Detection of lipid peroxidation in equine spermatozoa based upon the lipophilic fluorescent dye C1l-BODIPY581/591.
The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescence as detected by either fluorescence microplate reader or by flow cytometry. The addition of Fe2SO4 (0 to 0.5 mM), low concentrations of sodium ascorbate, and the addition of cumene hydroperoxide increased peroxidation of C11-BODIPY581/591. The addition of high concentrations (10 or 20 mM) of sodium ascorbate or alpha-tocopherol reduced peroxidation of C11-BODIPY581/591 during short-term incubations. During storage at 5 degrees C in a skim milk-based extender, equine spermatozoa demonstrated a progressive decline in motility and a small but significant increase in lipid peroxidation based upon ratiometric analysis of C11-BODIPY581/591. The addition of Fe2SO4 increased lipid peroxidation in cooled spermatozoa in a dose-dependent fashion and decreased sperm motility. The addition of alpha-tocopherol, however, did not reduce lipid peroxidation during cooled semen storage. These data demonstrate that the lipophilic fluorescent probe C11-BODIPY581/591 is a useful measurement of lipid peroxidation in equine spermatozoa and that there is an increase in lipid peroxidation during cooled storage of equine spermatozoa that is increased in the presence of ferrous promoters.
Ball
Barry A
BA
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis 95616, USA. baball@ucdavis.edu
Vo
Anthony
A
D000818:Animals; D001372:Aza Compounds / Q000494:pharmacology; D001555:Benzene Derivatives / Q000494:pharmacology; D002250:Carbon Radioisotopes; D003080:Cold Temperature; D004357:Drug Synergism; D005227:Fatty Acids / Q000494:pharmacology; D005456:Fluorescent Dyes; D006736:Horses / Q000378:metabolism*; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D009994:Osmolar Concentration; D016877:Oxidants / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D024502:alpha-Tocopherol / Q000494:pharmacology
D001372:Aza Compounds; C463799:BODIPY581; D001555:Benzene Derivatives; D002250:Carbon Radioisotopes; D005227:Fatty Acids; D005456:Fluorescent Dyes; D016877:Oxidants; D024502:alpha-Tocopherol; C007164:cumene hydroperoxide