Sperm cryopreservation of the Indian major carp, Labeo calbasu: Effects of cryoprotectants, cooling rates and thawing rates on egg fertilization

A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsever's solution (296 mOsmol kg−1) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80 °C) with two cooling rates (5 and 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia). Based on post-thaw motility, the 10 °C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P = 0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40 °C for 15 s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P = 0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P = 0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1 × 105:1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.