Effect of anti-GnRH vaccine on testicular tissues in stallions

Anti-GnRH Vaccine is known as an alternative to surgical castration. The main advantage is the reversibility of immunization in 90% of the cases (Bruyas et al. Reprod Dom Anim, p.55: https://doi.org/10.1111/rda.12800). Anti-GnRH immunization reduces fertility by inhibition of gonadotropin synthesis involved in steroid production and spermatogenesis. Only few papers report information on the recovery of sperm production after prime immunization (twice 4 weeks apart) without booster in some stallions. We aimed to assess the effect of the vaccination protocol (Primo immunization or/and boosters) and the delay between the last injection of anti-GnRH vaccine and surgical castration on testicular histology. Twelve unvaccinated (control) and 28 immunized Lusitanian stallions were castrated at different time points after the last immunization (range 0.5-14 months). Vaccination protocol was a primo vaccination by intramuscular injection of 1 mL IMPROVAC® twice 4 weeks apart, followed by booster(s) when stallion behavior returned or scrotal size re-increased (range 1-10 injections). Testes were fixed in formaldehyde (10%) and stained by HES (Hematoxylin-Eosin-Saffron) and Androgen Receptor immunohistochemistry. Testicular size and spermatogenesis were evaluated by 7 parameters: Johnsen scores, seminiferous tubule diameter, number and density of Sertoli cells, number and density of germ cells, and the ratio of germ to Sertoli cells.Statistical analysis was performed by ANOVA. Values are given as mean ±standard deviation. Testis size was larger for unvaccinated controls (88±5 mm; P<0.001) compared to vaccinated stallions (70 ±8 mm). The Johnsen scores were higher in control (9.3 ±0.3; P<0.0001) compared to vaccinated stallions (8.0 ±1.2). Seminiferous tubule diameters, number and density of germ cells, and the ratio of germ to Sertoli cells were larger in control stallions: respectively 173 ±10 µm (P<0.001), 180 ±34 germ cells per tubule (P<0.001), 6330 ±897 germ cells per mm² of tubule (P<0.001), and 8.2 ±1.5 (P<0.001) germ cells per Sertoli cell compared to vaccinated stallions: 135 ±22 µm, 83 ±47 germ cells per tubule, 4404 ±1454 germ cells per mm² of tubule, and 3.3 ±1.9 germ cells per Sertoli cell. Conversely, Sertoli cell numbers and density were larger in vaccinated: 27 ±4 Sertoli cells per tubule (P=0.005) and 1720 ±582 Sertoli cells per mm² of tubule (P<0.001) compared to 23 ±4 Sertoli cells per tubule and 817 ±165 Sertoli cells per mm² of tubule in controls. There was no significant difference among the vaccination protocols (e.g., number of boosters, delay between last injection and gonadectomy) on these parameters. In conclusion, multiple rounds of anti-GnRH immunization in stallions: (1) induced testicular hypoplasia is more dependent on stallion rather the vaccination protocol, and (2) had no reversible effects on testes (according to Johnsen scores).