A simple and efficient semen cryopreservation method to increase the genetic variability of endangered mediterranean brown trout inhabiting molise rivers

The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our "LIFE" Nat.Sal.Mo. project.