Evaluating the Reproductive Toxicity of Florasulam in Bulls: In Vitro Effects on Sperm Parameters and Testicular Cell Function

ABSTRACT This study investigates the in vitro effects of florasulam, a widely used herbicide with known environmental impact, on bull epididymal sperm and primary testicular cells. Epididymal spermatozoa were collected from the cauda epididymis attached to one testis of a paired set obtained from a local abattoir and diluted to a concentration of 1 × 10 8 spermatozoa/mL. The other testis was used to isolate testicular cells, which were then seeded onto 12‐well and 96‐well plates at the concentration of 5 × 10 5 and 5 × 10 4 cells per well, respectively. Sperm samples were exposed to various concentrations of florasulam (0–1000 μg/mL) for 2 h and evaluated for motility (M), plasma membrane integrity (PMI), acrosome integrity (AI), and mitochondrial membrane potential (MMP). Likewise, testicular cells were treated with different concentrations of florasulam for 48 h and assessed for cytotoxicity, apoptosis, steroidogenesis, and MMP. Statistical analyses were performed using ANOVA followed by Duncan's multiple range test. The results showed that florasulam exposure significantly reduced sperm motility and MMP at concentrations of 100–1000 μg/mL. Additionally, 10 μg/mL florasulam stimulated cell proliferation, whereas 10, 100, and 500 μg/mL inhibited steroid secretion in testicular cells. Apoptosis was significantly increased at 500 and 1000 μg/mL, and MMP was negatively affected at 1000 μg/mL ( p ≤ 0.05). These findings provide the first evidence that florasulam, even at sub‐toxic concentrations, can impair male reproductive function by reducing sperm motility and mitochondrial activity, and by inducing apoptosis and hormonal disruption in testicular cells. This highlights its potential risk to cattle fertility and broader environmental reproductive health.