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GnRH plays a pivotal role in reproduction by stimulating the release of gonadotrophins. Chemical substitutions in the GnRH molecule lead to analogues possessing antagonist or agonist activity (Paramo RM et al. 1993 J. Reprod. Fertil. Suppl. 47, 387?397). The highly potent agonist analogue, Buserelin, with up to 20 times of potency, by increasing binding affinity, desensitizing competitive receptors, and resisting metabolic degradation, shuts down rather than stimulates reproductive function (Bertschinger HJ et al. 2001 J. Reprod. Fertil. Suppl. 57, 275?283). In man, Buserelin is employed in several gonadal hormone-dependent diseases and for prostatic cancers. We suppress gonadal function in male dogs using Buserelin. Eight intact male German sheep dogs 20 months old were divided into two groups; A, 4 subjects treated for pharmacological castration (Buserelin acetate, 0.3 mg/each, s.c., every 8 h for 30 days) (Suprefact-Aventis Pharma, Italy); B, 4 subjects treated with placebo (NaCl, 0.9%, s.c., every 8 h for 30 days). Plasma testosterone concentrations were measured twice a week by RIA using commercial kits (Coat-A-Count, Los Angeles, USA). Clinical examination of the male genital tract was conducted by ultrasound monitoring. Before and after the pharmacological treatment, semen was collected and evaluated for macroscopic and microscopic parameters. After treatment, testicular specimens were collected by orchiectomy, fixed in Bouin's solution, and embedded in paraffin wax. Thin sections were cut and stained with hematoxylin/eosin. The presence of germ cells (spermatogonia to spermatozoa, Sertoli and Leydig cells number) were analyzed. Randomly selected fields of transverse and longitudinal sections of seminiferous tubules were observed and analyzed using a computer assisted image analyzer (MONO system, Italy). The images acquired were segmented and binarized in order to obtain the masks of the tubular profiles; the mean values of the area, major and minor axes, mean diameter, and perimeter occupied by the testicular tubules were calculated automatically. Data were analyzed by ANOVA test. After Buserelin, all dogs (group A) showed a reduction in testicular and prostatic diameters compared to group B. Azoospermia was observed in group A. Histological examination revealed a statistically significant cell reduction of the germinal line (spermatogonia and spermatocytes, P < 0.05; spermatids and spermatozoa, P < 0.001). GnRH pharmacological treatment induced a cessation of normal spermatogenesis at the spermatocyte level while no statistical difference in morphometric parameters of seminiferous tubules were observed. The basic testosterone level (3.2 1.3 ng/mL) rose to 12 3.7 ng/mL (21 day) and than shut down to 0.5 0.3 ng/mL (30 day), giving a long-term suppression. The present study demonstrates that Buserelin is an anti-fertility agent that gives suppression of reproductive function in male dogs. The method may have a clinical application. The utilization of a Buserelin depot will be a successive step.

The continuous administration of GnRH leads to the desensitization and down-regulation of GnRH receptors on pituitary gonadotropes (Parker and Schimmer 2001 in Goodman & Gilman's The Pharmacological Basis of Therapeutics, New York: McGraw-Hill, 1541–1562). In this study, we combined the use of a GnRH analogue with calcium/naloxone association (Sciorsci et al. 2000 Immunopharmacol. Immunotoxicol. 22, 575−626) to obtain a desensitizing effect on LH receptors in an attempt to regulate spermatogenesis in dogs. Eight post-pubertal mixed breed dogs were divided into two groups: (A) four subjects were pharmacologically treated with a 30 day administration of buserelin acetate (Suprefact Aventis Pharma, Italy) 0.3 mg/each s.c. TID, associated with 0.1-mL/kg/bw i.m. injection SID of calcium gluconate 20% (New ICC, Upjohn, Italy) in which 0.4 mg/mL of naloxone hydrochloride (Diosynth, The Netherlands) was dissolved; (B) four subjects received placebo (NaCl 0.9% s.c. TID for 30 days). At the end of the treatment, hemiorchidectomy was performed and 4 months later the second testis was removed. Clinical examination of the genital tract was done by ultrasound monitoring. Before and after pharmacological treatment and until total orchidectomy, semen was collected and evaluated for macroscopic and microscopic parameters. Plasma testosterone concentration was measured twice a week by RIA. Testicular specimens were fixed in formalin solution, embedded in paraffin wax and stained with hematoxylin–eosin. The presence of germ cells and the testicular tubule diameters were analyzed using a computer assisted image analyzer (MONO system, Italy). Data were analyzed by the ANOVA test. In the treated group, all dogs showed a reduction in testicular and prostatic diameters compared to the control group. Transient elevations in testosterone concentrations were observed in response to the early injections and reached baseline levels after 8–10 days. The testosterone decline resulted in lower ejaculate volume and, at the end of treatment, azoospermia was observed. At the end of treatment, testosterone values had progressively regained normal values over 2 months and, when measured immediately before total orchidectomy, sperm concentration and motility had returned to normal. At the histological observation, seminiferous tubules showed the block of spermatogenesis at the level of spermatocytes with a statistically significant cell reduction in spermatides and spermatozoa (P < 0.001) whereas the seminiferous tubular morphometry was normal. Total orchidectomy demonstrated that a normal histological testicular structure had been restored. In the control group, no modifications were observed. As observed in a previous study (Aiudi et al. 2005 Reprod. Fert. Dev. 17(2), 280–281), GnRH induced suppression of the reproductive function in male dogs. In this study, we demonstrated that GnRH analogue associated with calcium and naloxone induces the same effect in a shorter time and that this effect is reversible. These devices designed for the shutdown of gonadal steroidogenesis and spermatogenesis would be useful for treating sex hormone-dependent syndromes such as malignant and benign prostatic tumors.