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Viability assessment of mammalian sperm using SYBR-14 and propidium iodide
1School of Veterinary Medicine, University of Nevada, Reno 89557, USA.
Abstract:
The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using 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 nonmotile sperm that had lost their membrane integrity. Sperm from bulls, boars, rams, rabbits, mice, and men were stained and examined through use of fluorescence microscopy. The proportions of living and dead sperm were determined by first staining with SYBR-14 and PI and then assessing stain uptake by 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 staining was replaced by PI staining 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 through use of dual staining with SYBR-14 and PI and quantified through use of flow cytometry.
Insights
A dual staining technique using SYBR-14 and propidium iodide (PI) effectively distinguishes living, dead, and moribund sperm in mammals. This method accurately quantifies sperm viability across species using flow cytometry.
Area of Science:
- Reproductive Biology
- Cell Biology
- Biotechnology
Background:
- Assessing sperm viability is crucial for reproductive success and fertility studies.
- Traditional methods for sperm viability assessment can be time-consuming and subjective.
- Novel staining techniques are needed for accurate and efficient evaluation of sperm health.
Purpose of the Study:
- To evaluate a dual staining technique using SYBR-14 and propidium iodide (PI) for assessing sperm viability.
- To determine the reliability of this technique across six mammalian species.
- To quantify sperm populations (living, dead, moribund) using flow cytometry.
Main Methods:
- Utilized a dual staining technique with SYBR-14 (stains DNA in living cells) and PI (stains DNA in cells with compromised membranes).
- Examined sperm from bulls, boars, rams, rabbits, mice, and men using fluorescence microscopy.
- Quantified sperm viability by analyzing stain uptake via flow cytometry.
Main Results:
- SYBR-14 stained living sperm green, while PI stained dead sperm red.
- A distinct population of moribund sperm was identified, showing dual staining (green to red fluorescence transition).
- Consistent staining patterns and viability proportions were observed across all six mammalian species.
Conclusions:
- The SYBR-14/PI dual staining technique provides a reliable and accurate method for differentiating and quantifying living, dead, and moribund sperm.
- This technique is applicable across diverse mammalian species, offering a standardized approach to sperm viability assessment.
- Flow cytometry enables high-throughput quantification of sperm viability using this dual staining method.