In vitro preservation of morphofunctional integrity of domestic cat testicular tissue up to 22 days.

Domestic cat testicular tissue culture may support fertility preservation when mature spermatozoa are unavailable, but its evaluation requires suitable fixation and culture conditions. This study compared neutral buffered formalin, Bouin's fluid, modified Davidson's fluid, and form-acetic acid for the histological and immunohistochemical assessment of feline testicular tissue, and then evaluated juvenile testicular fragments cultured for 10 or 22 days in a rodent-derived basal medium alone or supplemented with retinol, taurine, or both. Fixation markedly affected tissue morphology and immunohistochemical signal detection. Bouin's fluid, modified Davidson's fluid, and form-acetic acid preserved seminiferous architecture and cellular details better than neutral buffered formalin, which caused more morphological artifacts but yielded stronger immunostaining. Form-acetic acid was selected for culture as the best compromise between morphology and antigen detectability. During culture, seminiferous tubular, fibrotic, and necrotic areas remained generally stable, without consistent effects of retinol, taurine or their combination. Caspase-3-positive cells showed limited time-dependent variation, whereas MCM-7-positive cells remained detectable throughout culture. PGP9.5-positive early germ cells were maintained for 22 days regardless of medium composition. SYCP3-positive spermatocytes, when present in fresh tissue, persisted in some samples at day 22 but did not increase over time. Thus, form-acetic acid is suitable for the morphological and immunohistochemical evaluation of domestic cat testicular tissue, and the organotypic culture system maintained tissue architecture, proliferative cells, and early germ cells for up to 22 days. However, retinol and taurine supplementation did not improve tissue preservation or promote germ cell progression, indicating that further optimization is required to support feline spermatogenesis in vitro.