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Curtis G Gravance1 , Julie Baumber1 , Barry A Ball1 *
Department of Population, Health and Reproduction, University of California, Davis, CA 1
The fluorescent probe JC-1, labels mitochondria with high membrane potential orange and low membrane potential green. JC-1 staining has been applied to bull and ram sperm mitochondria. JC-1 staining of equine sperm mitochondrial function was assessed by flow cytometry and fluorescence microplate assay. Semen samples were split and sperm prepared by Percoll gradient (PERC) or flash frozen (FF). The following ratios of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 1.0 mM JC-1 for 40 min. An aliquot of each sample was diluted 1:5 and assessed for orange and green staining by flow cytometry (FCM). Aliquots of each sample were placed in a microplate and analyzed for red and green using a fluorescence microplate assay. FCM analyses indicated that the mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments were 92.5, 72.8, 53.4, 27.3 and 8.3, respectively. A treatment effect (P<0.001) was observed between all groups. The expected percentage of sperm with high mitochondrial membrane potential (eg 100% in the 100 group) was correlated with the FCM determined percentage of orange labeled sperm (r=0.99). Green staining of sperm mitochondria was negatively correlated with expected levels of mitochondrial function (r=-0.99). The mean blank-corrected orange intensity, determined by microplate assay, was different (P<0.01) between groups. Blank-corrected orange fluorescence was correlated with the expected percent of sperm with high membrane potential (r=0.73) and with percentage of orange stained sperm determined by FCM (r=0.74). JC-1 staining can detect changes in mitochondrial membrane potential of equine sperm. These changes were detected using two assay methods.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.