Endocrine changes induced by GnRH immunization and re-stimulation of testicular function with a GnRH agonist in stallions

Resumption of testicular function after GnRH immunization varies among animals and some stallions regain fertility only after a prolonged time (Janett et al., 2009, Anim Reprod Sci 115:88e102). This study evaluated endocrine effects of GnRH immunization and subsequent re-stimulation with a GnRH agonist. We hypothesized that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. Shetland stallions were assigned to a GnRH vaccination group (Improvac; 400 µg/animal in weeks 0 and 4; n=6) or a saline control group (n=6). Blood was collected weekly for hormone analyses. Each vaccinated stallion was hemi-castrated together with an age-matched control when testosterone concentration was <0.3 ng/ml for two consecutive weeks (C1). Treatment of vaccinated stallions with the GnRH agonist buserelin (4 µg/day for 4 weeks and 8 µg/day for 6 weeks) started three weeks thereafter. Irrespective of treatment, the remaining testicle was removed when testosterone concentration increased to >0.5 ng/ml for two consecutive weeks in vaccinated stallions (C2; 9-10 weeks after start of buserelin treatment). Testicular tissues were stored at – 80°C and fixed in formalin for RT-qPCR and histology. Statistical comparisons were made by repeated measures ANOVA with group as between and time as within subject factor. Data on semen characteristics in these stallions have been published recently (Gautier et al., 2022, Theriogenology 194:27-34). Plasma testosteroneconcentration decreased after GnRH-vaccination and returned to control stallion concentrations during buserelin treatment (time p<0.001, time x group p<0.001, group p=0.001). Serum LH was undetectable after the second vaccination, increased during buserelin treatment but remained lower than in control stallions (time p=0.003, time x group p<0.001). Serum FSH concentration decreased after vaccination and increased with buserelin treatment in vaccinated but even more in control stallions after both hemi-castrations (time p<0.001, time x group p<0.001, group p=0.008). Relative mRNA abundance for LH receptor (LH-R) and aromatase (CYP19A1) were lower in testicular tissue of vaccinated than control stallions at C1 (p<0.001). At C2, LH-R mRNA abundance no longer differed between groups, whereas CYP19A1 mRNA remained lower in vaccinated than in control stallions (p<0.001). Relative FSH receptor (FSH-R) mRNA abundance was higher in vaccinated than in control stallions at C1 (p<0.001) and similar in both groups at C2. Immunolabelling for LH-R was lower in testes of GnRH-vaccinated compared to control stallions at C1 and C2 (p=0.007), whereas FSH-R protein expression at no time differed between groups. Aromatase immunostaining was reduced at C1 in vaccinated stallions and increased until C2 (time p=0.035, time x group p<0.0031, group p<0.001). In conclusion, endocrine testicular function can be advanced in GnRH-vaccinated stallions by daily low dose buserelin treatment.