Cryopreservation of brazilian flounder (paralichthys orbignyanus) sperm

Abstract

The Brazilian flounder, Paralichthys orbignyanus, is being considered for aquaculture due to its high demand and market price. Reproduction and larviculture studies have demonstrated the feasibility of massive fingerling production, and techniques that prolong life and increase gamete viability can assist in the culture development of this species. The aim of this study was to evaluate the efficiency of two different cryosolutions for cryopreservation of Brazilian flounder semen in order to improve broodstock management and consequently augment the potential for its culture.Two different cryosolutions were tested: a) glycerol–saline: glycerol solution (12% or 1.65 M) along a saline-based diluent (423 mM NaCl, 9 mM KCl, 9.25 mM CaCl2.2H2O, 22.92 mM MgCl2.6H2O, 25.5 mM MgSO4.7H2O and 2.15 mM NaHCO3; pH 8.2; osmolality 900 mOsmol/kg); and b) DMSO–sucrose: DMSO solution (10% or 1.40 M) along a sucrose-based diluent (110 mM Sucrose, 100 mM KHCO3 and 10 mM Tris-Cl; pH 8.2; osmolality 335 mOsmol/kg). Cryopreservation was made without equilibration time. First, 250 μl-straws were placed 6 cm above the surface of liquid nitrogen for 10 min, then they were maintained for 5 min on the surface of liquid nitrogen (1 cm) before being plunged into liquid nitrogen. The quality of cryopreserved sperm was assessed through the percentage of sperm motility and viability, fertilization capacity, hatching and larval viability. Motility was estimated with an arbitrary scale, ranging from 0 to 5. Spermatozoa viability was determined using a LIVE/DEAD® sperm viability kit. Motility of fresh sperm (3.5±0.2) was similar to frozen/thawed sperm with DMSO-sucrose (2.5±0.3) (PN0.05). On the other hand, the motility of frozen/thawed sperm with glycerol-saline (1.3±0.4) was lower than the other two treatments (Pb0.05). No difference was found in the percentage of live spermatozoa post-thawed between DMSO–sucrose and glycerol–saline solutions (Pb0.05).However, fresh sperm had a higher percentage of live spermatozoa than post-thawed sperm with glycerol-saline (Pb0.05). Sperm motility was positively correlated with the percentage of live spermatozoa (Adjusted R2=0.61, n=13). No difference was found for fertilization and hatching rates and larvae viability among the three treatments (PN0.05). This is the first report on successful cryopreservation of Brazilian flounder sperm.This procedure should improve broodstock management techniques for this species and consequently augment the potential for its culture.