Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa.
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, C(11)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 FeSo4/ascorbate (FeAc), because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); 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.
Guthrie
H David
HD
U. S. Department of Agriculture, Biotechnology and Germplasm Laboratory, Agricultural Research Service, Beltsville, MD, USA.
Welch
Glenn R
GR
D000818:Animals; D005434:Flow Cytometry / Q000379:methods*; D015227:Lipid Peroxidation / Q000502:physiology*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D034421:Sus scrofa
D008563:Membrane Lipids; D017382:Reactive Oxygen Species