Cryopreservation of sperm from Murray cod, Maccullochella peelii peelii

Freshwater fish sperm are inactive in the male reproductive tract and seminal plasma. They are activated under hypotonic conditions, with a brief period of motility at the time of fertilization. The aims were to find a dilution medium that would maintain sperm inactivity, to assess the cytotoxicity of a range of cryoprotectants, and test the effectiveness of selected diluents for cryopreservation of Murray cod sperm. Sperm remained immotile in ionic and non-ionic solutions above 300 mOsm kg(-1). Sperm motility after centrifugation and re-suspension in fresh water was better for solutions of 300 and 600 mOsm kg(-1) than 900 mOsm kg(-1). In the presence of cryoprotectants, dimethylsulfoxide, dimethylacetamide, and methanol, recovery was better at 300 mOsm kg(-1) compared to 600 mOsm kg(-1). Sperm diluted in all media containing glycerol remained inactive after centrifugation and re-suspension in water. A cryopreservation diluent composed of 300 mOsm kg(-1) D-Sorbitol (DS) solution with 10% methanol produced the best post-thaw motility (51.4 +/- 3.4%), followed by Tris-Sucrose-Potassium (TSK) solution with 10% methanol (32.9 +/- 4.7%), and Modified Kurokuras medium with 10% methanol (27.5 +/- 4.8%). Fluorescent staining for membrane integrity (SYBR-14 and Propidium Iodide) reflected the post-thaw motility results. Fertilization trials using sperm frozen in DS solution with 10% methanol produced a hatch rate of 11.0% (63.1 +/- 18.23% of a control fresh sperm hatch rate) and 6.4% (58.5 +/- 32.50% of fresh sperm hatch rate) using sperm frozen with TSK with 10% methanol. This represents the first successful cryopreservation of sperm from Murray cod. Sperm cryopreservation will facilitate both conservation and aquaculture of the Murray cod and related endangered MACCULLOCHELLA species. (C) 2008 Elsevier B.V. All rights reserved.