Horse spermatogonial stem cell cryopreservation: feasible protocols and potential biotechnological applications

The establishment of proper conditions for spermatogonial stem cells (SSCs) cryopreservation and storage represents an important biotechnological approach for the preservation of the genetic stock of valuable animals. This study demonstrates the effects of different cryopreservation protocols on the survival rates and phenotypic expression of SSCs in horses. The cells were enzymatically isolated from testes of eight adult horses. After enrichment and characterization of germ cells in the suspension, the feasibility of several cryopreservation protocols were evaluated. Three different cryomedia compositions, associated with three different methods of freezing (vitrification, slow-freezing and fast-freezing) were evaluated. Based on the rates of viable SSCs found before and after thawing, as well as the number of recovered cells after cryopreservation, the best results were obtained utilizing the DMSO-based cryomedia associated with the slow-freezing method. In addition, when isolated cells were cultured in vitro, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and immunofluorescence analysis indicated that the cryopreserved cells were as metabolically active as the fresh cells and were also expressing typical SSCs proteins (VASA, NANOS2 and GFRA1). Therefore, our results indicate that equine SSCs can be cryopreserved without impairment of structure, function, or colony-forming abilities.