Development and application of low-cost and environment-friendly techniques for fish sperm cryopreservation

Abstract

Fish sperm cryopreservation is a technique that can increase the reproduction in captive efficiency of freshwater and marine fishes. The technique is applied in environmental conservation programs and aquaculture. Over the last few decades, protocols for sperm cryopreservation from many fishes have been established. However, the researchers' main focus was successfully freezing and thawing sperm, neglecting the period in which the gametes remain in contact with the cryoprotective solution until fertilization. Exposure of sperm to cryoprotectant solutions after thawing can be harmful to the quality of the gametes since they can be toxic.The vast majority of the established protocols use ultra-resistant plastic containers to store sperm during the cryopreservation process. These containers usually are not reused, generating highly polluting waste for the environment. Furthermore, in some countries, the containers usually used are sold by a few industries, which makes acquisition difficult and increases the product's price. Thus, the main objective of the thesis was to create and test low-cost methodologies that enhance the use of fish post-thaw sperm and make the sperm cryopreservation process more environmentally friendly.The experiments in the Chapters 1 and 2 were developed in Brazil. We used the South American silver catfish Rhamdia quelen, a species considered an experimental model for native South American fishes. In vitro fertilization and sperm cryopreservation protocols for the species were already established; thus, we based our work on these protocols to apply innovations.In Chapter 1, we tested the use of post-thawing dilution to reduce the toxicity of the cryoprotectant solution. This technique is commonly used in mammalian sperm cryopreservation protocols but has never before been applied to post-thaw sperm of South American fishes. South American silver catfish post-thaw sperm samples were diluted in a saline extender (1.1% NaCl -325 mOsm kg -1 ; pH 7.6). The post-thaw sperm diluted samples showed higher velocities, straightness, progression, and flagellar beat frequency than SUMMARY 2 the cells of undiluted samples (control). The post-thawing dilution also provided higher fertilization and hatching rates than the control group. Thus, the post-thawing sperm dilution proved to be a simple, cheap, and efficient methodology that should be included in the silver catfish sperm cryopreservation protocol.