Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with C11-BODIPY 581/591

¹ Biotechnology and Germplasm Laboratory, Agricultural Research Service U. S. Department of Agriculture, Beltsville, MD 20705

^* To whom correspondence should be addressed. E-mail: dave{at}anri.barc.usda.gov.

	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, C₁₁BODIPY^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 (ROS) generators and metal chelators. Compared with no FeSO₄ and Na ascorbate, the combination of FeSO₄ and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from 1 to > 70% and increased (P < 0.05) BODIPY fluorescence intensity/cell 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 ROS generators tested, BODIPY oxidation was specific for FeAc as 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 presence of metal chelators EDTA or deferoxamine mesylate at 3 and 9 µM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17° 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. While incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were quite susceptible to FeAc-induced lipid peroxidation.

Key Words: C11-BODIPY^581/591, lipid peroxidation, hydroethidine, flow cytometry, motility