Consequences of in vitro exposure of chicken spermatozoa to the fungicide tebuconazole

Tebuconazole (TEB), a fungicide that inhibits 14α-demethylase (CYP51) and disrupts ergosterol synthesis, poses environmental and health risks due to its persistence and low biodegradability. This study examined TEB in vitro effects on rooster spermatozoa. In Experiment 1, semen from 10 Green-legged Partridge roosters was incubated with TEB (0, 0.1, 1, 10, 100 µM) at 36°C for 3 hours. Sperm motility was analyzed with Computer-Aided Sperm Analysis (CASA) system, while flow cytometry assessed membrane integrity, mitochondrial function, acrosome status, chromatin structure, intracellular calcium, apoptosis, caspase activity, and lipid peroxidation after 1 and 3 hours of exposure. Malondialdehyde (MDA) concentration and total antioxidant capacity (T-OAC) were measured by spectrophotometer. In Experiment 2, calcium channel blockers (SNX 325, MRS-1845, Nifedipine, HC-056456) were tested under the same conditions, focusing on motility, membrane integrity, calcium levels, apoptosis, caspase activity, and lipid peroxidation. Results in experiment 1 have shown that TEB (0.1, 1, 10 µM) reduced sperm velocity (VAP) after 3 hours (P < 0.01) without altering other motility parameters. Acrosome status, intracellular calcium level, and lipid peroxidation decreased significantly at all TEB concentrations (P < 0.01). Early apoptosis declined at 1 µM TEB (P < 0.01), while mitochondrial activity and membrane integrity remained stable. MDA levels were reduced (P < 0.01), with no effect on T-OAC. In Experiment 2, calcium channel blockers decreased motility parameters (VAP, VCL, VSL, MOT, PROG) and intracellular calcium levels (P < 0.01), but did not affect membrane integrity. Lipid peroxidation and caspase activity declined (P < 0.01), with no impact on early apoptosis. These findings underscore TEB's role in inhibiting calcium channels, reducing ion influx, blocking calcium-driven pore formation, thereby preserving membrane integrity. This mechanism mitigates early apoptosis and lipid peroxidation in chicken sperm, shedding light on TEB's impact on motility, calcium balance, and cell function.