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The aims of the present study are to clarify the effect of methyl-beta-cyclo-dextrin (MβCD) on the secretions of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL) and growth hormone (GH) from the anterior pituitary (AP) cells of prepubertal male cattle and to compare the characteristics of secretion to those of gonadotrophic-releasing hormone (GnRH), thyrotropin- releasing hormone (TRH) and growth hormone-releasing hormone (GHRH), respectively. The AP cells prepared from six castrated Holstein calves (age: 8-11 months) were incubated for 2 h with MβCD (10-3 M, 10-2 M), GnRH (10-8 M), TRH (10-8 M) and GHRH (10-8 M) or vehicle only as a control (CTL). The amount of LH, PRL and GH released in the medium was measured by a time-resolved fluoroimmunoassay (TRFIA). The amount of FSH released in the medium was measured by the radioimmunoassay (RIA). MβCD significantly (P<0.05) stimulated the secretion of LH, FSH, PRL and GH from the AP cells. Furthermore, GnRH significantly (P<0.05) stimulated the secretion of LH and FSH. Also, TRH and GHRH significantly (P<0.05) stimulated the secretion of PRL and GH, respectively. However, the potency of the MβCD was less compared to each respective hormone secretions in response to GnRH, TRH and GHRH (P<0.05).