Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid1

Abstract

Part of the reduction in boar sperm motility and fertility associated with hypothermic liquid storage and cryopreservation may be due to membrane lipid peroxidation. Lipid peroxidation was monitored by the shift from red to green fluorescence emission of 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), as measured by fluorescence-activated flow cytometry in live sperm (negative for propidium iodide). Experiments were conducted with Percoll-washed sperm to determine the specificity of BODIPY oxidation in the presence of different reactive oxygen species generators and metal chelators. Compared with no FeSO(4) and Na ascorbate, the combination of FeSO(4) and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from <or=1 to >70% and increased (P < 0.05) BOD-IPY fluorescence intensity/cell by 5- to 10-fold after a 30-min incubation. Motility was depressed (P < 0.05) after exposure to FeAc, but viability was not affected. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for 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 presence of the metal chelators EDTA or deferoxamine mesylate at 3 and 9 muM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17 degrees C for 1 and 5 d and the effect of freeze-thawing on basal and FeAc-induced BODIPY oxidation. Basal BODIPY oxidation (no FeAc) was low in liquid stored and thawed viable sperm (1.3 and 3.4%, respectively). Although the incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were susceptible to FeAc-induced lipid peroxidation.