Abstract: The study aimed to improve the efficiency and safety of spermatogonial stem cells (SSCs) transplantation recipients so that to decrease male sterility caused by busulfan toxicity, which is helpful in revealing the damage mechanism of busulfan on testis. Testicular biotin tracing experiments were performed on busulfan-treated mice to verify the integrity of the blood testis barrier (BTB); Liquid-mass spectrometry was used to target the content of busulfan in the semen of the epididymal tail to verify whether busulfan could directly pass through the testis BTB into the seminiferous tubules; ELISA determination of inflammatory factors was performed in mouse serum to verify whether its concentration had changed. The results showed that the earliest observable red fluorescence in the seminiferous tubules traced by biotin was detected at 24 h, suggesting the damage of blood testis barrier (BTB). And the highest proportion of busulfan in the epididymal semen reached a summit at 30 min post-treatment then gradually decreased. In serum, the concentration of TNF-α, IL-1β and IL-6 progressively increased by busulfan treatment(P<0.05), and at 36 h the amount of the three indexes was significantly higher than that in the control group. The values peaked at 21 d with TNF-α of (1 855.51±10.32) pg·mL^-1, IL-1β of (293.59±3.34) pg·mL^-1,and IL-6 of (340.30±12.55) pg·mL^-1, then gradually decreased. In summary, busulfan could freely cross over BTB and the biotracer could break through BTB within 24 h. The serum inflammatory factors TNF-α, IL-1β and IL-6 concentration gradually increased and reached significant levels, reaching the highest level at 21 d, then gradually decreased.

Key words: busulfan, blood-testis barrier, male sterility, inflammatory factor