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

Linhart, Otomar and Mims, Steve D. and Gomelsky, Boris and Cvetkova, Ludmila I. and Cosson, Jacky and Rodina, Marek and Horváth, Ákos and Urbányi, Béla (2006) Effect of cryoprotectants and male on motility parameters and fertilization rate in paddlefish (Polyodon spathula) frozen-thawed spermatozoa. Journal of Applied Ichthyology, 22 (s1). pp. 389-394. ISSN 0175-8659 (print), 1439-0426 (online)

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Abstract

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 oC and cooled from 0 Celsius to –5 Celsius at a rate of 3 Celsius.min-1, from –5 Celsius to – 15 Celsius at a rate of 5 Celsius.min-1, from –15 Celsius to –25 Celsius at a rate of 10 Celsius.min(-1), from –25 Celsius to –80 Celsius at a rate of 20 Celsius.min(-1). Thereafter the samples were held for 5 min at -80 Ceslius and finally transferred into LN(2) until next morning. The sperm was then thawed in a water bath at 40 Celsius for 105 s. Fertilization rate of control sperm was 81.5% (kept unfrozen; samples tested after 24-h storage at 3 Celsius), 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 (3.6.105 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.

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