Development of sperm cryopreservation protocol of endangered spiny eel, Mastacembelus armatus (Lacepede 1800) for ex-situ conservation

The study was conducted to develop sperm cryopreservation protocol for the endangered spiny eel, Mastacembelus armatus as well as to use the cryopreserved sperm in breeding. Activation of sperm was evaluated by observing motility in different osmolalities of NaCl solution (48-319 mOsmol/kg) and found that sperm motility decreased with increasing osmolality of the NaCl solution and it was totally inhibited at 319 mOsmol/kg. The highest motility and swimming duration of sperm were observed as 14.5 ± 0.5 min and 4.75 ± 0.25 min at 128 mOsmol/kg and 67 mOsmol/kg, respectively. The concentration of sperm was found to be 1.75 × 1010 to 2.25 × 1010 cells/ml. Evaluation of toxicity of cryoprotectants of dimethyl sulfoxide (Me2SO) and methanol to sperm was performed at 5, 10 and 15% concentrations during 5-30 min incubation period and found that cryoprotectants with 5 and 10% concentrations produced better motility up to 10 min incubation. In the selection of suitable diluent for cryopreservation of sperm, Alsever's solution with 10% Me2SO performed best producing 91.67 ± 1.67% and 65 ± 2.89% equilibration and post-thaw motility, respectively. In breeding trials, sperm preserved with Alsever's solution plus Me2SO produced more or less similar fertilization (82.25 ± 4.46%) and hatching (12 ± 0.65%) to those preserved with 0.9% NaCl solution plus Me2SO as 79.88 ± 4.67% and 11 ± 0.46% fertilization and hatching respectively. Fresh sperm used as control produced 87.63 ± 3.6% and 14 ± 0.5% fertilization and hatching respectively. Both cryopreserved and fresh sperm-derived fry performed well but the control fry showed a significantly better (P < 0.05) growth. Fry derived from four diluents did not show any significant (P > 0.05) growth difference but Alsever's solution plus Me2SO produced better results.