Network toxicology and multi-omics reveal the mechanism of testicular toxicity induced by chronic AFB1 exposure in male quails.

Aflatoxin B1 (AFB1) is a pervasive feed contaminant that poses a silent threat to male reproductive health under chronic low‑dose exposure. In this study, a male quail model was established with graded doses of AFB1 (25, 50, and 75 μg/kg). Integrated database prediction, multi‑omics profiling, and molecular docking revealed that AFB1 potentially targets AKT1, BCL2, TP53, and BID, disrupts testicular sphingolipid and steroid metabolism, and triggers the intrinsic mitochondrial apoptotic pathway. In vivo experiments confirmed that AFB1 exposure reduced body weight, sperm viability, and serum reproductive hormone levels, while inducing oxidative stress, inflammatory responses, and activation of the BAX‑Cyt‑c‑Caspase‑9/3 cascade. To move beyond correlative evidence, primary avian testicular Sertoli cells were employed for in vitro functional validation. Pharmacological interventions with GW4869 and all‑trans retinoic acid demonstrated that ceramide accumulation functions as an upstream driver of AFB1‑induced mitochondrial impairment and apoptosis. Collectively, these findings establish that AFB1 activates the sphingomyelin/ceramide‑dependent sphingolipid‑mitochondrial apoptotic axis, culminating in testicular cell death. This study provides novel mechanistic insights into AFB1‑induced male reproductive toxicity and offers a theoretical foundation for aflatoxin risk management in livestock production.