Skip to main content
ARS Home » Research » Publications at this Location » Publication #64412
Title: VIABILITY ASSESSMENT OF MAMMALIAN SPERM USING SYBR-14 AND PROPIDIUM IODIDE

Author
item GARNER, D - UNIVERSITY OF NEVADA-RENO
item Johnson, Lawrence

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The necessity for predicting the potential viability of a sample of semen becomes increasingly more important with the development of more assisted reproduction techniques. Current flow cytometry technology now allows us to measure the potential viability of sperm in more than 10,000 sperm in about a minutes time. Recently new stains have been developed that will attach to the DNA of sperm and can then be measured using flow cytometry. This study was designed to assess the efficacy of the new nucleic acid stain, SYBR-14 in 6 different species. Similar staining patterns were found in all species. Three populations were quantified, live, dead and those sperm which are in the process of degenerating. Overall, the three populations were readily identified in all species. These results will be used to carry out more specific experiments so that specialized protocols can be developed for each species.

Technical Abstract: The proportion of living sperm in semen from six representative mammals was assessed using a dual staining technique composed of the stains SYBR-14 and propidium iodide (PI). SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. Conversely, PI stained only sperm having degenerate nuclear membranes. Sperm from bulls, boars, rams, rabbits, mice and men were stained and examined using fluorescence microscopy. The proportion of living and dead sperm was determined by first staining with SYBR-14 and PI and then assessing stain uptake using flow cytometry. Similar staining patterns were observed in all six mammalian species tested. Three populations of sperm were identified; living-SYBR-14 stained, dead-PI stained and moribund-doubly stained. The SYBR-14 stainin was replaced by PI as sperm progressed from living to moribund. The transition from green (SYBR-14) to red (PI) fluorescence started at the posterior region of the sperm head and proceeded anteriorly. The proportions of living and dead sperm in mammalian semen were readily identified using dual staining with SYBR-14 and PI and quantified using flow cytometry.