Cryogenic preservation of sperm from lane snapper (Lutjanus synagris): Testing the effects of extenders and freezing rates on sperm quality

In this study, we assess the feasibility of cryopreservation of L. synagris sperm for short- (10 days) and long-term (12 month) storage using a combination of intracellular (DMSO or MeOH) and extracellular (egg yolk or milk) cryoprotectants, and two freezing protocols. Milt was mixed with a cryogenic solution (8% extracellular cryoprotectant, and 5%, 8%, 10% or 12% intracellular cryoprotectant) to a ratio of 1:2 (milt: medium), stored in 0.5 mL French straws and frozen using two protocols for each experiment: a Slow freezing curve (dry ice–nitrogen liquid vapor–liquid nitrogen, ca. 15 °C−min) and a Fast freezing curve (dry ice–liquid nitrogen, ca. 30 °C−min), for 10 minutes during each stage. Milt was then thawed in an ambient temperature water bath (28–30 °C). Activation of thawed milt was effective with either saline (450 nM NaCl) or bicarbonate (3.5% NaHCO3) solutions, but the best results were obtained with artificial sea water (ASW) at 870 mOsmol/kg. After 10 days or 12 months of storage, three-factor interactions were found between extenders, cryoprotectant concentrations and the two freezing curves for movement duration, % motility and % viability. All of the three post-thaw sperm components assessed were two to three times greater for the DMSO trials than for the MeOH trials. There was almost no difference in sperm viability and motility between the DMSO extenders with egg yolk or milk after 10 days or 12 months. Greater short- and long-term spermatozoa survival and post-thaw motility were found when semen was diluted 1:2 with extenders of 10% DMSO, 8% egg yolk/milk and 1% Ringer Solution, using a three-step cooling regime with a freezing rate of ca. − 15 °C−min. This study represents the first report of cryopreservation of L. synagris sperm, however further research is needed to improve the effectiveness of the cryopreservation protocol for broad-scale application, taking into account the effects of French straws of greater volume on cooling and freezing rates, and the effect of different thawing rates on spermatozoa properties and fertilization success.

► Lower intracellular cryoprotectant concentrations had less toxic effects on sperm
► Artificial sea water shows the best results in sperm activation after cryopreservation
► Post-thaw sperm components assessed were 2 to 3 times greater for the DMSO trials
► Cryoprotectants showed greater post-thaw semen features with a freezing rate of 15 °C−min
► We report a short- and long-term protocol for L. synagris sperm cryopreservation.