Short-term storage of cane toad (Bufo marinus) gametes

The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rave and endangered amphibians, to short-term storage at temperatures > 0 degreesC were studied. Whole excised testes were stored at 0 degrees or 4 degreesC for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degreesC and for 5 days at 4 degreesC. At 15 days, only spermatozoa stored at 0 degreesC still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 96 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 2:20 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degreesC in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degreesC immediately after ovulation lost viability when cooled rapidly to 5 degreesC and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degreesC. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.