Compound probiotics alleviate aflatoxin B1-induced reproductive toxicity in mice by remodeling gut microbiota and testicular metabolome via NRF2 pathway action

This study aimed to investigate the protective effects of compound probiotics (CP) comprising Saccharomyces cerevisiae , Lactobacillus casei , and Bacillus subtilis against aflatoxin B₁ (AFB₁) -induced reproductive toxicity in mice. Kunming mice (n = 60; male) were randomly assigned to six groups and treated via gavage for 28 d with control, AFB₁ alone (150 μg/kg), medium-dose CP (MCP) alone, or AFB₁ combined with low-, medium-, or high-dose CP. Notably, AFB₁ exposure significantly increased the integrated biomarker response version 2 individual (IBRv2i) value to 9.78, whereas medium- and high-dose CP reduced it to 3.59 and 3.22, respectively ( P < 0.05), with no significant difference between the MCP and HCP groups ( P > 0.05). This identified MCP (1.0 × 10⁶ CFU/mL of each strain) as the minimum effective dose. AFB₁ exposure disrupted gut microbiota by enriching opportunistic pathogens ( Staphylococcus , Atopostipes ) and altered the testicular metabolome by elevating indoleacetic acid and adenosine. These disturbances impaired spermatogenesis, as evidenced by downregulated meiotic marker SYCP3 alongside upregulated germ cell marker VASA, indicating meiotic arrest with accumulation of undifferentiated germ cells. Mechanistically, AFB₁ inhibited the testicular PI3K/AKT/NRF2 pathway, inducing oxidative stress and apoptosis. Conversely, MCP intervention remodeled gut microbiota by enriching beneficial bacteria (Lactobacillus , Carnobacterium ), elevated testicular antioxidant metabolites including 2-n-propylthiazolidine-4-carboxylic acid (PTCA) and L -cysteine, and activated the NRF2/p62 pathway, thereby restoring spermatogenic progression and reversing meiotic arrest. These findings offer new insights into probiotic interventions for environmental reproductive toxicity and highlight PTCA as a potential key signaling molecule in the "gut–testis axis" metabolic communication.