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

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Abstract

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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Abbreviations

DMEM:

Dulbecco’s Modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

EG:

Ethylene glycol

FBS:

Fetal bovine serum

GFRA1:

Glial cell line derived neurotrophic factor family receptor alpha 1

MTT:

Methylthiazol tetrazolium

NANOS2:

Nanos C2HC-type zinc finger 2

SSCs:

Spermatogonial stem cells

VASA:

DDX4, RNA helicase expressed in the germ cells

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Acknowledgments

Technical help from Mara Lívia Santos and Barbara Ramalho Ladeira Cardoso are highly appreciated. The support from the Image Acquisition and Processing Center (CAPI- ICB/UFMG) were of great importance.

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Authors and Affiliations

  1. Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil

    Guilherme M. J. Costa, Gleide F. Avelar, Samyra M. S. N. Lacerda, André F. A. Figueiredo, Amanda O. Tavares, José V. Rezende-Neto, Felipe G. P. Martins & Luiz R. França

  2. National Institute for Amazonian Research (INPA), Manaus, AM, Brazil

    Luiz R. França

Authors
  1. Guilherme M. J. Costa
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  2. Gleide F. Avelar
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  3. Samyra M. S. N. Lacerda
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  4. André F. A. Figueiredo
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  5. Amanda O. Tavares
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  6. José V. Rezende-Neto
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  7. Felipe G. P. Martins
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  8. Luiz R. França
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Correspondence to Guilherme M. J. Costa or Luiz R. França.

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Funding information

This study was funded by the National Council for Scientific and Technological Development (CNPq), Foundation to Support Research in the State of Minas Gerais (FAPEMIG), Coordination for the Improvement of Higher Education Personnel (CAPES) and PRPq-UFMG.

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The authors declare that they have no conflict of interest.

Research involving animals

All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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Costa, G.M.J., Avelar, G.F., Lacerda, S.M.S.N. et al. Horse spermatogonial stem cell cryopreservation: feasible protocols and potential biotechnological applications. Cell Tissue Res 370, 489–500 (2017). https://doi.org/10.1007/s00441-017-2673-1

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  • DOI: https://doi.org/10.1007/s00441-017-2673-1

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