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Theriogenology. 2011 Mar 15;75(5):951-61. doi: 10.1016/j.theriogenology.2010.10.037. Epub 2011 Jan 17.

Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm.

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1
Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, U S Department of Agriculture, Beltsville, Maryland, USA. dave.guthrie@ars.usda.gov

Abstract

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca(2+) ([Ca(2+)](i)), mitochondrial membrane potential (ΔΨ(m)), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O(2) atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca(2+)](i) marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl(2)-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨ(m) was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca(2+)](i) found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.

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