Abstract
Simple SummaryThe sperm cryobank is an effective strategy for protecting the biodiversity of the local brown trout population. Thus, the aim of the present work was to test, for the first time, the effectiveness of a semen cryopreservation protocol developed for cultivated salmonid fish on wild trout inhabiting the Molise rivers. Moreover, the effects of two different thawing rates (40°C/5 s and 10°C/30 s) were evaluated in vitro and in vivo. In addition, different sperm-to-egg ratios (6 × 105:1, 4.5 ×105:1 and 3 × 105:1) were also investigated in vivo in order to find an effective freezing protocol useful for the creation of the first European semen cryobank for the Mediterranean brown trout (S. macrostigma = S. cettii). From results obtained in vitro 40 °C turned out to be the best thawing rate, which in combination with a sperm-to-egg ratio of 4.5 × 105:1 was, in vivo, the highest fertilization rate. The important results obtained in the present work corroborated the remarkable validity and effectiveness of the freezing protocol used in this study on the native trout populations from Molise.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.
Highlights
Mediterranean brown trout is currently listed in Annex II of the Habitat Directive and its overall conservation status declared at the EU level as "bad" and “in decline”
European populations of this species. It is currently listed in the Italian IUCN Red List as critically endangered [1] under the accepted taxon for the Italian population, S. cettii. The decline of these native populations was mainly attributed to habitat loss, poorly regulated fishing and genetic introgression following the introduction of non-native strains
Recorded in brown trout, attracted our interest, and prompted us to test the effectiveness of this semen freezing protocol on the wild salmonid of the Mediterranean area (S. macrostigma = S. cettii)
Summary
Mediterranean brown trout is currently listed in Annex II of the Habitat Directive (under the taxon Salmo macrostigma) and its overall conservation status declared at the EU level as "bad" and “in decline” In such a contest, the situation of the Italian populations significantly contributes to the overall EU status because the Mediterranean trout populations largely represent the majority of the European populations of this species. The genetic introgression of native populations caused a loss of their ecological specialization characteristics, such as adaptation to global changes, resistance to diseases, etc
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