Document Detail

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.
MedLine Citation:
PMID:  16864869     Owner:  NLM     Status:  MEDLINE    
The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with 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. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.
H D Guthrie; G R Welch
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of animal science     Volume:  84     ISSN:  1525-3163     ISO Abbreviation:  J. Anim. Sci.     Publication Date:  2006 Aug 
Date Detail:
Created Date:  2006-07-25     Completed Date:  2006-10-19     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  8003002     Medline TA:  J Anim Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2089-100     Citation Subset:  IM    
Biotechnology and Germplasm Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA.
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MeSH Terms
Dose-Response Relationship, Drug
Flow Cytometry / veterinary*
Hydrogen Peroxide / pharmacology
Membrane Potentials / physiology
Mitochondrial Membranes / physiology*
Povidone / pharmacology*
Reactive Oxygen Species / metabolism*
Semen Preservation / methods,  veterinary
Silicon Dioxide / pharmacology*
Spermatozoa / cytology,  drug effects*,  metabolism
Swine / physiology*
Vitamin K 3 / pharmacology
Xanthine / metabolism
Xanthine Oxidase / metabolism
Reg. No./Substance:
0/Reactive Oxygen Species; 58-27-5/Vitamin K 3; 65455-52-9/Percoll; 69-89-6/Xanthine; 7631-86-9/Silicon Dioxide; 7722-84-1/Hydrogen Peroxide; 9003-39-8/Povidone; EC Oxidase

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