Abstract
The aim of this study was to test if a cryopreservation procedure using methanol as an internal cryoprotectant and trehalose as an external cryoprotectant could be applied to rainbow trout sperm (Oncorhynchus mykiss). Additionally, the effects of potassium ions and Tris-HCl on post-thaw sperm motility, viability, and fertilizing capacity were analyzed. In this study, we demonstrate for the first time that rainbow trout sperm can be cryopreserved using a trehalose and methanol based extender. Trehalose was found to be effective in rainbow trout sperm cryopreservation at concentrations ranging between 175 and 200mM when supplemented with 10% methanol (v/v). The most effective dilution ratio was 1:11.5, which resulted in a final sperm concentration ranging from 0.8 to 1.6×109 spermatozoa mL−1. A 15-min equilibration period of sperm in the extenders did not influence sperm motility parameters. The highest post-thaw sperm motility was found using extenders containing 175mM trehalose (77.4±10.8) and 175mM trehalose with 40mM KCl (79.3±9.2). The supplementation of the extender with Tris-HCl and the adjustment of the pH to 7.5 and 8.5 resulted in a decrease in post-thaw motility. The post-thaw sperm motility in samples cryopreserved in an extender containing 40mM of KCl did not decrease 60min after thawing. The fertilizing capacity of sperm cryopreserved in 175mM trehalose and 40mM KCl was the highest and did not differ from fresh sperm at a sperm-to-egg ratio of 200,000:1. In conclusion, our results indicate that rainbow trout sperm can be successfully cryopreserved with the application of a trehalose based extender. The supplementation of the cryopreservation extender with 40mM KCl resulted in prolonged post-thaw storage of the sperm, allowing prolonged use of the sperm over time for common fishery practices. Statement of relevanceThis work is related to fish production and conservation.
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