|Garner Duane L, - UNIVERSITY OF NEVADA|
|Yue S T, - EUGENE, OREGON|
|Roth B L, - EUGENE, OREGON|
|Haugland R P, - EUGENE, OREGON|
Submitted to: Journal of Andrology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 1994
Publication Date: N/A
Interpretive Summary: A rapid and accurate method for estimating the potential fertilizing capacity of semen is needed. Despite past efforts, past attempts to predict fertility of semen have been only partially successful. Therefore, additional means are needed to discriminate among quality factors in semen samples of agriculturally important animals. A new DNA stain combination using SYBR-14 to stain the living sperm bright green and propidium iodide, a traditional stain that stains the heads of dead sperm red, was examined as a means to assess sperm viability. This stain combination was found to be effective in determining the viability of fresh or cryopreserved bull sperm. These studies demonstrated that SYBR-14, when used in combination with PI, was effective for simultaneously visualizing both the living and dead populations of sperm. This staining technique was used effectively to assess the proportions of living and dead sperm in fresh and cryopreserved bovine semen. These experiments with bull sperm provide the necessary background so that this staining system can be adapted for use in other species including pigs, sheep, goats, and turkeys.
Technical Abstract: A new membrane permeant DNA stain, SYBR-14, was used in combination with 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 green. Aliquots containing nearly all living bovine sperm were prepared using glass woll/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, wer 85.1, 68.8, 39.8, 20.7 and 1.4 for rations of 100:0, 75:25, 50:50, 25:85 and 0:100 of filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% BSA and incubated for 0, 3, 6 and 24 hr at 36 C to assess changes in cell viability. As cell death occured during this incuabtion period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from 6 bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of thes samples, which had been processed and staored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.