The utilization of decellularized extracellular matrix (dECM) derived from animal testis tissue has demonstrated potential as a component of tissue-specific scaffolds. Current research is mostly centered around dECM as a natural resource for culturing testicular cells. This study aimed to assess firstly the comparison of Voytik-Harbin (VH) and Frytes protocol in creating Ram's dECM testis hydrogel and secondly the evaluation of the best protocol effect on in vitro spermatogenesis. Materials and

Using the VH protocol for producing ram TDH resulted in a firm hydrogel with a high frequency of repeat, which may be suited for testicular cell growth.

In this experimental study, the six testes of mature rams were decellularized and the hydrogel production was performed by i. The Frytes protocol utilized a concentration of 1 mg/mL of pepsin, dissolved in either 0.1 or 0.01 M HCl, and ii. The VH protocol was involved 10 mg of pepsin per 100 mg of ECM in 0.5 M of acetic acid. Subsequently, mouse testicular cells were cultivated on collagen hydrogel as the control and the more effective testicular-derived hydrogel (TDH) to evaluate the early stages of in vitro spermatogenesis.

While the Freytes protocol produced a homogeneous pre-gel solution with both HCl concentrations; elevating the pH to 7.4 loosened the hydrogel and made gelation problematic. In contrast, the VH protocol solidified the hydrogel and produced a strong hydrogel due to its gelation consistency. Furthermore, the prepared hydrogel by VH with 25 mg of dECM had a significantly higher priority in terms of rheology and structure (P<0.05). Following mouse testicular cell culture, TDH and collagen hydrogel did not differ significantly in terms of cell survival rates and the mRNA expression of early spermatogenesis genes.