Advanced Protocols in Oxidative Stress II pp 163-171

Part of the Methods in Molecular Biology book series (MIMB, volume 594)

Using Fluorescence-Activated Flow Cytometry to Determine Reactive Oxygen Species Formation and Membrane Lipid Peroxidation in Viable Boar Spermatozoa

  • H. David Guthrie
  • Glenn R. Welch
Protocol

Abstract

Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa loaded with, respectively, hydroethidine (HE) or the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C11BODIPY581/591 (BODIPY). ROS was detected by red fluorescence emission from oxidization of HE and membrane lipid peroxidation was detected by green fluorescence emission from oxidation of BODIPY in individual live sperm. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeSo4/ascorbate (FeAc), because menadione and H2O2 had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H2O2; FeAc had no effect. The incidence of basal or spontaneous ROS formation and membrane lipid peroxidation were low in boar sperm (<1% of live sperm) in fresh semen or after low temperature storage; however the sperm were quite susceptible to treatment-induced ROS formation and membrane lipid peroxidation.

Key words

C11-BODIPY581/591 Lipid peroxidation Hydroethidine Flow cytometry Motility 

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • H. David Guthrie
    • 1
  • Glenn R. Welch
    • 1
  1. 1.U. S. Department of Agriculture, Biotechnology and Germplasm LaboratoryAgricultural Research ServiceBeltsvilleUSA

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