Flow cytometry evaluation of DNA integrity of alpaca sperm after cryopreservation with analogues of superoxide dismutase

Authors

  • Alexei Santiani A. Laboratorio de Reproducción Animal, Facultad de Medicina Veterinaria, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
  • Shirley Evangelista V. Laboratorio de Fisiología de la Reproducción Animal, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
  • Carolina Cheuquemán A. Centro de Biotecnología de la Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile
  • Alejandra von Baer H. Criadero Llamas del Sur, Temuco, Chile
  • Jennie Risopatrón G. Centro de Biotecnología de la Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile
  • Raúl Sánchez G. Centro de Biotecnología de la Reproducción (CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile

DOI:

https://doi.org/10.15381/rivep.v23i2.898

Keywords:

cryopreservation, alpaca sperm, superoxide dismutase, flow cytometry, DNA, antioxidants

Abstract

DNA integrity in alpaca spermatozoa was evaluated by flow cytometric analysis in sperm cryopreserved using antioxidants analogues of superoxide dismutase (Tempo and Tempol). Twelve alpaca semen samples were frozen using an extender based on skim milk, fructose, egg yolk, and ethylene glicol. Each sample was divided into three aliquots: Control group, Tempo group (1 mM), and Tempol group (1 mM). Antioxidants were added during cooling at 10 °C. After thawing, samples were fixed using 2% formaldehyde solution and permeabilizated using 0.8% Triton X-100 solution. TUNEL assay and Iodure propidium (PI) were used for evaluation of DNA integrity and cell permeability. Spermatozoa labeled with TUNEL and PI was classified as cells with DNA damaged. Samples were analyzed by flow cytometric using a 488 nm laser, counting at least 10 000 cells per sample, and by epifluorescene microscopy. Frequency of DNA damaged sperm in control group (38.8 ± 16.2%) was significantly higher (p<0.05) than in the Tempol group (16.7 ± 8.4%), whereas the results in the Tempo group (25.4 ± 5.8) were similar to the control and Tempol groups. It was concluded that the addition of superoxide dismutase analogue Tempol (1 mM) during cooling (10 °C) in alpaca sperm cryopreservation partially prevents sperm DNA damage.

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Published

2012-06-29

Issue

Vol. 23 No. 2 (2012)

Section

Artículos Primarios

License

Copyright (c) 2012 Alexei Santiani A., Shirley Evangelista V., Carolina Cheuquemán A., Alejandra von Baer H., Jennie Risopatrón G., Raúl Sánchez G.

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

AUTHORS RETAIN THEIR RIGHTS:

a. Authors retain their trade mark rights and patent, and also on any process or procedure described in the article.

b. Authors retain their right to share, copy, distribute, perform and publicly communicate their article (eg, to place their article in an institutional repository or publish it in a book), with an acknowledgment of its initial publication in the Revista de Investigaciones Veterinarias del Perú (RIVEP).

c. Authors retain theirs right to make a subsequent publication of their work, to use the article or any part thereof (eg a compilation of his papers, lecture notes, thesis, or a book), always indicating the source of publication (the originator of the work, journal, volume, number and date).

How to Cite

Santiani A., A., Evangelista V., S., Cheuquemán A., C., von Baer H., A., Risopatrón G., J., & Sánchez G., R. (2012). Flow cytometry evaluation of DNA integrity of alpaca sperm after cryopreservation with analogues of superoxide dismutase. Revista De Investigaciones Veterinarias Del Perú, 23(2), 182-191. https://doi.org/10.15381/rivep.v23i2.898