ABSTRACT. Whether the fungicide carbendazim affects the meiotic spermatocytes and consequently induces chromosome aberrations in the spermatids was determined in the adult rat testis using the micronucleus test. Round spermatids containing micronuclei (MN) were significantly increased in number at stages I and V on days 1 and 4.5 after treatment with carbendazim (100 mg/kg), respectively (p<0.05). Immunocytochemistry indicated that approximately 68% of the carbendazim-induced MN contained kinetochores. These results suggest that carbendazim induces chromosome aberrations in spermatids with a high incidence of aneuploidy.-KEY WORDS: carbendazim, micronucleus, spermatid.J. Vet. Med. Sci. 61(5) 573-576, 1999 vascular perfusion technique. The tissue blocks were postfixed in 1% OsO 4 , dehydrated in ethanol, and embedded in Quetol 812. Sections cut at a thickness of 1 µm were stained with toluidine blue and observed using a × 100 oil immersion objective. For studies of other end points, unfixed seminiferous tubules were processed as follows. Testes were collected at the individual post-treatment intervals, and the seminiferous tubules were immediately teased apart and isolated in phosphate-buffered saline (PBS). The stages of the isolated tubules were determined under a transilluminating stereomicroscope according to the criteria of Parvinen et al. [13,14], and segments of tubules at specific stages were cut out. The segments were transferred to glass slides, overlaid with cover glasses, and gently squashed to squeeze the germ cells from the tubules. After confirmation of the steps of round spermatids under a phase-contrast microscope, the specimens were frozen in liquid nitrogen, and the cover glasses were removed. They were then fixed with 10% acetic acid in ethanol for 10 min at room temperature, followed by DNA staining with Hoechst 33258 (3 µg/ml PBS, Sigma, St. Louis, MO, U. S. A.) for 1 hr. To study the dose-dependency, 1,500 step 1 spermatids per animal were examined for the presence of MN using an Olympus BX50F4 fluorescence microscope, and their frequencies were compared among doses. To study the survival of the MN, 1,500 round spermatids per animal prepared at specific post-treatment intervals were examined. For kinetochore immunocytochemistry, tubular segments at stage I were fixed with 10% acetic acid in ethanol for 10 min. The specimens were rinsed in PBS containing 0.1% Tween 20, and then incubated initially in CREST serum (Binding Site, Birmingham, U. K.) diluted at 1:50 with PBS containing 0.1% Tween 20 overnight at room temperature. CREST serum is derived from scleroderma patients and contains the antibody to kinetochores [3,10]. As controls, sections were incubated without CREST serum. Following rinses in PBS, the specimens were incubated in FITC-conjugated rabbit anti-human IgG (1:50, Dako, Glostrup, Denmark) for 2 hr. Following counterstaining with Hoechst 33258, kinetochore
