| Autor: |
Costa GMJ; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil. gmjc@ufmg.br., Avelar GF; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., Lacerda SMSN; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., Figueiredo AFA; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., Tavares AO; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., Rezende-Neto JV; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., Martins FGP; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil., França LR; Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil. lrfranca@icb.ufmg.br.; National Institute for Amazonian Research (INPA), Manaus, AM, Brazil. lrfranca@icb.ufmg.br. |
| Abstrakt: |
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. |
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