DUAL DNA STAINING ASSESSMENT OF BOVINE SPERM VIABILITY USING SYBR-14 AND PROPIDIUM IODIDE

A new membrane-permeant DNA stain, SYBR-14, was used in combination wi th propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright gre en. Aliquots containing nearly all living bovine sperm were prepared u sing glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-tha wing and used to provide mixed aliquots containing known ratios of liv ing and dead sperm. Flow cytometry was used to assess the green and re d fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20 .7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEP ES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours a t 36 degrees C to assess changes in cell viability. As cell death occu rred during this incubation period, a relatively rapid transition of s taining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 an d PI to assess the proportion of living and dead cells. Flow cytometri c analyses of these samples, which had been processed and stored in ho mogenized milk, indicated that this stain combination was useful in as sessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viabi lity of fresh or cryopreserved sperm.