The Native Mediterranean brown trout (S. macrostigma) population inhabiting the Molise rivers (South of Italy), is declining as a result of river pollution, poorly regulated fishing activities and the introduction of allochthonous strains for recreational purposes, causing genetic introgression. The conservation status of S. macrostigma in the Mediterranean biogeographical area, according to the Italian report is considered as “critically endangered” by IUCN. The overall status at EU level, taking into account the Mediterranean area, is "bad" (U2) and “in decline”. The status of Italian populations significantly contributes to the overall EU status because the Mediterranean trout populations largely represent the majority of the European population of this species. Semen Cryobanking is a valuable tool in preserving the genetic resources of endangered fish species and it plays an important role in biodiversity conservation and restocking programmes. The semen cryobanks provide the opportunity to preserve representative samples and further reconstruct the original strain, population, or diversity. Finding an efficient freezing protocol for the Molise authotrout will allow the creation of a sperm cryobank. The sperm cryobank of autochthonous Mediterranean trout populations with high genetic variability represents an action within our financed “LIFE” project focusing on the recovery and conservation of this native trout in Molise rivers. Many efforts have been made by our research group in order to find an effective semen protocol and promising results have been obtained so far, we aim to obtain a freezing procedure that reports values of post-thaw quality and fertilization similar to fresh semen (Iaffaldano et al., 2016; Di Iorio et al., 2019; Rusco et al., 2019). The aim of the present work was to test the effectiveness of semen cryopreservation protocol developed recently by Polish researchers for other salmonids (Ciereszko et al., 2017; Judycka et al., 2018) and adptading it for the authoctonus trout from Molise. Two different thawing rates were evaluated. Semen of five individual males was diluted to a final extender concentration of 0.15 M glucose and 7.5% methanol) and loaded into 0.25 mL plastic straws, obtaining a final sperm concentration of 3.0 × 109 sperm/mL. Subsequently, the straws were placed on a 3 cm high frame and equilibrated for 15 min on ice. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above liquid nitrogen level for 5 min and were then placed in liquid nitrogen. The straws were then thawed by immersion in a water bath at 40°C for 5 sec or at 10°C for 30 sec. Sperm motility (CASA system), spermatozoa movement duration (SMD) and sperm viability (SYBR-PI) were then assessed. Motility, SMD and viability were significantly higher in fresh semen compared to the frozen one. However, a significant effect of thawing rate was observed for the motility: higher value were found in semen thawed at 40°C. According to the results obtained, the thawing rate at 40°C for 5 sec emerged as more appropriate. However, this cryopreservation protocol seems to be more effective compared to other cryopreservation protocol developed previosuly in our laboratory. Further studies are necessary to confirm this encouraging results in vivo. Our findings provide a valid contribution for the creation of a sperm cryobank aiming at the restoration of this authochtonous trout in Molise rivers.
The safeguard and conservation of S. macrostigma through the first european semen cryobank
IAFFALDANO N.;RUSCO G.;DI IORIO M.;IAMPIETRO R.;MANCHISI A.;COPPOLA R.
2019-01-01
Abstract
The Native Mediterranean brown trout (S. macrostigma) population inhabiting the Molise rivers (South of Italy), is declining as a result of river pollution, poorly regulated fishing activities and the introduction of allochthonous strains for recreational purposes, causing genetic introgression. The conservation status of S. macrostigma in the Mediterranean biogeographical area, according to the Italian report is considered as “critically endangered” by IUCN. The overall status at EU level, taking into account the Mediterranean area, is "bad" (U2) and “in decline”. The status of Italian populations significantly contributes to the overall EU status because the Mediterranean trout populations largely represent the majority of the European population of this species. Semen Cryobanking is a valuable tool in preserving the genetic resources of endangered fish species and it plays an important role in biodiversity conservation and restocking programmes. The semen cryobanks provide the opportunity to preserve representative samples and further reconstruct the original strain, population, or diversity. Finding an efficient freezing protocol for the Molise authotrout will allow the creation of a sperm cryobank. The sperm cryobank of autochthonous Mediterranean trout populations with high genetic variability represents an action within our financed “LIFE” project focusing on the recovery and conservation of this native trout in Molise rivers. Many efforts have been made by our research group in order to find an effective semen protocol and promising results have been obtained so far, we aim to obtain a freezing procedure that reports values of post-thaw quality and fertilization similar to fresh semen (Iaffaldano et al., 2016; Di Iorio et al., 2019; Rusco et al., 2019). The aim of the present work was to test the effectiveness of semen cryopreservation protocol developed recently by Polish researchers for other salmonids (Ciereszko et al., 2017; Judycka et al., 2018) and adptading it for the authoctonus trout from Molise. Two different thawing rates were evaluated. Semen of five individual males was diluted to a final extender concentration of 0.15 M glucose and 7.5% methanol) and loaded into 0.25 mL plastic straws, obtaining a final sperm concentration of 3.0 × 109 sperm/mL. Subsequently, the straws were placed on a 3 cm high frame and equilibrated for 15 min on ice. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above liquid nitrogen level for 5 min and were then placed in liquid nitrogen. The straws were then thawed by immersion in a water bath at 40°C for 5 sec or at 10°C for 30 sec. Sperm motility (CASA system), spermatozoa movement duration (SMD) and sperm viability (SYBR-PI) were then assessed. Motility, SMD and viability were significantly higher in fresh semen compared to the frozen one. However, a significant effect of thawing rate was observed for the motility: higher value were found in semen thawed at 40°C. According to the results obtained, the thawing rate at 40°C for 5 sec emerged as more appropriate. However, this cryopreservation protocol seems to be more effective compared to other cryopreservation protocol developed previosuly in our laboratory. Further studies are necessary to confirm this encouraging results in vivo. Our findings provide a valid contribution for the creation of a sperm cryobank aiming at the restoration of this authochtonous trout in Molise rivers.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
