Individual study: Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species
Shishova N.R., Uteshev V.K., Kaurova S.A., Browne R.K. & Gakhova E.N. (2010) Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology, 75, 220-232
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Freeze sperm or eggs for future use
A replicated, controlled study in 2009 of European common frogs Rana temporaria in the Moscow Region, Russia (Shishova et al. 2010) found that recovery of sperm after cryopreservation was high with certain cryoprotectants. Sperm motility was significantly greater with the cryoprotectant dimethyl formamide (motility: 65%; fertilization: 90%) compared to dimethyl sulphoxide (36–44%; 82–90%). High concentrations of dimethyl sulphoxide (6 vs 2–4%) significantly reduced hatching (54 vs 80%) and larval survival (49 vs 70–76%), but not fertilization (80 vs 86–90%). Motility-inhibiting saline and glycerol cryoprotectant resulted in low motility (28%) and zero fertility. Tris buffer in cryoprotectants did not significantly increase motility (43–48 vs 45%) or fertilization (70–81 vs 84%). Maximum fertilization was achieved with spermic urine from hormonally induced males (luteinizing hormone-releasing hormone) at concentrations of 15 x 106/ml (93%). Spermic urine or macerated testes from wild frogs were mixed with simplified amphibian Ringer solution or saline and cryodiluents: 2–12% dimethyl sulphoxide or 12% dimethyl formamide or motility-inhibiting saline and 5% glycerol, with 2.5, 6.5 or 10% sucrose with or without Tris buffer or 5–10% egg yolk. Spermic urine (1.0 x 108 cell/ml) and cryodiluents were frozen at 5–7°C/minute and then stored in liquid nitrogen. Thawing was in a 40°C water bath. Spermic urine, sperm from macerated testes (different concentrations) or thawed sperm in cryodiluents were added to eggs from hormonally induced wild females. Fertilization was assessed after 4–6 hours.