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United States Department of Agriculture

Agricultural Research Service


item Garner, D
item Dobrinsky, John
item Welch, Glenn
item Johnson, Lawrence

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Development of reliable methods to determine the viability of sperm in the laboratory has been a long sought goal. Fluorescent stains in combination with flow cytometry offer potential for reliable methodology. SYBR-14 is fluorescent stain that stains living sperm very brightly and is highly correlated with sperm viability. To determine the extent of its value we inseminated swine with semen treated with it and recovered eggs and embryo to see if there was any detrimental effects to fertilization. We found tha fertilization was reduced somewhat but no more than with other fluorescent stains (Hoechst 33342) that we currently use. Embryo development progresse normally once fertilization took place. These results aid us in seeking to establish a method for estimating viability of sperm in the laboratory. Future experiments will be devised using this information.

Technical Abstract: The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine its effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead sperm in ejaculated and epididymal semen. Flow cytometric analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained sperm in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained sperm. Gilts were inseminated with unstained sperm or sperm stained with two levels of SYBR-14 or two levels of the reference stain, Hoechst 33342. Embryos were recovered at 42-48 hr post-insemination were morphologically evaluated and only 8-cell embryos were continued in culture. Overall, fluorescent staining of boar sperm with SYBR-14 or Hoechst 33342 did not affect ability to fertilize oocytes, nor the developmental competence of the resultant embryos.

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