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− 1d-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.