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United States Department of Agriculture

Agricultural Research Service

Title: Determination of Intracellular Reactive Oxygen Species and High Mitochrondrial Membrane Potential in Viable Boar Sperm Using Fluorescence Activated Flow Cytometry

Authors
item Guthrie, Howard
item Welch, Glenn

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 1, 2006
Publication Date: July 1, 2006
Citation: Guthrie, H.D., Welch, G.R. 2006. Determination of intracellular reactive oxygen species and high mitochrondrial membrane potential in viable boar sperm using fluorescence activated flow cytometry. Journal of Animal Science. 84:2089-2100.

Interpretive Summary: The use of frozen semen in the swine industry is limited by problems with viability and fertility compared to liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species. Chemiluminescence measurements of reactive oxygen species are not possible in live cells and are problematic because of poor specificity. We developed an alternative approach, flow cytometry, to identify sperm containing reactive oxygen species utilizing the dyes hydroethidine and 2,7-dichlorodihydro-fluorescein diacetate as oxidizable substrates and impermeant DNA dyes exclude dead cells. Sperm were incubated with and without reactive oxygen species generators and free radical scavengers. Basal reactive oxygen species formation was low (less than 4%) and did not differ between viable fresh and thawed boar spermatozoa. In addition, fresh and thawed sperm were equally susceptible to intracellular formation of reactive oxygen species produced by xanthine/xanthine oxidase, 94.4 and 87.9% of sperm, respectively. Hydrogen peroxide was likely the primary cause of dye oxidation and inhibition of sperm motion for the reactive oxygen species generators tested.

Technical Abstract: The use of frozen semen in the swine industry is limited by problems with viability and fertility compared to liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. We developed an alternative approach, flow cytometry, to identify viable boar sperm containing reactive oxygen species utilizing the dyes hydroethidine and 2’,7’-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes exclude dead sperm. Sperm were incubated with and without reactive oxygen species generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ between viable fresh and thawed boar sperm. In addition, fresh and thawed viable sperm were equally susceptible to intracellular formation of reactive oxygen species produced by xanthine/xanthine oxidase, 94.4 and 87.9% of sperm, respectively. Catalase and superoxide dismutase (SOD) treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not SOD, was capable of attenuating ROS induced inhibition of motility. While basal intracellular hydrogen peroxide formation was low in viable fresh and thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.

Last Modified: 12/21/2014
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