Effects of ozone exposure time on aflatoxin concentration, diet composition, growth, carcass traits, and intestinal morphology in broilers fed contaminated diet

Introduction: Mycotoxins are toxic chemical substances generated as secondary metabolites by specific types of filamentous fungi (Conte et al., 2020). Aflatoxins are a significant group of mycotoxins, predominantly produced by specific fungi species, especially Aspergillus flavus and Aspergillus parasiticus (Caceres et al., 2020). Aflatoxins adversely impact poultry by decreasing growth, feed efficiency, and egg production, while causing liver fat accumulation, reduced serum protein levels, carcass bruising, poor pigmentation, liver damage, impaired digestion, and immune suppression (Murugesan et al., 2015). Aflatoxin B1 is the most toxic and biologically active form of aflatoxins, known for its widespread occurrence (Min et al., 2021). One effective detoxification method is treating mycotoxins with oxidizing agents, which alter their molecular structure (Liu et al., 2022). Ozone, approved by the food and drug administration for food use, has advantages over other oxidants, such as its residue-free application in gaseous or aqueous form, abundant precursors, and the ability to be generated on-site (Peivasteh-Roudsari et al., 2022). The study by Demirci et al. (2023) showed that ozonation at 10 mg/L (60 min) or 3.33 mg/L (90 min) effectively reduced aflatoxins in hazelnuts. This study aimed to investigate the effects of ozone treatment time on aflatoxin-contaminated feed by examining detoxification efficiency and changes in feed chemical composition, as well as evaluating its impact on growth performance, carcass traits, and intestinal absorptive capacity in broiler chickens consuming the contaminated diet. Material and method: The first experiment was conducted in a completely randomized design with 4 treatments and 6 replicates. The experimental treatments were as follows: (1) a diet contaminated with aflatoxin (0.1 mg/kg) without ozone gas exposure, (2) an aflatoxin–contaminated diet exposed to ozone gas for 30 min, (3) an aflatoxin–contaminated diet exposed to ozone gas for 60 min, and (4) an aflatoxin–contaminated diet exposed to ozone gas for 90 min. The samples analyzed in this study were starter feed for broiler chickens. The levels of total aflatoxins and aflatoxin B1 were quantified by HPLC following the Iranian National Standard Method INSO 6782:2003. All feed samples were also analyzed for dry matter, ash, crude protein, ether extract, crude fiber, gross energy, calcium, and phosphorus content. In the second experiment, a total of 180 one-day-old male Ross 308 broilers were randomly assigned in a completely randomized design with 3 treatments and 6 replicates: control diet, diet contaminated with 0.1 mg/kg aflatoxin, and contaminated diet treated with ozone (10 mg/L for 60 minutes). Parameters measured included performance, carcass characteristics, and morphology of different sections of the small intestine. Data from both experiments were analyzed using a completely randomized design with ANOVA in SAS (4 treatments × 6 replicates for experiment 1; 3 treatments × 6 replicates for experiment 2). Tukey’s test identified significant differences at 𝑃<0.05. Polynomial regression was used to evaluate the effect of ozone exposure duration on the parameters. Results and discussion: Ozone treatment significantly reduced total aflatoxins and aflatoxin B1 in feed, with longer exposure causing greater decreases (P<0.01). Regression analysis also showed a significant linear decrease (P<0.01) in total aflatoxins and aflatoxin B1 levels as ozone exposure duration increased. Ozone treatment for 30 and 60 min did not affect (P>0.05) nutrient levels, while 90 min significantly reduced dry matter (DM), crude protein (CP), and gross energy content (GE; P<0.05). Regression analysis showed that DM, CP, ether extract (EE; P < 0.05), phosphorus (P < 0.05), and GE decreased linearly (P < 0.01) as ozone exposure increased from 0 to 90 minutes. Luo et al. (2014) found that ozonation of maize at 90 mg/L for 20 and 40 minutes significantly reduced aflatoxin B1 levels from 83 µg/kg to 12.18 µg/kg and 9.9 µg/kg. Similarly, Torlak et al. (2016) reported 74.3% and 86.4% reductions in AFB1 in poultry feed after 240 minutes of ozone exposure at 2.8 mg/L and 5.3 mg/L. Ozonation breaks down aflatoxin by reacting with the C8-C9 double bond on the furan ring and the lactone ring, destroying the toxin (Salsabila et al., 2025). Asadnejad et al. (2023) similarly found that longer ozone exposure (0 to 48 hours) at 10 g/hr caused a linear decrease in DM and EE in feather meal, due to ozone’s strong oxidative effects. The reduction in GE content may be attributed to the decrease in the percentages of DM, CP, and EE in the feed samples. Feeding aflatoxin-contaminated feed, either with or without ozone treatment, significantly reduced (P<0.01) body weight gain (BWG) and European performance efficiency factor (EPEF), and increased (P<0.01) feed conversion ratio (FCR), while feed intake (FI) was not affected (P>0.05). No significant (P>0.05) differences in relative weights of breast, leg, heart, gizzard, or spleen were observed. Carcass yield and bursa weight were higher (P<0.05) in the control diet compared to aflatoxin-contaminated feed but similar to the ozone-treated group. Liver weight increased (P<0.05) with aflatoxin contamination but normalized after ozone treatment. The Villus height: crypt depth ratio was significantly (P<0.01) better in the control and ozone-treated groups compared to the untreated aflatoxin group, indicating that ozone mitigated the negative effects of aflatoxin on intestinal structure. The mitigating effects of processing aflatoxin-contaminated feed on the relative weights of the carcass, liver, and bursa, as well as the improvement of small intestine morphology, can be attributed to the role of ozone in degrading aflatoxin into less toxic or non-toxic compounds. Generally, ozone treatment effectively degraded aflatoxin in contaminated feed and mitigated its adverse effects on carcass traits and intestinal morphology. However, the lack of improvement in overall performance suggests that residual aflatoxin levels or ozone-induced alterations in feed composition may still influence bird growth. Overall, ozone processing can be considered a promising detoxification approach when properly optimized to balance toxin removal and feed quality.