Inefficacy of Thymus satureioides Essential Oil in Cryopreserved Beni Arouss Buck Semen: Post-Thaw Quality Hinges on Antibiotics for Bacterial Control

Growing concerns about antibiotics in semen extenders have driven interest in natural antimicrobial alternatives. The primary objective was to evaluate whether Thymus satureioides essential oil (EO) could replace antibiotics in a skim-milk extender while controlling bacterial growth and maintaining the post-thaw quality of Beni Arouss buck semen. Eighty ejaculates from eight bucks were pooled per collection and assigned to 12 treatments: EO at 0–0.05% (v/v), each with or without penicillin–streptomycin. After cryopreservation, extenders were assessed for oxidative balance, and semen was evaluated for sperm quality, lipid peroxidation, and aerobic bacterial load. Anti-oxidant activity increased and lipid peroxidation in the extender decreased as EO concentration rose whereas antibiotic-containing treatments showed higher lipid peroxidation. In semen, EO at 0.01–0.02% caused no significant changes in motility, viability, morphology, or membrane integrity compared to the control. Antibiotics better preserved sperm quality and reduced bacterial growth, but within 0–0.02% EO they were linked to higher lipid peroxidation. At ≥0.03%, EO showed significant toxicity, sharply reducing sperm quality while bacterial growth correlated negatively with motility, viability, and membrane integrity and positively with abnormalities. These findings suggest that oxidative stress may not have been the primary driver of sperm damage, as much of the measured MDA likely originated from extender lipids. In addition, significant EO × antibiotic interactions for total motility, abnormalities, and bacterial growth indicate that the response to EO depended partly on antibiotic supplementation. Overall, the tested EO concentrations did not reproduce the combined bacterial control and post-thaw sperm preservation observed with antibiotics and therefore did not support replacing antibiotics under these conditions.