Semen cryobank plays a valuable role in biodiversity preservation of fish species at risk of extinction. According to the Italian freshwater fish Red list, the Mediterranean trout, Salmo cettii, is listed as critically endangered. In this regard, the ‘Nat.Sal.Mo.’ project aims to recover and conserve the native S. cettii populations of Molise rivers. The creation of a sperm cryobank is a project milestone, therefore the development of a semen cryopreservation protocol was an important goal to achieve. However, since the sampling sites in the project area are often not easily accessible, distant from each other and from the laboratory, the wide time that elapses between collection and processing of semen could negatively affect its freezability. In light of these considerations, two possible scenarios were developed to evaluate the effect of cool storage time intervals (1 h and 6 h) on both fresh and cryopreserved semen motility parameters and post-thawed fertilizing ability. Eggs and semen samples were collected by stripping. Each ejaculate (n = 10) was split into two aliquots and stored on ice for 1 h and 6 h. After each time interval, the sperm 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 (LN2) vapor 3 cm above the LN2 level for 5 min. The semen was thawed at 40 °C/5 s. Fresh and post-thawing sperm motility was evaluated by the CASA system. Fertilization trials were performed using three groups of eggs (N −~ 90) inseminated with fresh sperm, and sperm frozen 1 and 6h post-collection. In fresh semen significant decreases (p < .05) from 1 to 6 h of storage were recorded for total motility (93.7 vs. 57.3%), movement duration (36.1 vs. 28.6 s) and beat cross frequency (6.4 vs. 4.6 Hz). When the sperm was frozen, only the total motility was significantly reduced (p < .05) from 1 to 6h (52.1% vs. 39.8%). No significant differences of fertilization rates (% eyed eggs) between the two storage times using frozen sperm (59.5% vs. 57.4%) were found. In conclusion, we showed that after 6 h of cool storage time post-collection, the post-thawing semen quality is preserved, and its fertilizing capacity is not compromised. However, the cool storage time significantly affects the freezability of fresh semen, eliminating the most cool-sensitive populations.
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