Effects of osmolality on inner mitochondrial transmembrane potential and ATP content in spermatozoa recovered from the testes of striped bass (Morone saxatilis)

The objective was to determine the effects of osmolality on the energy status of testicular spermatozoa of striped bass incubated in a TRIS free base-NaCl medium (pH 8) adjusted to either 300 (T300) or 600 mOsm/kg (T600) with NaCl. High mitochondrial inner transmembrane potential (ΔΨm) was assessed (flow cytometry) with the mitochondrial probe 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) and ATP was measured with a luciferin–luciferase assay. Spermatozoa maintained on ice were equally viable (>95% for T300 and T600) for up to 80 min, whereas sperm viability in artificial fresh water (FW) at 27 mOsm/kg decreased (P < 0.05) to 67% after 5 min, with only 3.5% viability at 25 min. After 20 min of staining, more spermatozoa (P < 0.05) maintained a high ΔΨm 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 number). The Jagg fluorescence was a function of oxidative phosphorylation; 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. After incubation for 30 min in the absence of CCCP, sperm ATP concentration was greater (P < 0.05) in T300 than in T600 (2.0 vs. 0.2 pmol/106 cells), but was below detectability in the presence of CCCP in either medium. In conclusion, we developed a unique approach to assess the energetic status of striped bass spermatozoa during storage and after activation, and concluded that the effects of osmolality must be considered in the design of activating and storage extenders to maintain striped bass sperm motility, viability, and fertility in vitro.