Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Cryobanking is an important tool to preserve the genetic resources of fish species. Semen cryopreservation has been extensively used in conservation programs for endangered species. Here, we aimed to find an effective cryopreservation protocol for the autochthonous Mediterranean brown trout inhabiting the Biferno river (south Italy), in order to create a sperm cryobank. Low-density lipoproteins and sucrose were tested as non-permeating cryoprotectants (NP-CPAs) to replace the egg yolk. Moreover, the thawing rate (10 °C for 30 s vs. 30 °C for 10 s) was also studied. From results obtained in vitro and in vivo, egg yolk emerged as the best NP-CPA and the lower thawing rate recorded better post-thaw semen quality in vitro and higher fertilization and hatching rates in vivo. These findings are important because they will contribute to the creation of a sperm cryobank for Molise’s native trout, which is a milestone of our European project (Life Nat.Sal.Mo)