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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 osmolality on testicular striped bass (Morone saxatilis) sperm energy status

Authors
item Guthrie, Howard
item Woods, L - UNIVERSITY OF MARYLAND
item Long, Julie
item Welch, Glenn

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 20, 2008
Publication Date: May 1, 2008
Citation: Guthrie, H.D., Woods, L.C., III, Long, J.A., Welch, G.R. 2008. Effects of osmolality on inner mitochondrial transmembrane potential and ATP content in spermatozoa recovered from the testes of striped bass (Morone saxatilis). Theriogenology. 69:1007-12.

Interpretive Summary: Hybridized striped bass are an important component of the fish industry. The problems in this industry are sperm motility is maintained for only 60 minutes after activation and in vitro fertilization is inefficient because viability can be maintained for only a few hours during low temperature storage of striped sperm. An experiment was conducted to determine the effect of osmolality on the energy status of testicular sperm of striped bass incubated in a TRIS free base-NaCl medium (pH 8) adjusted to 300 (T300) and 600 mOsm/kg (T600) with NaCl. High mitochondrial inner transmembrane potential was measured by flow cytometric analysis of the fluorescence of a mitochondrial fluorescent probe and ATP was measured by a luciferin-luciferase assay. Sperm remained equally viable (> 95%) for up to 80 min at 24 oC in T300 and T600 while viability in sperm incubated in artificial fresh water (FW) at 27 mOsm/kg decreased (P < 0.05) after 5 min and only 3% of cells were viable at 25 min. More sperm (P < 0.05) were energized in terms of maintaining a high mitochondrial transmembrane potential in T300 than in T600; 80% and 50%, respectively. The high mitochondrial inner transmembrane potential was shown to be a function of oxidative phosphorylation because the percentage of cells maintaining a high transmembrane potential was decreased to 3% in the presence of an uncoupler of cell respiration and oxidative phosphorylation, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Sperm ATP concentration was greater (P < 0.05) at 300 mOsm/kg (2 pmoles/million cells) than in T600 (0.2 pmoles/million cells) and was reduced to non-detectable levels in the presence of CCCP in either medium. In conclusion these results show that the effects of osmolality must be considered in the design and use of activating and storage/freezing solutions to maintain striped bass sperm motility, viability, and fertility in vitro.

Technical Abstract: An experiment was conducted to determine the effect of osmolality on the energy status of testicular sperm of striped bass incubated in a TRIS free base-NaCl medium (pH 8) adjusted to 300 (T300) and 600 mOsm/kg (T600) with NaCl. High mitochondrial inner transmembrane potential (''m) was measured by flow cytometric analysis of the fluorescence of the molecular aggregates of a mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) and ATP was measured by a luciferin-luciferase assay. Sperm remained equally viable (> 95%) for up to 80 min at 24 oC in T300 and T600 while viability in sperm incubated in artificial fresh water (FW) at 27 mOsm/kg decreased (P < 0.05) to 67% after 5 min and only 3.5% of cells were viable at 25 min. More sperm (P < 0.05) maintained a high ''m in T300 than in T600; 80% and 50%, respectively. Sperm JC-1 aggregate (Jagg) fluorescence intensity was also greater (P < 0.05) in T300 than in T600 (10 and 5 channel units, respectively). The Jagg fluorescence was shown to be a function of oxidative phosphorylation because, the percentage of cells containing Jagg fluorescence decreased to 3% in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) an uncoupler of cell respiration and oxidative phosphorylation. Sperm ATP concentration was greater (P < 0.05) in T300 (2 pmoles/106 cells) than in T600 (0.2 pmoles /106 cells) and was reduced to non-detectable levels in the presence of CCCP in either medium. In conclusion these results show that the effects of osmolality must be considered in the design and use of activating and storage/freezing solutions to maintain striped bass sperm motility, viability, and fertility in vitro.

Last Modified: 10/1/2014
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