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Title: Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa

Author
item Guthrie, Howard
item Welch, Glenn

Submitted to: Advanced Protocols in Oxidate Stress
Publication Type: Book / Chapter
Publication Acceptance Date: 11/17/2008
Publication Date: 1/1/2010
Citation: Guthrie, H.D., Welch, G.R. 2010. Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa. Methods in Molecular Biology (Advanced Protocols in Oxidative Stress II). 594:163-171.

Interpretive Summary: The Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa. ROS was detected by red fluorescence emission from oxidization of hydroethidine and membrane lipid peroxidation was detected by green fluorescence emission from oxidation of C11-BODIPY-581/591 in individual live sperm. The spontaneous formation ROS and membrane lipid peroxidation were low during storage in boar sperm. Only when reactive species generators were added did ROS formation and membrane lipid peroxidation become wide spread in the sperm population. These analyses are important advances in the monitor the quality of sperm during processing and storage.

Technical 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, C11-BODIPY-581/591 (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 FeAc, because menadione and hydrogen peroxide had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and hydrogen peroxide; FeAc had no effect. The incidence of basal or spontaneous ROS formation and membrane lipid peroxidation were low in boar sperm (< 1 percent of live sperm) in fresh or after low temperature storage; however the sperm were quite susceptible to treatment-induced ROS formation and membrane lipid peroxidation.