The effect of freezing rate on the quality of striped bass sperm.

Several studies have been conducted in an attempt to determine the optimal freezing rate for cryopreservation of striped bass (Morone saxatilis) sperm. In this study, the effects of freezing rate (-10 °C, -15 °C, -20 °C, and -40 °C/min) on gamete quality was examined, using Sybr-14 and propidium iodide to determine viability (sperm cell membrane integrity), ATP concentration using a luciferin-luciferase bioluminescence assay, and a CEROS computer-assisted sperm analysis system to characterize striped bass sperm motion. Adult male striped bass (N = 12) were sampled once a week for 5 weeks. Collected samples were extended, cryoprotected using a 7.5% (vol/vol) dimethyl sulfoxide final concentration solution, and frozen using a Planer Kryosave controlled-rate freezer. Samples were stored in liquid nitrogen for 49 days, and sperm quality was re-evaluated after thaw (same methods). Sperm cryopreserved at -40 °C/min resulted in means for total motility (10.06%), progressive motility (7.14%), ATP concentration (0.86 pmol/10(6) cells), and sperm viability (56.5%) that were greater (P < 0.05) than those for slower cooling rates. Therefore, -40 °C/min was the optimal freezing rate (among those tested) for cryopreservation of striped bass sperm.

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

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.