Slow freezing and 3D

This study This study aimed to evaluate two conservation times (2h vs 6h) at 4 ºC of prepubertal goat testicles for subsequent slow freezing and 3D in vitro culture. Testicular fragments from six animals were preserved at 4 ºC for 2 or 6 h. After 2 or 6 h of conservation, the fragments were randomly fixed, constituting the control (CTR2; CTR6), cultured in vitro (IVC2; IVC6), frozen (SFZ2; SFZ6), or frozen and cultured in vitro (SFZIVC2; SFZIVC6). All fragments were analyzed by classical histology (morphology), immunohistochemistry (PCNA), immunofluorescence (β-catenin), PCRq-RT (Oct4, C- kit, Bax, Bcl-2), transmission (TEM), and scanning (SEM) electron microscopy, respectively. The histomorphological data showed that regardless of the conservation time, whether followed by cryopreservation or not, epithelial (Ealt) and nuclear (Nalt) alterations increased after 48 h of IVC (p<0.05). Immunostaining for β-catenin was lower (p < 0.05) in SFZIVC2 and SFZIVC6 fragments compared with IVC2 and IVC6, respectively. Oct4 expression in SFZ6 was lower than that in CTR6 (p < 0.05). Conversely, C-kit expression was similar among CTR2, CTR6, SFZ2, and SFZ6 fragments. Bax and Bcl-2 expression were also similar among IVC2, IVC6, SFZIVC2, and SFZIVC6 fragments. The Bax/Bcl-2 ratio in IVC2 and SFZIVC6 was higher than that observed in CTR2 and SFZ6, respectively. In conclusion, our results demonstrate that controlling the duration of testicular storage at 4 °C before slow freezing and/or in vitro culture directly affects the viability and quality of testicular tissue, thereby influencing the success of reproductive function preservation.