Development of Sperm Cryopreservation Protocol of Koi Anabas Testudineus

This study was dealt with the development of sperm cryopreservation protocol of koi (Anabas testudineus) and a series of experiments were conducted. Sperm was collected by sacrificing males and suspended in extenders. Activation of sperm motility was evaluated in different osmolalities and found that sperm motility decreased with the increase of osmolality of the extending media and it was completely inhibited above 319 mOsmol/kg. The toxicity of cryoprotectant (DMSO, methanol and ethanol) to sperm was tested at different concentrations (5%, 10%, 15%) and incubation time (5-40 min) and found that cryoprotectants with 5% and 10% concentrations produced better motility during 5 and 10 min incubation. 15% concentration seemed toxic to sperm. Three extenders Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were used for preservation of sperm. Alsever’s solution with 10% DMSO showed highest performance, produced 67.5±5.45% and 50±6.12% equilibration and post-thaw motility respectively. The fertilization and hatching rates were 51.04±2.44% and 33.38±3.82% respectively, for Alsever’s solution plus DMSO which was higher than that of Alsever’s solution plus methanol (40.96±3.85% fertilization and 20.54±0.81% hatching). In contrast, fresh sperm produced 77.96±4.05% fertilization and 60.92±5.33% hatching respectively.