Amides as cryoprotectants for the freezing of Bryconorbignyanus sperm

The objective of this study was to evaluate the use of different amides as an alternative for the cryopreservation of sperm from the endangered species, Bryconorbignyamus, an important fish for aquaculture in Brazil. The cryoprotectants which were combined with Beltsville Thawing Solution™ extender, each at the concentrations of 2.5%, 5%, 7.5%, and 10%, were as follows: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), and (3) methylformamide (MF). The cyoprotectant methylglycol (MG, 10%) was used as a control group. Therefore, 13 treatment groups were performed. The sperm were diluted in each cryoprotectant, loaded into 0.25-mL straws, frozen in a nitrogen vapor vessel (dry shipper), and stored in liquid nitrogen at–196 °C. The following sperm-movement parameters were measured using CASA: total motility (TM), progressive motility (PM), curvilinear speed (VCL), rectilinear speed (VSL), average path velocity (VAP), curved line distance (DCL), straight line distance (DSL), average path distance (DAP), straightness (STR), linearity (LIN), wobble (WOB), cross beat frequency (BCF) and sperm movement duration (SMD). In order to assess DNA fragmentation index, lipid peroxidation, cell integrity, integrity of plasma membrane, membrane fluidity, mitochondrial functionality, and reactive oxygen species, flow cytometry was used. The best results were obtained with 7.5% DMF, among all the amides tested, with sperm motility (66%) significantly higher than the other cryoprotectants evaluated (P < .05). DMF (7.5%) exhibited higher velocities and distances, and lower ROS production and lipoperoxidation, in comparison to MG (P < .05). It also exhibited high membrane integrity (84.5%) and cell integrity (98.7%), and low membrane fluidity (20.3%) (P < .05). MG (10%) exhibited only 38% motility, lower mitochondrial protection (39.8%), and higher DNA fragmentation index (0.07), despite the high membrane integrity observed (85.6%). The DMF concentration of 7.5% demonstrated greater protection of sperm function and cellular structure, and is therefore recommended for the cryopreservation of B. orbignyamus spermatozoa.