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.