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Isolation and enrichment of type A spermatogonia from pre-pubertal buffalo ( Bubalus bubalis) testis

Authors

  • T. Rafeeqi,

    1. Department of Biochemistry, National Dairy Research Institute, I.C.A.R., Karnal, Haryana, India
    Current affiliation:
    1. Centre of Animal Biotechnology, SKUAST University, Srinagar, J&K, India
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  • G. Kaul

    Corresponding authorCurrent affiliation:
    1. King Edward Memorial Hospital, University of Western Australia, Subiaco, Perth, Australia
    • Department of Biochemistry, National Dairy Research Institute, I.C.A.R., Karnal, Haryana, India
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Correspondence

Dr. Gautam Kaul, Incharge S.B.Lab-I, Department of Biochemistry, National Dairy Research Institute, I.C.A.R., Karnal-132001, Haryana, India.

Tel.: +91 184 2259133;

Fax: +91 184 2250042;

E-mail: gkndri@gmail.com

Summary

The aim of this study was to isolate and subsequently enrich type A spermatogonia from pre-pubertal buffalo testis. Two-step enzymatic digestion was used to isolate spermatogonia from 10 to 14 months pre-pubertal buffalo calves, resulting in maximal release of spermatogonia from the seminiferous tubules. After enzymatic digestion, the type A spermatogonia were subsequently enriched by differential plating and Percoll gradient centrifugation. The identity of type A spermatogonia was determined by light microscopy and further characterised by Dolichos biflorus agglutinin, a specific marker for bovine type A spermatogonia by fluorescence-activated cell sorting analysis. After enzymatic isolation, the cell suspension contained about 27% of type A spermatogonia, which was enriched up to 71% with >70% cell viability. Further flow cytometric analysis showed the presence of THY1+ cells (cells expressing thymocyte differentiation antigen 1), suggesting that THY1 is a conserved marker of the undifferentiated spermatogonial cells in buffalo. The isolation of the enriched type A spermatogonia from buffalo testis opens ways to study the further biochemical characteristics of this important class of germ cells in this species.

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