Optimization of sperm cryopreservation protocol for Basa catfish (Pangasius bocourti)

Abstract

The aim of this study was to optimize a sperm cryopreservation protocol for Basa catfish (Pangasius bocourti). Key factors for the efficiency of cryopreservation, including cryoprotectants, thawing conditions, equilibration times, dilution ratios and freezing methods, were investigated. The suitable time for post-thaw storage as well as pre-freezing cool storage was also examined. Five cryoprotectants (dimethyl sulfoxide, Me2SO; dimethylformamide, DMF; ethylene glycol, EG; propylene glycol, PG; N-methylacetamide, MA) at different final concentrations (5, 10 and 15%) were tested, 10% PG provided the best cryoprotective effect. Thawing temperature at 30–40 °C yielded significantly higher post-thaw motility than 20, 25, 50 or 60 °C. No obvious effect on sperm motility was detected either in equilibrated or thawed samples during a 2-h equilibration. Regarding dilution ratio (semen/cryomedium, v/v), percentage of motile spermatozoa was significantly higher at ratios of 2:1, 1:1 and 1:3 than those with higher ratios (1:5, 1:7 and 1:9). Thawed sperm was sensitive to post-thaw storage, but no reduction in motility was detected within 30 min. Further evaluation of the effective pre-freezing storage time indicated that sperm in diluted form had more advantage in maintaining its freezability, which could be chilled for 24 h before freezing without compromising post-thaw sperm motility. P. bocourti sperm could be successfully cryopreserved with both a programmable freezer and the floating frame technique (frozen 5 or 7 cm above the surface of liquid nitrogen). Cryopreserved sperm (77.5 ± 5.1%) fertilization was not significantly different from fresh sperm (80.9 ± 4.7%) at the ratio of 2 × 105 spermatozoa per egg. Our results provided more detailed suitable conditions for P. bocourti sperm cryopreservation than previous studies. Standardizing the cryopreservation protocol and storage time would be helpful in facilitating artificial reproduction in this species.