Protocol

Advanced Protocols in Oxidative Stress II

Volume 594 of the series Methods in Molecular Biology pp 163-171

Date:

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

  • H. David GuthrieAffiliated withU. S. Department of Agriculture, Biotechnology and Germplasm Laboratory, Agricultural Research Service
  • , Glenn R. WelchAffiliated withU. S. Department of Agriculture, Biotechnology and Germplasm Laboratory, Agricultural Research Service

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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