The cryobank of the semen proved to be an effective strategy to protect the biodiversity of some fish species and the cryopreservation protocol, originally developed for farmed salmonids, was tested for the first time on the sperm of wild native trout that populate the Molise rivers by the scientific project staff.
Thus was born, in Molise, the first European cryobank for the genetic recovery of the Mediterranean trout, with an innovative and effective sperm cryopreservation protocol published in some prestigious international scientific journals.
Between in vitro and in vivo evaluations, the various stages of the freezing and thawing process were tested as well as the optimal ratio of sperm / egg quantity.
From the results obtained in vitro, 40 ◦C for a time of 5 seconds turned out to be the best thawing rate which, in combination with a sperm / egg ratio of 4.5 × 105: 1 produced, in vivo, the highest fertilization rate.
However, even the freezing process has been developed at maximum efficiency: it is known that this process puts the semen at risk, especially the very delicate one of salmonids. The risk of cell damage (due to osmotic or oxidative stress or the formation of ice crystals) is remarkably high.
To reduce the damage and preserve the fertilizing capacities close to fresh sperm as much as possible it is necessary to dilute the sperm to a final extender concentration of 0.15 M glucose and 7.5% methanol, load it into plastic straws of 0, 25 mL, to obtain a final sperm concentration of 3.0 × 109 sperm / mL. Subsequently, the straws are placed on a 3 cm high frame and balanced for 15 min on ice. At this point freezing occurs by exposure to nitrogen vapor for 5 minutes at temperature of -196 °C. The study showed that the process performed correctly returns sperm with motility greater than 80%, an excellent result, very useful for the “reserves” of fish species at risk of extinction.
(excerpt from: Giusy Rusco, Michele Di Iorio, Roberta Iampietro, Stefano Esposito, Pier Paolo Gibertoni, Maurizio Penserini, Alessandra Roncarati, and Nicolaia Iaffaldano, A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers in Animals – 29 February 2020)