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Cryopreservation of Sperm of Spotted Seatrout (Cynoscion nebulosus)

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Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks' balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCl2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/l NaCl(424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45ºC/min, yielding a retention of initial motility of 31%. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications.

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