Single Intraperitoneal Busulfan Injection Induces Long‐Term Reproductive Dysfunction and Reduces Male Offspring Ratio in BALB/c Mice

This study aims to assess the long‐term effects of busulfan on male reproductive function in BALB/c mice to provide insight into its toxicological effects. A total of 80 BALB/c mice were randomly assigned to a control group ( n = 20) and three experimental groups ( n = 20 each), which received a single intraperitoneal injection of busulfan at 15, 20, and 25 mg/kg, respectively. A subset of mice was assessed at multiple time points for spermatogenesis. Additionally, fertility experiments with both sexes served as a vital measure of fertility potential. Results revealed that relative testicular weight and semen quality in the experimental groups were significantly decreased compared with those in the control group ( p < 0.05). Abnormal sperm morphology showed a significant increase ( p < 0.05), with a notable correlation observed between abnormal sperm neck morphology and reduced sperm motility ( p < 0.05). Fertility trials indicated that busulfan‐treated males had a reduced ability to impregnate females, as evidenced by reductions in pregnancy rates and litter sizes. Remarkably, the male offspring ratio in the busulfan‐treated group (7 out of 17) was significantly lower than that in the control group (21 out of 42). In conclusion, a single intraperitoneal injection of busulfan led to sustained fertility impairment in mice for at least 13 weeks, with potential transgenerational effects, including alterations in the sex ratio of male descendants.