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Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i‐based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα‐1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+/GFRα‐1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll‐based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα‐1, PLZF, anti‐apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c‐kit and pro‐apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2O2‐induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2‐mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm. [ABSTRACT FROM AUTHOR]