Untitled

Iampietro

et al. 2020

Animals

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.

Section: Discussionmentioning

confidence: 99%

A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers

Rusco

Iampietro

et al. 2020

Animals

“…The semen was extended 1/1000 (v:v) with 3% NaCl (w:v), and sperm counts were carried out in duplicate, at a magnification of 400× and expressed as ×10 9 /mL. Sperm motility was subjectively evaluated as reported in our previous paper [5,7]. Briefly, 1 μL of semen was placed on a glass microscope slide, and 10 μL of 0.3% NaCl or 1% NaHCO 3 as an activation solution was added for fresh semen and frozen semen, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Sperm viability was assessed using the LIVE/DEAD Sperm Viability Kit (Molecular Probes, Inc.), which contained the fluorescent stains SYBR-14 and propidium iodide (PI), following the same procedure described in our previous works [5,7].…”

Section: Methodsmentioning

confidence: 99%

“…In this regard, we attempted to find an effective semen cryopreservation protocol for the Mediterranean brown trout ( Salmo cettii ), a native species that inhabits the rivers of Molise [5]. Currently, this species is listed in the Italian IUNC Red List as critically endangered [6], due to river pollution, uncontrolled fishing, and hybridization following the introduction of non-native strains that have drastically reduced the number of the native species in recent centuries [7].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Rusco

Gibertoni³

et al. 2019

Animals

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

“…The decrease of these native populations is the result of a series of anthropogenic activities, such as the river pollution, habitat deterioration, water withdrawals, largely unregulated fishing activities and the introduction of alien species (Iaffaldano et al, 2016a;Iaffaldano et al, 2016b;Lorenzoni et al, 2019;Carosi et al, 2020). In Italian freshwaters, including Biferno and Volturno rivers (Molise region), the introgressive hybridization with the alien trout species is the most important threat to the native species survival (Lorenzoni et al, 2019;Carosi et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

The role of semen cryobanks for protecting endangered native salmonids: Advantages and perspectives as outlined by the LIFE Nat.Sal.Mo. project on Mediterranean brown trout (Molise region – Italy)

Rusco²,

Esposito³

et al. 2023

Front. Mar. Sci.

The Mediterranean brown trout is one of the most endangered freshwater species. A complicated network of climate and human influences has severely harmed its biodiversity. The introduction of alien trout is one of the most serious threats to native populations’ intraspecific diversity. In Molise region (south-Italy) an important conservation program (LIFE Nat.Sal.Mo project) has recently been proposed to preserve the genetic integrity of native Mediterranean trout. This project, alongside safeguarding and re-establishing the habitats’ usefulness aims to restore the genetic integrity of the autochthonous population. This is one of the major goals, and it is accomplished by employing frozen wild breeder semen in conjunction with proper fertilization techniques to carry out artificial reproduction to enhance genetic diversity in the progeny and maintain fitness within self-sustaining populations. In this regard, the implementation of the first European semen cryobank has played a strategic role for conserving extant genomic diversity of native population. The goal of this review is to outline the procedures developed and guidelines established for the creation of a Mediterranean trout sperm cryobank. Here, we specifically provide an overview of some of the main challenges associated with the implementation of semen cryobank, the results achieved, the prospects for restoring genetic integrity in native populations, and lastly, future views for hatchery management to preserve the wild biodiversity of native salmonid species. During the project timeframe 1,683 semen doses, from 150 native breeders were stored inside the cryobank. Our results clearly showed the efficiency of the freezing procedure used, both in vitro and in vivo. In fact, we recorded satisfactory values of post-thaw sperm motility and viability that ranged from 40% to 80%, and excellent fertilization rate in vivo, which ranged from 64% to 81%.