Spermatogenesis originates from a small population of spermatogonial stem cells, which have the ability to both self-renew and produce differentiated germ cells. We previously established a surrogate broodstock technique using xenotransplantation of spermatogonia in salmonids. This technique promised to be an efficient tool for producing target seeds that are valuable to markets or endangered species. We have been attempting to establish a technique to produce seeds by transplanting spermatogonia proliferated in culture dishes. However, our previous methods for culturing spermatogonia had several defects. First, residual testicular somatic cells infiltrated excessively proliferating cultures and eventually outcompeted spermatogonia. Second, the total number of spermatogonia gradually decreased during culture periods even though mitosis was confirmed. Third, the cultured spermatogonia were less able to be incorporated into the recipient gonads following transplantation as compared to the ability of intact spermatogonia. To overcome these defects, in the present study we improved upon spermatogonia culture conditions. The overgrowth of testicular somatic cells could be suppressed by adjusting fetal bovine serum concentration in the medium to 1%. The addition of soluble factors, such as bovine serum albumin, adenosine, and salmonid serum, to the medium would enhance spermatogonial survival, mitotic activity, and transplantability. Under newly developed conditions, we extended the culture periods. Furthermore, a transplantation assay showed that spermatogonia cultured in the modified medium for 42 days still possessed their transplantability. The present study represents valuable steps toward establishing a culture method enabling spermatogonia to expand in vitro for use in seed production with surrogate broodstock technology.