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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT OF NEW TECHNOLOGIES AND METHODS TO ENHANCE THE UTILIZATION AND LONG-TERM STORAGE OF POULTRY, SWINE AND FISH GERMPLASM Title: Effects of hypothermic storage of striped bass (Morone saxatilis) sperm on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and viability

Authors
item Guthrie, Howard
item Welch, Glenn
item Theisen, D -
item Woods, L -

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 23, 2010
Publication Date: March 20, 2011
Citation: Guthrie, H.D., Welch, G.R., Theisen, D., Woods, L.C. 2011. Effects of hypothermic storage of striped bass (Morone saxatilis) sperm on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and viability. Theriogenology. 75:951-961.

Interpretive Summary: Aquaculture of the Moronidae family of fish requires a hybrid line such as produced by in vitro fertilization of eggs from white bass females using semen from striped bass males. Semen must be collected from wild striped bass males during spring spawning or from captive males hormonally induced to spermiate during the spawning season of white bass females. The use of striped bass semen is complicated by the lack of a good method of storage of the semen before use. Experiments were conducted to determine the effect of hypothermic 24 h storage of striped bass sperm cells on viability, intracellular Ca2+ content, mitochondrial membrane potential, and reactive oxygen species (ROS) formation, motion activation, and ATP concentration. Semen was stored for 1 or 24 h at 4 oC in an O2 atmosphere undiluted (raw) or diluted 1:4 (one volume semen with 3 volumes) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH = 8). Compared to 1 h storage for 24 h caused an increase in intracellular calcium and decreased viability; however mitochondria transmembrane potential and ATP did not change. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation. The inclusion of EGTA in T350 blocked increased intracellular calcium and decreased the incidence of cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased to 30% after 24 h. However, activation failed in the presence of EGTA at 1 or 24 h. The increased intracellular calcium found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.

Technical Abstract: Experiments were conducted to determine the effect of hypothermic 24 h storage of striped bass sperm cells on viability, intracellular Ca2+ ([Ca2+]i), mitochondrial membrane potential (D'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 oC in an O2 atmosphere undiluted or diluted 1:4 (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma (SP) 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 intracellular [Ca2+]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 in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM EGTA blocked CaCl2-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased to 30% after 24 h. However, activation failed in the presence of EGTA at 1 or 24 h. During storage D'm was not affected by storage time or treatment. In contrast, sperm ATP was greater at 1 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 intracellular calcium found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.

Last Modified: 10/25/2014
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