscosity in this phase. In a similar study, Dänicke et al. [19
] fed a control diet resulting in high ileal viscosity (above 25 mPa-s, i.e., 25 cP), which was considerably higher than that observed in our study. Viscosity can be increased by including rye and excluding NSP enzymes [20
] as we did in the present study. An increase in viscosity will negatively influence nutrient digestibility and absorption, decreasing BWG and increasing FCR. Therefore, it is not surprising that this diet, combined with moderate DON contamination, impaired broiler performance. Nonetheless, differences in DON levels and dietary composition do not allow a comparison between the study of Dänicke et al. [19
] and the current study. Also, the wheat batch we used was naturally contaminated with a mixture of mycotoxins and not submitted to induced infection with a specific Fusarium
strain, and nutritional composition may vary among batches. Recently, we showed that broiler chickens fed a corn-based diet (low in NSP) contaminated with ~3500 ppb DON and ~50 ppb of its derivatives 3+15 Ac-DON presented an impaired FCR, indicating that intestinal viscosity is not crucial to observe the negative impact of DON on performance [21
]. Likewise, a longitudinal study also showed that mixtures of mycotoxins below EU recommendation levels impair the performance of broiler chickens [22
Intestinal morphometric changes in the first 14 days of exposure were negligible and limited to an increase in the VH:CD ratio in the ileum of broilers fed the MD diet, regardless of the presence of activated charcoal, indicating that cell proliferation decreased in broilers fed the MD diet without resulting in immediate villus shortening. This was expected, since DON decreases cell proliferation and the crypt is responsible for cell renewing and maintenance of villus length. After 28 days, however, broilers fed the MD diet presented a significantly lower villus height and VH:CD ratio than those fed the LD diet. This leads us to infer that birds fed the MD diet were probably trying to maintain villus height by a compensatory increase in the proliferation in the crypt, which required extra energy. Besides this, shortened villi and deeper crypts will result in suboptimal nutrient absorption and impaired animal performance [23
]. This fits with the observed decreased BWG and increased FCR at D28. Besides, the ileum presented a higher VH:CD ratio due to an increase in crypt depth, showing that in this intestinal section, cell proliferation was increased to keep villus height similar to the expected (LD diet) levels. Such a compensatory mechanism costs energy and will result in suboptimal performance. At D35, no differences were observed, because the finisher diet had negligible levels of DON and intestinal cell turnover takes around 48 h to 96 h [25
]. The number of goblet cells was not affected by the treatments. In a study with pigs, it was shown that DON causes oedema in the lamina propria
and contact loss between lamina propria
and enterocytes [26
]. However, in the present study, the tested DON levels were not able to cause such effects. Based on the morphometric analysis and lesion scores in the jejunum and ileum, it can be confirmed that the jejunum was the intestinal section more sensitive to DON than the ileum, as reported before for broilers submitted to DON exposure [27
] or other sources of stress [28
In conclusion, broiler chickens fed a diet containing moderate levels of DON (2300 ppb) will perform inefficiently. Further, the influence of mycotoxins on poultry performance should be assessed not only on its toxicity per se, but also considering animal age, dietary composition, and the presence of other types of additives, such as NSP enzymes and anticoccidials.