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Effect of cryoprotectants and male on motility parameters and fertilization rate in paddlefish ( Polyodon spathula ) frozen‐thawed spermatozoa
Author(s) -
Linhart Otomar,
Mims Steve D.,
Gomelsky Boris,
Cvetkova Ludmila I.,
Cosson Jacky,
Rodina Marek,
Horvath Akos,
Urbanyi Bella
Publication year - 2006
Publication title -
journal of applied ichthyology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 62
eISSN - 1439-0426
pISSN - 0175-8659
DOI - 10.1111/j.1439-0426.2007.00992.x
Subject(s) - biology , bella , cryoprotectant , fishery , zoology , cryopreservation , physics , embryo , nuclear physics
A sperm cryopreservation method using different cryoprotectants\udand sperm from different males was developed. Different percentages\udof pure cryoprotectants (methanol and DMSO) were added to\udextender 1 or 2 (20 mM tris pH 8 + 30 mM sucrose + 0.5 mM KCl\udor 20 mM tris pH 8 + 50 mM sucrose + 0.5 mM KCl, dilution 1:1)\udor non-extended sperm and every 1 ml of mixture was transferred\udto a 2-ml cryotube. The cryotubes were directly transferred to a\udpre-programmed PLANER Kryo 10 series III freezer at 0 oC and\udcooled from 0 Celsius to –5 Celsius at a rate of 3 Celsius.min-1, from –5 Celsius to –\ud15 Celsius at a rate of 5 Celsius.min-1, from –15 Celsius to –25 Celsius at a rate of 10\udCelsius.min(-1), from –25 Celsius to –80 Celsius at a rate of 20 Celsius.min(-1). Thereafter\udthe samples were held for 5 min at -80 Ceslius and finally transferred\udinto LN(2) until next morning. The sperm was then thawed in\uda water bath at 40 Celsius for 105 s. Fertilization rate of control sperm\udwas 81.5% (kept unfrozen; samples tested after 24-h storage at 3\udCelsius), indicating that the gametes were of good quality. Percentage\udand the velocity of motile sperm from were evaluated in fresh and\udpost-thawed sperm using video frames and subsequent image analysis.\udThe results on hatching rates were significantly correlated\udwith post-thawed sperm motility (r=0.49, P=0.035) and velocity\ud(r=0.55, P=0.014) and not correlated with velocity of post-thawed\udspermatozoa (r=0.32, P=0.177). The best fertilization rates were\udobtained with 64-75 % in post-thawed sperm (3.6.105 spermatozoa\udper egg) when sperm were treated either without any extender or\udwith both extenders with methanol concentrations of 8 or 10 %.\udThese results were not significantly different compared with those\udobtained using fresh sperm control samples. Hatching rate was\udvery low, only 8-15 %, when sperm was frozen with 8 or 10 %\udDMSO. ANOVA showed a significant effect of males on sperm\udmotility, velocity and fertilization rate in post-thawed sperm