| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Biotechnology and Germplasm Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705
2 Corresponding author: dave{at}anri.barc.usda.gov
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.
Key Words: 5,5',6,6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide • cryopreservation • flow cytometry • hydroethidine • motility
This article has been cited by other articles:
|
|
 |
|
  R.J. Aitken and G.N. De Iuliis On the possible origins of DNA damage in human spermatozoa Mol. Hum. Reprod., January 1, 2010; 16(1): 3 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Awda, M. Mackenzie-Bell, and M. M. Buhr Reactive Oxygen Species and Boar Sperm Function Biol Reprod, September 1, 2009; 81(3): 553 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cheng, C. A. Corzo, N. Luetteke, B. Yu, S. Nagaraj, M. M. Bui, M. Ortiz, W. Nacken, C. Sorg, T. Vogl, et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein J. Exp. Med., September 29, 2008; 205(10): 2235 - 2249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. D. Guthrie and G. R. Welch Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid J Anim Sci, June 1, 2007; 85(6): 1402 - 1411. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
