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We evaluated the influence of different media plus various concentrations of Glial cell line‐derived neurotrophic factor (GDNF) during the in vitro culture (IVC) of testicular tissues from prepubertal collared peccary. Testes from 5 individuals were collected, fragmented and cultured for 28 days (34°C and 5% CO2). Culture media were Dulbecco's modified essential medium (DMEM) or stem cell serum free media (StemPro‐34™ SFM), both supplemented with various concentrations of GDNF (0, 10, or 20 ng/mL). Fragments were cultured on the flat surface of 0.75% agarose gel and were evaluated every 7 days for fragment area, histomorphology, cellular viability, and proliferative activity. Data were expressed as mean ± standard error and analyzed by Kruskal–Wallis's and Tukey test. Fragments area decreased over the 28 days‐culture, regardless of the treatment. For morphology, the StemPro‐37 SFM medium plus 10 ng/mL GDNF provided higher scores at all time points in comparison to DMEM using any GDNF concentration (p <.05). After 28 days, similar cellular viability (~70%) was observed in all treatments (p >.05). For proliferating cell nuclear antigen assay, only DMEM plus 10 ng/mL GDNF improved (p <.05) cellular proliferation on Days 14 and 28. Looking at argyrophilic nucleolar organizing regions, after 28 days, there were no differences among treatments regarding cell proliferative capacity for both spermatogonia and Sertoli cells (p >.05). In summary, the DMEM and StemPro‐34 SFM are adequate medium for IVC of prepubertal peccary testicular tissue. Supplementation with GDNF, especially at a 10 ng/mL concentration, appears to be essential for the maintenance of cell survival and proliferation. [ABSTRACT FROM AUTHOR]