The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty-four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica—Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin β1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA+) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA+) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca.
Bibliographical noteFunding Information:
This study was supported by Grant PIBA15A106 and B17100101 Universidad Nacional Mayor de San Marcos and National Council of Science Technology and Innovation N° 313-2011-CONCYTEC-OAJ. This article includes results of Martha Valdivia ?octoral Thesis of Sciences with mention of Physiology, Universidad Peruana Cayetano
- epididymal sperm mobility
- molecular markers