Title: 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
Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 4, 2007
Publication Date: May 20, 2007
Citation: Guthrie, H.D., Welch, G.R. 2007. 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. Journal of Animal Science. 85:1402-1411.
Interpretive Summary: Boar sperm gradually lose the ability to fertilize eggs and begin dying during low temperature liquid storage of three days or more or stored in frozen form. This experiment was conducted to determine if this loss of function and viability were caused by storage induced plasma membrane lipid peroxidation. Lipid peroxidation was monitored by the change in fluorescence emission from red to green after the oxidation 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 BODIPY581/591 (BODIPY). The percentage of live sperm cells with BODIPY fluorescence and their fluorescence intensity was measured by fluorescence-activated flow cytometry. Fresh, liquid stored, and thawed boar sperm were very sensitive to the induction of lipid peroxidation using FeSO4/Na ascorbate treatment with increased BODPIY oxidation and loss of sperm movement without increases cell death. However, storage-induced BODIPY oxidation in the absence of FeSO4/Na ascorbate treatment was low in liquid stored and thawed viable sperm 1.3% and 3.4%, respectively. These results indicate that basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing and not a major cause of the increased death and loss of fertilizing capacity associated with storage.
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 change in 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 BODIPY581/591 (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 to no FeSO4/Na ascorbate (FeAc), the presence of FeAc increased (P < 0.01) the percentage of sperm containing oxidized BODIPY 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 microM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17C for 1 and 5 days 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 membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were quite susceptible to FeAc-induced lipid peroxidation.