Aflatoxin B₁ (AFB₁) is a highly toxic mycotoxin, primarily produced by Aspergillus flavus and Aspergillus parasiticus, and it poses a significant concern in poultry farming, especially for turkey poults (1, 2). Thus, extensive research has been conducted to investigate the harmful effects of AFB₁ in animals, including their carcinogenic, mutagenic, teratogenic, and growth-inhibitory properties (3, 4). Contamination with aflatoxins leads to reduced feed quality and animal performance due to poor nutrient conversion and various issues, such as reproductive abnormalities (5). Aflatoxicosis in poultry manifests as listlessness, decreased appetite, reduced growth rate, inefficient feed utilization, decreased egg production, and increased mortality. Additionally, aflatoxin contamination in feed can impair both humoral and cellular immune responses, rendering birds more susceptible to environmental and infectious agents (4).

Previous studies have extensively reported the presence of potential aflatoxin-producing strains and AFB₁ molecules in commercial poultry feeds. As a result, there is an urgent need for practical and cost-effective methods to decontaminate aflatoxin-contaminated feed on a large scale (6). Several physical, chemical, and biological techniques have been employed to decontaminate agricultural commodities with good results, but their implementation has been limited by uncertainty about their in vivo effectiveness (7). One practical approach is the utilization of non-nutritive adsorbent materials, which can effectively bind mycotoxins and inhibit their absorption in the gastrointestinal tract (8), thereby reducing their toxic impact on poultry and minimizing the carryover of these fungal metabolites into poultry products such as meat-end eggs (9). However, not all adsorbents exhibit significant effectiveness, and some of them have been found to hinder nutrient utilization. An optimal adsorbent material should exhibit a high affinity for aflatoxins, facilitating the formation of stable complexes with minimal risk of dissociation. Furthermore, it should possess a substantial binding capacity to prevent saturation (10).

Humic substances (HS) are widely distributed and can be found in areas undergoing decomposition or sedimentation, such as soils (11). Within the HS, three distinct components are distinguished based on their solubility: fulvic acids (FA), humic acids (HA), and humins (12). These substances have demonstrated a strong ability to bind various compounds, including heavy metals, herbicides, mutagens, aromatic compounds, minerals, and bacteria (13). Recent studies have shown that incorporating HS into poultry diets promotes growth by enhancing energy digestibility and nutrient retention (14, 15). Although the specific mechanisms behind this growth promotion are not fully understood, the inclusion of HA in the chicken diet leads to elevated intestinal viscosity and improved intestinal integrity (16, 17). In a recent study, HA derived from vermicompost demonstrated remarkable efficacy in adsorbing AFB₁ in an in vitro model that simulates the digestive tract of birds (18). Therefore, this study aimed to evaluate the effects of HA on performance parameters, relative organ weight, liver histological lesions, and serum biochemical and hematological analyses in AFB₁-exposed turkey poults.

Table 2 summarizes differences in BW, BWG, FI, and FCR in turkeys consuming a corn-soybean-based diet contaminated with AFB₁ (250 ng AFB₁/g) added with HA. The differences in BW started decreasing compared to the positive control group (221.92 g) and both HA treatments (249.49 and 241.09 g for HA and HA+AFB₁, respectively) at day 14 (P < 0.0001). At day 21, the BW of both HA treatments (428.35 and 390.10 g for HA and HA+AFB₁, respectively) was higher than the AFB₁ treatments (348.78 g) (P < 0.0001). Similarly, at day 28, the BW of both HA treatments (637.47 and 571.95 g for HA and HA+AFB₁, respectively) was higher than the AFB₁ treatments (428.14 g) (P < 0.0001). Differences in BWG were observed from the second week (7–14 days). The BWG of both HA treatments was higher than the AFB₁ treatments (P < 0.0001). Furthermore, the BWG of HA treatment was 178.85 g in the third week (14–21 d) (P < 0.0001) and 209.12 g in the fourth week (21–28 days) (P < 0.0001), followed by HA+AFB₁ treatment (149.01 and 181.85 g, respectively) compared to positive control treatment (126.86 and 85.14 g, respectively). When comparing from 0–28 days, the BWG of both HA treatments remained significantly higher (581.13 and 515.25 g for HA and HA+AFB₁, respectively) compared to the positive control treatment (371.76 g) (P < 0.0001). Furthermore, during the last week (21–28 days), the FI was significantly higher in both HA treatments (288.00 and 266.80 g for HA and HA+AFB₁, respectively) when compared to the positive control treatment (141.75 g) (P < 0.0001). With respect to the FI from 0–28 days, it was significantly higher in both HA treatments (764.04 and 706.76 g for HA and HA+AFB₁, respectively) compared to the positive control treatment (141.75 g) (P < 0.0001). Moreover, the FCR was consistently higher in the positive control group than in all the other treatments throughout the entire duration of the experiment. Finally, the FCR from 0–28 days was significantly higher in the negative control (1.56), positive control (1.52), and ZEO+AFB₁ (1.51) treatments compared to both HA treatments (1.31 and 1.37 for HA and HA+AFB₁, respectively) (P < 0.0001), denoting a better FC in the treatments added with HA.

Table 3 shows the relative weight of the liver, spleen, bursa of Fabricius, and intestine in poults consuming diets contaminated with AFB₁ and HA. The relative weight of the liver was lower (P < 0.0001) in the positive control group (3.72 g) compared to the negative control group (2.89 g) and the HA (3.35 g) poults, while it was similar to the zeolite+AFB₁ group (2.98 g) and higher than the HA+AFB₁ (2.56) poults. The AFB₁ inclusion in the positive control (0.15 g) caused an increase in the relative weight of the spleen (P < 0.006) compared to the negative control (0.12), and it showed similar values compared to the rest of the treatments. The negative control (0.17 g) poults showed a similar relative weight of the bursa of Fabricius compared to the positive control (0.19 g) and the zeolite+AFB₁ (0.20 g) treatments, while it was lower compared to the HA (0.22 g) and HA+AFB₁ (0.22 g) poults. The relative weight of the intestine of the positive control (8.25 g) was similar to that of the negative control (8.66 g) and the zeolite+AFB₁ (8.07 g) treatment, which in turn were higher (P < 0.0001) than that of the HA (6.96 g) and HA+AFB₁ (6.42 g) treatments. No significant differences were observed in the bursa/spleen ratio among treatments.

Table 4 presents the blood cell counts of poults consuming diets contaminated with AFB₁ and HA. No significant differences were observed in the white cells, hematocrit, heterophils, lymphocytes, basophils, or heterophils/lymphocyte ratio. The monocyte counts in the negative control (1,500/μL), positive control (2,423/μL), and HA+AFB₁ (2,208/μL) poults were similar, and the counts were higher (P < 0.0001) between the HA (6,127/μL) and zeolite+AFB₁ (5,648/μL) treatments.

Table 5 presents the serum biochemical parameters in poults consuming diets contaminated with AFB₁ supplemented with HA. The inclusion of AFB₁ the positive control induced a significant decrease in serum levels of total protein (P < 0.0001), AST (P < 0.001), GLDH (P < 0.02), creatinine (P < 0.0001), BUN (P < 0.0009), glucose (P < 0.002), Ca (P < 0.0001), and P (P < 0.0002) compared to the negative control treatment. In poults receiving the diet with added HA, there was a partial restoration of total protein, creatinine, and Ca compared to the positive control, and there was a lower concentration of ALT, ALP, AST, BUN, glucose, P, and uric acid, but higher Na, Cl, and CO₂ concentrations compared to the negative control. All the biochemical parameters were similar between HA+AFB₁ and the positive control poults, with the exception of CO₂, which was higher in the former group. In zeolite+AFB₁ poults, the total protein, ALT, creatinine, and BUN were higher, and Ca concentrations were lower than the positive control.

Table 6 summarizes the hepatocellular degeneration responses in poults consuming diets contaminated with AFB₁ and supplemented with HA. The positive control treatment, combined with AFB₁, showed significant increases (P < 0.05) in cellular degeneration and fibrosis compared to the negative control. In HA poults, lower (P < 0.05) vessel congestion was found compared to the positive and negative control groups, and lower bile duct hyperplasia was observed compared to the positive control group. The findings were corroborated by histopathology studies, which showed that the liver of turkeys from the AFB₁ group had substantial cellular degeneration and fat deposition (Figures 1A–E). In HA+AFB₁ poults, the cell degeneration was higher compared to the negative control, while the rest of the variable responses were similar compared to the positive and negative controls and the HA poults.

Regarding the zeolite+AFB₁-fed poults, the cell degeneration was higher than the negative control. Meanwhile, vacuolar degeneration, congestion vessels, lymphocytic infiltration, and bile duct hyperplasia were similar to positive, negative, and HA poults. Conversely, fibrosis exhibited the lowest occurrence compared to all the other treatments.

The exposure of turkeys to AFB₁ negatively impacts productivity, causing significant economic losses and health problems in the poultry industry because of its toxicity (24). In an earlier study, a significant decrease in BWG and FCR was observed in poults fed diets with quantities equal to or higher than 400 ppb from 2 to 7 weeks of age (25). In poults fed naturally contaminated whole corn kernels with AFB₁ from 1 to 21 days of age, reduced BWG was observed (26). Reduced FI and BW were also seen in poults from 1 to 21 days of age fed diets added with 100 ppb of aflatoxins and from 1 to 42 days of age when diets contained 200 ppb of aflatoxins (27). In poults fed 100 ppb aflatoxin, reduced BWG was observed, and at higher inclusions of aflatoxins, reduced FI and higher mortalities were found (28). Other recent publications have reported reduced BWG and increased FCR in poult-fed aflatoxin-contaminated feeds (29, 30). The reduced BW and BWG and increased FCR in poults fed the positive control diet in the present study agree with previous findings, which show that the growth performance of poults is negatively affected by the consumption of AFB₁.

Conversely, poults fed with HA showed increased BW, BWG, and FI, and reduced FCR, which is consistent with previous findings on broiler chickens supplemented with HS derived from worm composts (15). It is worth noting that HA-fed poults outperformed the negative control ones in BW, BWG, FI, and FCR by 31.8, 36.2, 14.3, and 16.0%, respectively. Interestingly, HA+AFB₁-fed poults also improved the BW, BWG, FI, and FCR by 18.3, 20.7, 5.7, and 12.2%, respectively, compared to those of the negative control group. These results indicate that HA-fed turkeys not only restored the productive parameters that were adversely affected by AFB₁ in the positive control group but also improved the productivity of the negative control group. To the best of our knowledge, this is the first study where HA supplementation is reported to improve the productivity of unchallenged and AFB₁-challenged poults.

HS has demonstrated a strong ability to bind various compounds, including heavy metals, herbicides, mutagens, aromatic compounds, minerals, and bacteria (13). In a recent study, HA derived from vermicompost was characterized and found to be highly effective in adsorbing AFB₁ in an in vitro model simulating a chicken's digestive tract (18). Due to its highly hydrophobic surfaces and a wide range of negatively charged functional groups (31), interactions between HA and AFB₁ can occur through various mechanisms. Tan (9) has identified seven potential binding mechanisms for HA with gaseous, liquid, and solid components, which include (i) physical forces, (ii) chemical forces, (iii) hydrogen bonds, (iv) hydrophobic interactions, (v) electrostatic interactions, (vi) coordination reactions, and (vii) ligand exchange. Among these mechanisms, electrostatic interactions and hydrogen bonding play crucial roles in the HA-AFB1 interaction.

Additionally, other interactions, such as π- π stacking and hydrophobic interactions, can also be considered. Although this phenomenon is still being studied, it seems that aromatic structures and functional groups such as OH and COOH are also involved. Further details about the hypothetical mechanism by which HA binds the AFB₁ molecule are found in a previous study by our research group (18).

Another important result was that zeolite+AFB₁ poults had BW, BWG, and FI similar to those in the positive control but lower than the negative control group. The FCR was similar among the positive control, negative control, and zeolite+AFB₁-fed poults. These results indicate that the protective effect of zeolite against AFB₁ was overlooked in the present study. Zeolite is known to bind AFB₁, preventing its absorption into the body, and has been shown to be partially protective against high levels of AFB₁ (32, 33). In the literature, no studies looking at the protective effects of zeolite against AFB₁ on the performance of turkey poults were found. Nonetheless, it was recently demonstrated that supplementation with zeolite in the diets of female and male turkey poults had a positive effect on growth performance compared to the control group (34). This topic deserves further investigation.

In terms of relative liver weight, the negative control treatment had the highest, followed by the treatments with HA, HA+AFB₁, zeolite+AFB₁, and the positive control. Similar to these findings, in poults fed AFB₁-contaminated corn, a reduction in liver weight was reported, while in poults fed uncontaminated corn, increased liver weight was observed (26). Reduced liver weight in poults fed aflatoxin-contaminated feeds was also reported compared to a control group fed non-contaminated feed (30). Rauber et al. (27) conducted a study on poults with different aflatoxin levels (20–1,000 ppb). The liver weight of control poults and those fed diets with aflatoxin levels between 20 and 500 ppb showed no significant difference, with the exception of the group fed 50 ppb, which displayed a lower liver weight. However, poults that were fed diets containing 1,000 ppb of aflatoxins demonstrated an increase in liver weight (27). Furthermore, enlargement of the liver was reported in poults fed AFB₁-contaminated feeds (35). These discrepancies regarding the effect of AFB₁ feeding on the liver of poults need further research.

A larger spleen was reported in poults fed aflatoxin-contaminated corn (26) and rations (35), while spleens of similar weight between poults fed aflatoxin-contaminated and non-contaminated feeds were reported (30). In this research, the larger spleen was found in zeolite+AFB₁ and positive control poults and the smallest in the negative control group. These results agree with research in which larger spleens were seen in poults fed aflatoxin-contaminated feeds. Concerning the bursa of Fabricius, both HA treatments demonstrated higher weights, and the positive control showed lower weights. Different from these findings, significant atrophy and degenerative changes were observed in the bursa in a study involving 7-day-old female turkey poults challenged with 0.2 mg AFB₁/kg; the changes included degeneration, necrosis, and depletion of lymphoid cells (36). The higher weight of the bursa of Fabricius in poults fed aflatoxin-contaminated corn was also reported (26), while in other reports, a lower bursa weight was recorded in poults fed aflatoxin-contaminated feed (30, 35). In HA and HA+AFB₁-fed poults, the weight of the intestine was lower; this was surprising since, in other animal species, it has been reported that HS increases the size of the intestine, the viscosity in the lumen, the mucosal surface area, and the number of goblet cells, which leads to increased intestinal weight (17, 37).

No significant differences were found in the number of cells, hematocrit, heterophils, lymphocytes, basophils, or hetero/lymph ratio. Significant differences were observed only in monocyte counts in the HA treatment. Monocytes are derived from the bone marrow and released into the bloodstream. They have a brief circulation in the blood before migrating into the tissues and differentiating into tissue macrophages. Macrophages play critical roles in various physiological functions, including inflammation, wound healing, immune regulation, and metabolic homeostasis. Therefore, a decrease in the number of peripheral blood monocytes in birds fed contaminated diets suggests a potential reduction of synthesis in the bone marrow or increased clearance from the bloodstream (38). Our findings corroborate the results of a previous study conducted by Bujnák et al. (39), which also reported no significant changes in red and white blood cell parameters with the addition of HS on blood variables in finishing pigs.

In the AFB₁ treatment group, a notable decrease in serum levels of total protein, AST, CK, GLDH, creatinine, BUN, glucose, Ca, and P was observed when compared to the negative control treatment. These findings indicate that AFB₁ exposure significantly impacts various serum markers, suggesting potential alterations in liver function and metabolic processes. The inclusion of AFB₁ in the feed had detrimental effects on serum protein, cholesterol, glucose, and uric acid levels, while the activity of AST and ALT enzymes increased in the turkey poults and poultry chickens (40). Furthermore, the activities of γ-glutamyl transferase, lactate dehydrogenase, and aspartate aminotransferase were found to be elevated in poultry exposed to AFB₁ (1, 35).

HA exhibited positive effects on certain serum enzyme activities and helped alleviate the serum biochemical changes associated with aflatoxin toxicity (41). Moreover, there were reductions observed in the levels of serum albumin, total protein, uric acid, and cholesterol with HA supplementation (42). The protection of HS against liver injury was also evaluated in several studies in which hepatotoxicity was induced using lipopolysaccharide, carbon tetrachloride, and ethanol in rats, reporting reduced AST, ALT, and ALP serum concentrations (43, 44).

Compared to the negative control, the positive control exhibited a significant increase in cellular degeneration and fibrosis. No significant differences were observed between the positive control and the HA+AFB₁ treatment. The histopathology studies provide further support for the findings, demonstrating that the liver of turkeys from the AFB₁ treatment exhibited significant cellular degeneration and fat deposition. The observed enlargement and weight increase of the liver can be attributed to an accumulation of lipid content, leading to a condition known as a friable and fatty liver in the liver of chickens affected by aflatoxicosis (45). Moreover, in aflatoxicosis, the presence of severe inflammatory cell infiltrates, primarily composed of lymphocytes and heterophils, is frequently observed. This inflammatory response is believed to be a mechanism by which the body responds to the degeneration and vacuolation of hepatocytes caused by AFB₁ exposure (46).

One of the main future challenges is to develop new procedures that may achieve comparable decontamination efficiencies in a broad spectrum of feed matrices because no general all-purpose decontamination methods could be broadly employed. Notably, the novel emerging decontamination technologies should not change the physical–chemical properties of the treated feed products significantly (47–49). The utilization of HS as feed additives has been extensively researched and has shown diverse advantages. These benefits are often observed when HS is combined with other feed additives, leading to synergistic or enhanced effects. The potential economic advantage for livestock and poultry producers lies in utilizing HA from high-quality sources and being administered according to dosage recommendations supported by research. This approach ensures the optimal use of HS and its associated benefits.

The results of this study provide confirmation that the presence of 250 ng AFB₁/g had severe toxic effects on various aspects of performance, organ health, biochemical and hematological parameters, and hepatic injury in turkey poults over 28 days. The current research employed HA as a dietary inclusion in relatively small amounts, demonstrating its effective use as an additive. The findings strongly suggest that HA derived from worm compost has the potential to serve as an effective adsorbent for mitigating AFB₁ toxicity in turkey poults. Despite these positive findings, it is important to note that research on HA as mycotoxin binders is still relatively limited, and further studies are needed to fully understand their long-term effects and optimal inclusion levels in feed formulations. Nonetheless, their promising efficacy makes them a valuable area of research for addressing mycotoxin-related challenges in poultry production. At present, our research team is conducting experiments to study the effects of HA throughout poultry production.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by all animals handing procedures complied with the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (Protocol No. 22020). The study was conducted in accordance with the local legislation and institutional requirements.

JM-G: Conceptualization, Writing—review and editing, Methodology, Writing—original draft. MN-R: Conceptualization, Methodology, Writing—original draft, Investigation. SG-R: Writing—original draft, Conceptualization, Validation, Writing—review and editing, Funding acquisition, Investigation, Project administration. MÁ: Methodology, Writing—original draft, Funding acquisition, Investigation, Project administration. BS-C: Methodology, Writing—original draft. DH-P: Methodology, Writing—original draft. RM-G: Conceptualization, Software, Writing—original draft. XH-V: Writing—review and editing. JH-R: Formal analysis, Methodology, Software, Writing—review and editing. IL: Software, Writing—review and editing. RS-C: Software, Writing—review and editing. JL: Formal analysis, Writing—review and editing. AV-D: Writing—review and editing, Formal analysis, Investigation. XD: Software, Writing—review and editing. AM-A: Conceptualization, Investigation, Writing—review and editing, Validation. BH: Funding acquisition, Project administration, Writing—review and editing, Validation. GT-I: Conceptualization, Funding acquisition, Project administration, Writing—original draft, Writing—review and editing, Investigation, Validation.