A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad ( Bufo marinus) sperm

Abstract

Previous studies on cane toad ( Bufo marinus; Bufonidae; Anura) sperm cryopreservation were extended to compare the effects of cryopreservation in established sucrose (non-ionic) diluents with cryopreservation in ionic diluents containing amphibian Ringer solutions (with and without egg-yolk). In addition, methanol was tested as a cryoprotectant for B. marinus sperm for the first time. Twenty-seven cryoprotective solutions were trialled, with each containing one of the three diluents [10% (w/v) sucrose, simplified amphibian Ringer (SAR) or SAR/egg-yolk], with one of the three cryoprotectants (Me 2SO, glycerol, or methanol) at one of the three concentrations (10%, 15%, or 20% v/v). Sperm were collected by maceration of testes into cryoprotective solutions with post-thaw recovery assessed as the percentage of motile sperm and the degree (vigour) of motility. Percentage motility was the most sensitive measure of post-thaw recovery. The recovery of motility was lowest in Ringer (SAR) diluents and highest in sucrose diluents, with improved motility in SAR diluents when egg-yolk was added. Methanol was the poorest cryoprotectant and Me 2SO the most effective. Methanol at high concentrations was shown to support recovery in sucrose diluent but not in SAR, although its effectiveness in SAR was improved by egg-yolk. Overall, the efficacy of diluents in supporting a high percentage of sperm recovery was in declining order: sucrose > SAR/egg-yolk > SAR diluents, and with cryoprotectants: Me 2SO > glycerol > methanol. In conclusion, SAR offers less potential as a diluent than sucrose, presumably due to the presence of inorganic ions.