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

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

Development of Cryoprotective Media for Low-Temperature Freezing of Sterlet (Acipenser Ruthenus) Sperm

“…2). The obtained results confirm the data of other scientists who observed a lower quality of the thawed sperm of sturgeons and salmonids, which was diluted by the cryoprotective solution based on methanol [11,24,25].…”

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants, antioxidant, membrane stabilizer, equilibration time and dilution ratio on sperm motility and fertility

“…; Linhart et al . ). Acrosomal reaction activation in sperm samples cryopreserved with DMSO is thought to contribute to this phenomenon (Lahnsteiner et al .…”

“…) and paddlefish (Horvath et al . ; Horvath, Urbányi, Mim, Bean, Gomelsky & Tiersch ; Linhart, Mims, Gomelsky, Cvetkova, Cosson, Rodina, Horvath & Urbanyi ) sperm. In most studies, DMSO has been reported the best CPA, probably due to its small molecular size, fast penetration into spermatozoa and by its interaction with the phospholipids of the sperm membrane (Suquet, Dreanno, Fauvel, Cosson & Billard ).…”

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

“…; Adams, Smith, Roberts, Janke, King, Tervit & Webb ), rainbow trout (Lahnsteiner, Weismann & Patzner ; Conget et al . ) and paddlefish (Linhart, Mims, Gomelsky, Cvetkova, Cosson, Rodina, Horvath & Urbanyi ; Horváth, Urbányi, Wang, Onders & Mims ), where a higher CPA concentration was required for the programmable freezing technique.…”