ORIGINAL ABSTRACT

Glyphosate-based herbicides (GBHs) are widely used, yet their effects on rooster spermatozoa and epigenetic regulation remain poorly understood. This study evaluated the in vitro effects of glyphosate (GLP), a commercial GBH formulation, and the model surfactant polyethoxylated tallow amine (POEA) on rooster sperm function and global methylation. Pooled rooster semen samples were exposed to increasing concentrations of GLP or GBHs for 1 h and 3 h to assess dose- and time-dependent effects (Experiments 1 and 2). Based on these findings, a third experiment compared GLP, Roundup®, and POEA at the highest concentration after 1 h of incubation. Sperm motility was evaluated by CASA, whereas functional parameters were assessed by flow cytometry. Global DNA methylation was measured using a colorimetric assay. GLP had limited effects on motility and did not impair plasma membrane integrity or mitochondrial membrane potential, but altered DNA fragmentation, intracellular Ca²⁺ levels, caspase activity, and lipid peroxidation. In contrast, GBH induced broader alterations, including reduced motility by approximately 30-50%, impaired membrane integrity, increased DNA fragmentation, altered apoptotic responses, decreased intracellular Ca²⁺ and lipid peroxidation, and reduced mitochondrial activity under the most severe conditions. POEA alone affected selected functional endpoints, including DNA fragmentation, caspase activity, intracellular Ca²⁺ levels, lipid peroxidation, and acrosome status. Global DNA methylation decreased by approximately 70-80% compared with the control, with the strongest reduction observed after POEA exposure. These findings demonstrate that the GBH formulation exerts stronger adverse effects on rooster spermatozoa than GLP and POEA alone and underscore the importance of evaluating complete herbicide formulations, rather than active ingredients, in reproductive risk assessment.