Cryopreservation of summer flounder, Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high-value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose-based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (-5, -10 and -15 degrees C min(-1)) were evaluated using differential staining to assess post-thaw sperm survival. Seven combinations of the factors examined reduced post-thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 +/- 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at -5 degrees Cmin(-1) (38.4 +/- 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.