Received December 21, 2016
Accepted June 10, 2017

Objective: To compare a new vitrification protocol with reduced cryoprotectant exposition time with the slow freezing protocol in the cryopreservation of prepubertal rat testicular tissue. Methods: Five sexually immature male Wistar rats were submitted to bilateral orchiectomy. Tissue samples from each testicle were fragmented into small pieces and randomly separated into three groups: Group A, fresh tissue (control); Group B, slow programmable freezing (SPF); and group C (vitrification). Frozen/thawed, vitrified/warmed, and fresh testicular tissue were histologically compared. The morphology (cell differentiation, nuclei and epithelium) of 10 seminiferous tubules of each testicle (totalling 100 tubules per Group) were assessed by a pathologist blinded to the procedures. Results: Differentiation between Sertoli and spermatogonial stem cells and the visualization of nucleoli could be easily performed in all tubules assessed in all three groups. Small alterations in tissue architecture were observed in the control group as a result of tissue handling. Moderate alterations of the epithelium with the formation of small gaps and cell detachment from the basal membrane were observed in 28% of frozen and 9% of vitrified tubules. Condensed nuclei involving a small proportion of cells was observed in 6 and 3 tubules of the frozen and vitrified group, respectively. Despite the alterations, 97% of frozen and 99% of vitrified tubules were considered well preserved. Conclusions: The findings indicate that the vitrification protocol tested in this study adequately preserved the morphological integrity of prepubertal testicular tissue in the rat model. Further studies are now required to confirm testicular tissue functionality after grafting.