Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation

Abstract

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0°C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me₂SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me₂SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%–40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me₂SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me₂SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me₂SO added prior to storage (mean (±SEM) 46 ± 7% relative to initial; 39 ± 6% absolute). Sperm were also stored for 6 days at 0°C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.