Evaluation of a Broad-Spectrum Mycotoxin Binder Developed Using the Isothermal Method on Growth Performance and Liver Health in Broilers Fed Multi-Contaminated Feed

Artun Reman Temiz, Pınar Saçaklı, Nuran Yüce, Özcan Erdoğan

Abstract

This study was conducted to determine the effects of a newly developed broad-spectrum mycotoxin binder on performance parameters and the liver in broilers consuming multi-contaminated feeds. A clay mineral with high, broad-spectrum mycotoxin-binding capacity was identified by the in vitro isothermal method and by the EURL verification test. The most effective combination of the selected clay mineral with yeast cell wall rich in mannan and β-glucan was determined, and a plant extract was added to impart hepatoprotective activity to the developed binder. Using an isothermal method simulating the avian digestive tract, the binder’s efficacy (spectrum of activity, binding level, binding rate, and interaction with nutrients) was defined and then tested in an in vivo animal trial. A total of 240 day-old ROSS 308 male chicks were randomly allocated to 6 experimental groups with 8 replicates of 5 birds per pen. The Negative Control (T-1) feed contained neither toxin nor binder; the T-2 group received only ToxFinder Defence (1 kg/t). Positive Control diets (T-3) were contaminated with 0.3 ppm aflatoxin B1 + 0.3 ppm ochratoxin A + 0.3 ppm T2 toxin + 0.3 ppm ochratoxin A and 0.3 ppm zearalenone. Other contaminated-feed groups (T-4, T-5, T-6) received, respectively, ToxFinder Defence (Kartal Kimya A.Ş.) and two different commercial binders (Commercial A and Commercial B) at 1 kg/t. Results showed that the adverse effects of mycotoxins on performance indices can be mitigated by using toxin binders in feed. The newly formulated binder exhibited high, mycotoxin-specific and broad-spectrum binding efficacy and showed a hepatoprotective effect. This work was supported by TÜBİTAK under the Industrial R&D Projects Support Programme, Project No. 3192009.

Keywords: mycotoxin, multi-contamination, toxin binder, broiler, performance

Introduction

Mycotoxins are secondary metabolites produced by moulds during early harvesting and during the processing, storage or transport of feeds under adverse conditions, further exacerbated by global climate change. With carcinogenic, mutagenic, teratogenic, nephrotoxic, immunosuppressive, neurotoxic and oestrogenic effects, mycotoxins negatively impact animal health and performance (1, 2). Fungi commonly found in animal feeds and their typical mycotoxins include Aspergillus (aflatoxins, AFs; ochratoxin A, OTA), Penicillium (OTA) and Fusarium (trichothecenes, fumonisins, FBs; and zearalenone, ZEA) (3). Among preventive strategies against mycotoxins in feed, the use of binders is the most effective and economical. Clay minerals in binders are mostly effective against AFs, whereas yeast cell walls (YCW) are more often effective against ZEA, OTA, FBs and DON (4). Some non-selective adsorbents such as activated charcoal may also adsorb nutrients, potentially reducing the bioavailability of minerals, trace elements and some vitamins (5). YCW can bind various mycotoxins via different mechanisms and, through MOS and β-glucans, support gut function and immunity (4, 6). To help preserve liver function, the organ responsible for homeostasis and detoxification, binder efficacy may be supported with botanical extracts. Among the most preferred due to their high hepatoprotective and antioxidant effects are Silybum marianum and Ginkgo biloba (7, 8). Phenolic plant compounds participate in many stages of oxidative reactions, scavenge free radicals and, by increasing reduced glutathione, support organ physiology, notably the liver (9). The effects of binders combining clay, yeast cell wall (YCW), and phenolic compounds on broiler performance and liver health are of current practical interest. This study investigated the effects on growth performance and liver health in broilers fed multi-contaminated feed when using a broad-spectrum mycotoxin binder that, according to in vitro results obtained by the isothermal method, showed high activity against various mycotoxins.

Materials and Methods

To select the clay mineral for the formulation, four different clays from various regions of Türkiye (activated charcoal, zeolite, bentonite and clinoptilolite) were collected. Their AFB1-binding levels at pH 3 and pH 7 were measured by LC-MS/MS using an isothermal method adapted from Joannis-Cassan et al. (10) simulating the avian digestive system. According to the EU Reference Laboratory (EURL) method (EC 1060/2013), AFB1-binding efficacy was evaluated. Other components of the binder, including yeast cell wall (YCW) and botanical extracts, as well as different combinations of clay and YCW at varying inclusion levels, were evaluated using the isothermal method to determine binding efficiency. The formulation showing the highest binding activity of clay and YCW was further supplemented with hepatoprotective botanical extracts to produce the final binder used in the animal trial. For this most active, broad-spectrum formulation, the spectrum of action, binding rate, and binding stability (desorption test) were assessed (11). Possible interactions with selected nutrients (lysine, methionine, threonine, calcium, and phosphorus) were also examined (5).

Guided by the in vitro data, 240-day-old Ross 308 male broilers were used. Diets based on maize and soybean meal were formulated to Aviagen specifications (12) (3,100 kcal ME/kg; 22% crude protein). After a 3-day adaptation, birds were fed ad libitum to day 21 with contaminated feed (0.3 ppm AFB1 + 0.3 ppm T2 toxin + 0.3 ppm OTA + 0.3 ppm ZEA). Six treatments were arranged with 8 replicates of 5 birds each. Standard commercial husbandry was applied. Body and feed were weighed at the start and day 21; body weight (BW), BW gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were calculated. On day 21, two birds per replicate (n = 96) were slaughtered. One liver per replicate was scored macroscopically for haemorrhage, steatosis and contour. Performance data were analysed by one-way ANOVA in SPSS 22.0.

Results and Discussion

Numerous clays (zeolite, bentonite, clinoptilolite, activated charcoal) are used as feed additives due to adsorptive properties, but efficacy varies by shape, size, porosity, ion charge, polarity, composition and inclusion rate, as well as by source and seam (1). In vitro AFB1-binding supported this variability. Among the four clays tested, AFB1 binding (high to low) was: at pH 3 (Figure 1) activated charcoal > zeolite > bentonite > clinoptilolite; and at pH 7 (Figure 2) activated charcoal > bentonite > zeolite > clinoptilolite. Although activated charcoal binds multiple mycotoxins (AFB1, ZEA, OTA, DON, FB1), its non-specific adsorption limits practical use (13).

Figure 1. AFB1 adsorption (%) of clays at pH 3

Figure 2. AFB1 adsorption (%) of clays at pH 7

The EURL test assesses the acceptability of feed additives as “binders” by AFB1-binding capacity under defined conditions. A binder should show ≥90% efficacy in a buffer at pH 5.0 at 0.02% inclusion with 4 mg/L AFB1. Under these criteria, bentonite and activated charcoal were acceptable (Figure 3).

Figure 3. AFB1-binding levels (%) of clays by the EURL method

Because binding can be affected by diet composition, ions and molecules (including proteins, enzymes and vitamins) and pH, clays may interact with amino acids, vitamins and minerals, impairing absorption and performance (14). Bentonite was therefore selected for the binder. Alongside clays, the organic adsorbent YCW is effective in multi-contaminated feeds, notably against OTA, ZEA and FBs (4). In vitro, combining bentonite with YCW broadened the spectrum; the most effective result was with Formula F4 (Figure 4).

Figure 4. Mycotoxin-binding (%) of clay, YCW and their combinations

Mycotoxins induce oxidative stress, apoptosis, inflammation and acute/chronic hepatic lesions, in addition to carcinogenic, teratogenic and endocrine effects. Silymarin, a flavonolignan- and flavonoid-rich extract of milk thistle (Silybum marianum), protects hepatocytes and also scavenges free radicals and modulates immune responses (15). Thus, a sufficient level of active botanical extract was added for hepatoprotection. When all components were combined, total binding increased, indicating synergy (Figure 5). The developed binder showed higher binding by the isothermal method than other commercial binders.

Figure 5. Mycotoxin-binding (%) by isothermal method: ToxFinder Defence and components vs. other binders

The formulated binder bound some mycotoxins more strongly at low pH. Because some binders may release bound toxins as pH changes, risking acute toxicosis or sudden mortality (12, 16), desorption (stability) was tested: AFB1 bound at pH 3 was not released at pH 7, showing high stability. The binder did not adversely interact with lysine, methionine, threonine, Ca or P at the same inclusion level.

An effective binder should rapidly adsorb toxins and retain them through the gut to prevent systemic absorption. ToxFinder Defence showed high activity within one minute at different pH values (Figure 6), and activity did not decline over time (except for T-2 toxin).

Figure 6. Time-dependent mycotoxin binding (%) of ToxFinder Defence

Mycotoxins detrimentally affect immunity, gut health, nutrient absorption and performance. In this study, the contaminated-feed (PK) group showed worsened BW, BWG, FI and FCR vs. Negative Control (NK) (P < 0.05) (Table 1).

Even low-level contamination can impair broiler performance (17). Here, performance impairment by mycotoxins was confirmed; however, inclusion of a binder mitigated these effects (P < 0.05). Among contaminated groups, FCR was numerically improved with ToxFinder Defence.

Table 1. Performance parameters by treatment (g; means; SEM; P)

Protective effects on the liver were more evident. Macroscopic assessment recorded steatosis, haemorrhage and contour. Only in the contaminated-only (PK) group were severe (25%) and moderate (12.5%) lesions detected (Table 2). No moderate or severe lesions occurred in the PK + ToxFinder Defence group, suggesting liver protection via the botanical components.

Table 2. Liver lesion scores by treatment (n and %): Normal / Mild / Moderate / Severe

Conclusion

Using the isothermal method in combination with LC-MS/MS supports the development of sustainable and comprehensive mycotoxin analysis in the feed and feed additive sector. Guided by in vitro results, a specific broad-spectrum, high-capacity mycotoxin binder was formulated from a Türkiye-sourced clay mineral and a yeast cell wall (YCW) fraction rich in mannan and β-glucan, and further enriched with flavonoid-rich botanical extracts possessing free radical–inhibiting activity, conferring multifunctionality. At 1 kg/t, ToxFinder Defence mitigated performance losses and liver lesions caused by mycotoxins. Considering both in vitro and in vivo results, ToxFinder Defence appears to be an effective solution to mycotoxin risk, supporting animal health and production.

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