Novel aflatoxin oxidase CotA alleviates aflatoxin B1 toxicity in broilers by reducing hepatic AFB1 residues and inhibiting oxidative stress-mediated ferroptosis.

Aflatoxin B1 (AFB1) is a common mycotoxin present in animal feed. This study systematically examined the protective effects of the novel aflatoxin oxidase CotA in alleviating the adverse impacts of AFB1 on broilers. A total of 432 one-day-old Arbor Acres Plus broiler chicks were randomly assigned to four experimental groups. Experimental diets consisted of a control (CON) group fed the basal diet, an AFB1 group supplemented with 200 μg/kg aflatoxin B1, a CotA group supplemented with 5 U/kg aflatoxin oxidase CotA laccase, and a CotA+ AFB1 group supplemented with both 200 μg/kg aflatoxin B1 and 5 U/kg aflatoxin oxidase CotA. Compared with the control group, broilers exposed to AFB1 exhibited a 10.8 % reduction in 42-day body weight and a 13.2 % increase in the F/G ratio from 22 to 42 d (P < 0.05). Furthermore, AFB1 exposure significantly compromised meat quality, as evidenced by a 25.4 % increase in drip loss and an 18.4 % increase in cooking loss in the breast muscle (P < 0.05). However, these adverse effects were effectively mitigated by aflatoxin oxidase CotA supplementation, which restored the 42-day body weight to 2298.45 g and reduced the drip loss and cooking loss to 1.59 % and 35.48 %, respectively. Notably, the architectural integrity of hepatocytes in the livers of broilers fed AFB1-contaminated feed was compromised, characterized by pronounced vacuolar degeneration and infiltration of inflammatory cells. The liver index in the AFB1 group was also significantly higher, accompanied by marked increases in serum activities of aminotransferases (ALT and AST) and alkaline phosphatase (ALP) (P < 0.05). The incorporation of aflatoxin oxidase CotA into AFB1-contaminated feed effectively mitigated growth retardation and enhanced meat quality. Furthermore, compared with the AFB1 group, aflatoxin oxidase CotA significantly improved the hepatic activities of antioxidant enzymes, including total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) (P < 0.05). Simultaneously, dietary aflatoxin oxidase CotA supplementation effectively alleviated AFB1-induced hepatic oxidative stress and ferroptosis in broilers. Mechanistically, aflatoxin oxidase CotA activated the Nrf2 signaling pathway, as demonstrated by the upregulated mRNA expression of Nrf2, NQO1, and HO-1 (P < 0.05). Regarding ferroptosis inhibition, aflatoxin oxidase CotA treatment significantly reversed AFB1-induced iron overload by downregulating the expression of iron-acquisition genes (TFR1, DMT1, and STEAP3) and reduced Fe2+ content (P < 0.05). Furthermore, aflatoxin oxidase CotA suppressed ACSL4/LPCAT3-mediated lipid peroxidation and enhanced the antioxidant system by upregulating the key ferroptosis defense genes GPX4, SLC7A11, and FSP1, leading to increased GSH levels and decreased MDA content (P < 0.05). In comparison to the AFB1 group, the addition of aflatoxin oxidase CotA markedly decreased the accumulation of AFB1, AFBO residues, and AFB1-DNA adducts in the liver (P < 0.05). Concurrently, dietary supplementation with aflatoxin oxidase CotA effectively alleviated AFB1-induced intestinal villi disruption and significantly enhanced the villus height-to-crypt depth (V/C) ratio by approximately 25 % in both the jejunum and ileum (P < 0.05). Notably, aflatoxin oxidase CotA significantly preserved mucosal barrier integrity through the 1.5 to 2-fold upregulation of essential tight junction proteins. These results suggest that aflatoxin oxidase CotA holds potential as a novel feed additive to counteract the harmful impacts of AFB1 in broilers.