The safety of zinc gluconate administration in dogs has been confirmed before [5, 7]. In this study the vital signs before and after treatment was recorded to be within normal limits. In other studies, such as the Cedillo et al. , similar results were observed and only 3.1% of dogs treated with zinc gluconate neutralized with arginine revealed ulcers or fistulas on the scrotum; other dogs showed a mild inflammatory response. Also, researchers observed an initial inflammatory response 15 days after the intratesticular injection of zinc gluconate as the acute phase of inflammation . In most studies, mild testicular swelling and inflammation were reported to occur within a few days after treatment which usually resolved spontaneously [5, 7, 21, 28, 29]. Moreover, intratesticular administration of high concentrations of zinc caused an inflammatory reaction that was accompanied by the presence of macrophages, neutrophils, and predominantly CD8-positive lymphocytes . In our study, systemic inflammatory response was not significant and significant difference was observed between dogs only in lymphocytes.
We did not find any significant change in the testosterone concentrations among dogs at the beginning and end of the study (37 days). Similarly, Vanderstichel et al.  did not observe any significant difference in circulating concentrations of testosterone between control and treated dogs between 4 and 6 months after the treatment. Also, in another study, the basal concentration of testosterone decreased initially, but after 2 years, it was not significantly different from untreated dogs . Others  reported that testosterone concentration did not change in dogs after the first injection and during the experiment, but after the second administration of zinc gluconate, testosterone concentrations were lower than the normal range for the untreated male dogs in two different times of experiment (45 and 135 days). Oliveira et al.  reported a decline (40–60%) in the level of testosterone after the injection of a zinc-based solution, especially during the first 30 days, but differences were not significant between the treated and control dogs and the concentration of testosterone was in the normal range for dogs. These contradictory results could be due to differences in concentrations of testosterone among and within dogs, as well as differences in concentrations that can occur throughout the day, due to the pulsatile secretion of luteinising hormone in dogs which results in fluctuations in concentrations of testosterone . In addition, variation in the number of zinc gluconate administrations, chemical composition of treatments, dose, testicular size, and age of dog could also have affected concentrations of testosterone after treatment [15, 26, 31].
Our findings also showed that there was no sign of regeneration in the Leydig cells at the end of the study (37 days) while others reported that the administration of zinc-based solution in the testis of dogs could cause necrosis, lipid degeneration, and death of Leydig cells 5 months after the treatment . However, Vanderstichel et al.  observed a reproliferation and repopulation of Leydig cells after the testicular damage and that the testosterone levels would have increased later . Moreover, unlike in surgical castration, testes were not removed following the intratesticular administration of zinc gluconate in dogs, so the source of testosterone was not eliminated completely .
Others indicated that libido in dogs was not significantly reduced following the injection of zinc gluconate in testes . Many dog owners may prefer the preservation of some behaviors (e.g. guarding behavior) following gonadectomy. Administration of higher doses and repeated injections of zinc gluconate could preserve such behavior, but may cause some reduction in sexual aggression, mounting, libido, and spraying if concentrations of testosterone are reduced . However, it is unlikely to completely suppress male-like behaviors such as roaming, sexual aggression, marking, or mounting as some elevation in concentrations of testosterone above basal concentrations remains likely [5, 6].
In this study, ultrasonography revealed normal structure and pathologic conditions such as abscess of testes. The use of ultrasonography for evaluation of echotexture, focal and diffuse anomalies and determination of testicular volume has been confirmed in dogs [33, 34]. So, it is a useful technique for monitoring changes that may occur following intratesticular injection.