Effect of cryoprotectants and male on motility parameters and fertilization rate in paddlefish (Polyodon spathula) frozen-thawed spermatozoa

A sperm cryopreservation method using different cryoprotectants and sperm from different males was developed. Different percentages of pure cryoprotectants (methanol and DMSO) were added to extender 1 or 2 (20 mM tris pH 8 + 30 mM sucrose + 0.5 mM KCl or 20 mM tris pH 8 + 50 mM sucrose + 0.5 mM KCl, dilution 1:1) or non-extended sperm and every 1 ml of mixture was transferred to a 2-ml cryotube. The cryotubes were directly transferred to a pre-programmed PLANER Kryo 10 series III freezer at 0 degrees C and cooled from 0 degrees C to -5 degrees C at a rate of 3 degrees C. min(-1), from -5 degrees C to 15 degrees C at a rate of 5 degrees C. min(-1), from -15 degrees C to -25 degrees C at a rate of 10 degrees C. min(-1), from -25 degrees C to -80 degrees C at a rate of 20 degrees C. min(-1). Thereafter the samples were held for 5 min at -80 degrees C and finally transferred into LN2 until next morning. The sperm was then thawed in a water bath at 40 degrees C for 105 s. Fertilization rate of control sperm was 81.5% (kept unfrozen; samples tested after 24-h storage at 3 degrees C), indicating that the gametes were of good quality. Percentage and the velocity of motile sperm from were evaluated in fresh and post-thawed sperm using video frames and subsequent image analysis. The results on hatching rates were significantly correlated with post-thawed sperm motility (r=0.49, P=0.035) and velocity (r=0.55, P=0.014) and not correlated with velocity of post-thawed spermatozoa (r=0.32, P=0.177). The best fertilization rates were obtained with 64-75 % in post-thawed sperm (3.6.10(5) spermatozoa per egg) when sperm were treated either without any extender or with both extenders with methanol concentrations of 8 or 10 %. These results were not significantly different compared with those obtained using fresh sperm control samples. Hatching rate was very low, only 8-15 %, when sperm was frozen with 8 or 10 % DMSO. ANOVA showed a significant effect of males on sperm motility, velocity and fertilization rate in post-thawed sperm.