Cryopreservation of spermatozoa obtained postmortem from the European common frog Rana temporaria

Abstract

Cryopreserved spermatozoa offers a reliable, efficient and cost-effective means to perpetuate the genetic variation of endangered amphibian species in concert with conservation breeding programs. Here we describe successful cryopreservation of testicular spermatozoa of the common frog Rana temporaria, preliminarily stored in the carcasses of decapitated animals at +4°C for 0, 1 and 4 days. The motility, membrane integrity and fertilisation capability of fresh testicular spermatozoa treated with cryoprotective medium supplemented with 15% dimethylformamide (DMF) or 15% dimethylsulfoxide (DMSO) were examined. DMSO had a significantly greater toxic effect on fresh frog spermatozoa than DMF. Low levels of DNA fragmentation were seen in spermatozoa stored in the testis for different times and then treated with DMF (mean (±s.e.m.) 8.2±0.7% and 18.2±1.8% after 0 and 4 days storage respectively). After 1 day of storage in frog carcasses, the quality of spermatozoa cryopreserved with DMF was not significantly different from that of control spermatozoa (0 days of storage). After 4 days of storage, the quality of frozen–thawed spermatozoa was significantly lower in the DMF-treated than control group: 35% of the spermatozoa cryopreserved with DMF retained motility, 25% maintained the ability to fertilise fresh oocytes and 80% of fertilised oocytes survived to hatch.