1. Introduction
Agriculture represents a vital part of the global economy, with animal production adding considerably to gross domestic product. The contamination of agricultural products with mycotoxins is one of the issues encountered in this sector [
1]. Depending on harvesting, storage, and procedures, contamination might occur both before and after harvest. When circumstances are appropriate, the fungus of the genus Aspergillus (A), primarily
Aspergillus flavus and
Aspergillus parasiticus, generate aflatoxins (AFs). Broilers are the most susceptible species to AFs [
2]. Poor feed conversion ratio (FCR) and growth in broilers, increased mortality rate, anorexia, and movement difficulties are some of the effects, in addition to concern for public health due to the risk of AF residue in broiler meat [
3]. For the detoxification of mycotoxin-contaminated feedstuffs, a range of technologies have been explored, including thermal inactivation, physical separation, microbial degradation, irradiation, and other treatments [
4]. The use of adsorbent materials in the diet, which can be organic (microbial) or inorganic (primarily clay minerals), is a method for the mycotoxin detoxification. Biological products to reduce mycotoxin availability have received much attention; the most frequent technique for preventing and treating mycotoxicosis in birds is the use mycotoxin adsorbents [
5]. Medicinal herbs have recently received a lot of interest as feed additives for reducing the negative effects of mycotoxins.
The medicinal plant milk thistle (MT) (
Silybum marianum) is commonly used to treat liver disorders. Different flavonolignans, such as silybin, are found in MT extract. The majority of the active compounds in MT are found in seed, which contains about 70–80% silymarin. The incorporation of MT in broiler chickens’ diets has led to improved growth performance and health status [
6,
7,
8,
9,
10,
11,
12,
13]. Silybin shows significant biological activities, including in birds affected by AFB1 [
14]. Although there are several commercial adsorbents available in the form of feed additives to mitigate these naturally occurring toxins, there are few data on the prevalence of AFs in commercial broiler diets and on birds’ performance.
Toxofix Arka (Noavaran Arka Tejarat Kabudan. Co., Urmia, Iran) is a novel multi-component toxin-binder with a strong affinity for polar and nonpolar toxins, such as AF,
Fusarium, and
Zearalenone. It has uses three key effective strategies of adsorption, biotransformation and bioprotection to remove the vast variety of mycotoxins.
Spirulina platensis (SP) is important due to it medicinal properties, and its high nutritional value has initiated new perspectives of research on different feed additives to the diets of animals. These algae may operate as immune stimulants or performance enhancers, hence increasing the feed quality for specific animal species. Aćimović [
15] and Tufarelli et al. [
16] discovered that nutritional supplementation with algae improved the hepatic and intestinal antioxidant capacity of old laying hens and helped to detoxify aflatoxin-contaminated diets [
17]. It was reported by Suwarno et al. [
18] that carotenoid compounds including beta-carotene and beta-cryptoxanthin significantly reduced aflatoxin production by
Aspergillus flavus in an in vitro study. Another in vitro study found that commercially obtained carotenoid inhibited aflatoxin biosynthesis by >70% for 38
Aspergillus genotypes isolated from maize. Algae, and in particular
Spirulina platensis, naturally contain high amounts of carotenoids.
Because of the importance of AF detoxification in poultry diets, the current study investigated growth performance, carcass characteristics, antibody titers, serum blood indices, and cecal microbial population in broiler chickens fed with an aflatoxin-contaminated diet.
3. Results
The effects of the MT, TB and SP powders on the BWG, FI and FCR of broiler chickens exposed to AFB1 are shown in
Table 4. Broilers fed AFB1-contaminated diets had substantially decreased BWG and FI (
p < 0.05). Birds fed an AFB1-contminated diet exhibited poor FCR (
p < 0.05). Furthermore, MT, TB, and SP powders reduced the deleterious effects of AFB1 on BWG, FI, and FCR in the chickens (
p < 0.05).
Table 5 summarizes the relative weights of carcass traits. In comparison to the control birds and other treatments, broilers given an AFB1-contaminated diet had a higher relative weight of abdominal fat (
p < 0.05).
Table 6 shows the blood serum biochemical characteristics of broiler chicks exposed to AFB1. Dietary supplements had no effect on blood serum glucose, total protein, triglyceride, cholesterol, or uric acid (
p > 0.05). When compared to the control, AFB1 diets resulted in a substantial increase in AST and ALT activity (
p < 0.05). MT, TB, and SP powders significantly lowered serum AST and ALT activity in chickens (
p < 0.05).
Table 7 shows the outcomes of experimental treatments in broiler chickens exposed to AFB1 in response to the phytohemagglutinin skin challenge. The AFB and MT groups had the lowest skin thickness 24 h after injection (
p < 0.05). According to the findings of the phytohemagglutinin injection, TB and SP appear to be more effective than other additives at removing toxins from feed (
p < 0.05). The results of the skin challenge with phytohemagglutinin 48 h after injection revealed no significant changes between treatments (
p > 0.05).
Table 8 shows the impact of experimental treatments on SRBC response and immunoglobulin titers in broiler chickens exposed to AFB1. At 28 and 35 days of age, there was no significant change in IgM and IgG titers across treatments (
p > 0.05). The SRBC titer was considerably lower in the AFB group compared to the control group at 28 days of age (
p < 0.05). The SRBC titer in treatments with different additives was intermediate between the control and AFB groups (
p < 0.05). However, by inhibiting AFB1, the TB group showed a closer titer to the control with a higher effectiveness than the other treatments.
Table 9 shows the results of the cecal microbial population of broiler chickens exposed to AFB1. When compared to the control, there was an increase (
p < 0.05) in cecal
Coliform population in chicks given an AFB1-contominated diet; moreover, adding MT, TB, and SP powders to the AFB1-contaminated diet significantly reduced cecal
Coliforms (
p < 0.05).
4. Discussion
The AFs, as the most common mycotoxins, represent a great problem for poultry producers. Mycotoxins have been found to infect over 25% of global crops [
23], and it is expected that the AFB1 contamination rate of feed components in many countries will reach 60% in the coming years, posing major public health concerns. The concentration of AFB1 in meals was established at 0.6 mg/kg in this study, which resulted in lower BWG and FI, as well as poor FCR in broiler chickens at 42 days of age, indicating that the birds exhibited subclinical aflatoxicosis. These findings are consistent with those of Barati et al. [
21] and Nazarizadeh and Pourreza [
5], who found that broilers fed an AFB1-contominated diet had lower feed consumption and reduced FCR. On the other hand, Siloto et al. [
24] found no variations in FI in birds fed an AFB1-contaminated diet (1 mg/kg feed). Broilers fed diets including MT, TB, and SP powders may have had fewer negative effects on their overall performance. Silymarin has been shown to improve FI and feed efficiency in broilers when used with MT [
14]. In broilers, Fani Makki et al. [
8] found that adding silymarin alone enhanced FI and BWG compared to a group fed a diet contaminated with AFB1, but there was no effect on FCR. Supplementing broilers fed an AFB1-contaminated diet with MT enhanced FI and raised BWG. This was in agreement with Chand et al. [
6]. By lowering free radicals and enhancing antioxidant enzymes, MT may be able to encourage detoxification. It also binds toxins and restricts their absorption into the hepatocyte by occupying binding sites [
14].
The inclusion of TB in AFB1 diets enhanced growth performance and reduced undesirable effects in broilers. The addition of TB (1.0 g/kg of feed) to an AFB1-contaminated diet (0.3 g/kg of feed) decreased the growth inhibitory effects on broiler chicks fed contaminated diets for 21 days, according to a previous study [
5]. Cation exchange capabilities, allowing TB to trap molecules inside their pores, were one of the most critical characteristics of the TB employed in this study. The porous nature of TB allows it to attract AFB1 in the gastrointestinal tract, and thus reduces its deleterious effect on broiler chicken performance [
20]. Another property of TB is that it contains phenolic compounds from plant extracts which have high antioxidative capabilities. Some botanical extracts have been reported to have antifungal effects [
25]. TB, on the other hand, is high in probiotics and some yeasts, such as
Saccharomyces cerevisiae, which have the ability to reduce the amount of AFB1 in the environment through a mechanism involving the thick peptidoglycan layer of the Gram-positive bacterial cell wall, which interacts with AFB1 and increase its excretion [
26]. In the current study, the content of SP was shown to be able to compensate the negative effects of AFB1 in the broilers. Previous research has revealed that the marine alga has a wide range of therapeutic qualities and biological activities, including immunomodulatory, antioxidant, and anti-inflammatory capabilities. The findings of this study correspond with those of Subhani et al. [
17], who found that algae might reduce the detrimental effects of AFB1 and have a positive influence on broiler chicken health. Algae’s excellent antioxidant properties are one of its most important characteristics. Broilers fed a diet supplemented with SP had reduced oxidative stress levels, leading to better antioxidant capacity [
16]. Data obtained from in vitro and in vivo studies suggest that the vitamins in algae are very effective in preventing mycotoxin-induced damages in cells. For example, different forms of vitamin A inhibited AFB1–DNA adduct [8-hydroxydeoxy-guanosine (8-OHdG)] formation by regulating the metabolism of AFB1 conducted by the cytochrome P450 (CYP450) enzyme system [
27]. A previous study found that chicks fed AFB1 at 80 g AFB1/kg feed had considerably greater liver weight than those fed a diet without adsorbents [
28]. In the present study, the greater liver weight of birds fed AFB1 was related to increased fat retention, validating the findings of Siloto et al. [
24]; moreover, adding MT, TB, and SP to the diet was effective in binding AFB1. When hepatocytes are damaged, transaminases are leaked into the blood, raising serum transaminases activity. Elevated blood AST and ALP concentrations suggest cellular (hepatocyte) damage, such as necrosis or changes in cell membrane permeability, as well as muscle damage caused by AFB1-induced lipid peroxidation [
20]. AFB1-contaminated diets increased serum hepatic enzymes including ALT and AST, which were reduced when MT, TB, and SP were supplied. Abou-Shehema et al. [
29] reported similar findings, claiming that MT containing silymarin reduced the cytochrome P450 system, limiting AFB1 activation. The TB was composed of complex indigestible carbohydrates (glucomannans and peptidoglycans), phenolic compounds, probiotics, and aluminosilicates such as bentonite, which inhibited mycotoxins from causing damage to the digestive tract. These findings are consistent with those of Subhani et al. [
17], who found that marine algae exhibited hepatoprotective properties and lowered liver enzymes, hence reducing the harmful effects of AFB1.
It has also been suggested that ascorbic acid, which is high in SP, protects animals from the acute toxicity of AFB1 by activating AFB1–epoxide hydroxylase, aldehyde reductase, and CYP3A enzymes located in the enterocytes [
27]. Additionally, Simonich et al. [
30] reported that chlorophyll and chlorophyll b were potent protective agents against AFB1 carcinogenesis in rat liver and colon, providing supporting evidence that both agents offer protection by inhibiting carcinogen uptake from the gut, thus reducing the availability of AFB1 to the target organ. Increased liver weight is a sign of aflatoxicosis since the main effects of AFs are related to liver damage. Broilers need a strong immune system, especially as birds are frequently exposed to many infections. The immune system is known to be extremely susceptible to AFB1 [
5]. Adding AFB1 to broiler diets has been shown to reduce skin reactions to phytohemagglutinin. According to Alhidary et al. [
14], silymarin boosted the immune system by quenching free radicals. It has the capacity to protect glutathione supply and to reduce oxidation, as well as having a direct influence on immune cells. Using probiotics, phenolic compounds and yeast may boost immunoglobulin levels and boost the immune system, according to the findings of the TB group. According to a previous study, the beneficial effects of probiotics include lowering gut pH, which prevents bacteria from colonizing through competitive exclusion, the production of organic acids, the production of antibacterial mucin and enzymes, and competition for nutrients in the gut [
21].
The SP is a powerful antioxidant that helps to prevent lipid peroxidation. Multiple biological effects of SP polysaccharides have been demonstrated, including anti-inflammatory, antibacterial, antiviral, immunomodulatory, and free radical scavenging. It has the capacity to improve the immunological condition of birds when their immune systems have been compromised by mycotoxin exposure [
31]. We detected a considerable increase in
Coliform populations with the AFB1-contaminated diets, which was reversed by adding MT, TB, and SP into the diets. In chickens, exposure to AFB1 has been demonstrated to impair resistance to many bacterial, viral, and protozoan diseases. The presence of phenolic compounds and silymarin in MT, which have antibacterial and antifungal properties, regulate growth and metabolic factors and lower the amount of harmful bacteria in the gut, have been shown to have a key role in minimizing the negative effects of AFB1 [
32].
The use of TB in chicken diets has been pioneered due to its positive effects. The most essential aspect in lowering intestinal bacteria is the inclusion of probiotics, plant chemicals, and mannano-oligosaccharides (MOS) in the composition of TB. The MOS also improves villus height and the amount of anaerobic and cellulytic bacteria in the gut, which improves lactate consumption and the pH of the stomach. Hydrogen bonds and van der Waals forces bind mycotoxin to yeast wall glucomannan, and this binding is persistent throughout the gut [
33]. According to previous studies, probiotics exhibit antibacterial action against
E. coli and the capacity to adsorb AFB1 [
34,
35]. It is well known that including algae in the diet can help to reduce the negative effects of mycotoxins. Furthermore, according to some experts, algae play an important function in animal performance and health by regulating the gut’s ecological balance [
17,
23].