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

Abstract

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.

Vital Analysis of Cryopreserved Sperm of Marbled Flounder, Pseudopleuronectes yokohamae

“…Five sexually mature male marbled flounder were collected from Yeosu special fisheries market (Yeosu, Korea) in January and February of 2020 during the natural peak of its breeding season. Three to five individuals were previously used for the successful sperm cryopreservation of Atlantic halibut, Hippoglossus hippoglossus (Babiak et al, 2006); sterlet, Acipenser ruthenus (Xin et al, 2018b); striped catfish, Pseudoplatystoma magdaleniatum (Herrera-Cruz et al, 2019); and brown Trout (Rusco et al, 2020). Before the sperm collection, the area surrounding the genital pore was dried with a paper towel.…”

Genotyping of Two Mediterranean Trout Populations in Central-Southern Italy for Conservation Purposes Using a Rainbow-Trout-Derived SNP Array

“…Nevertheless, over the years, resource managers have adopted massive allochthonous restocking initiatives for balancing the effects of recreational overfishing. This had dramatically compromised the genetic variability of many Mediterranean trout native populations [ 3 , 12 ], thus several conservation projects have been recently proposed to restore native trout intraspecific biodiversity [ 3 , 5 , 15 , 16 , 17 ].…”

Could the Storage Time Post-collection Affect the Post-thaw Motility and Fertilizing Ability of Mediterranean Brown Trout Semen?

“…Fresh spermatozoa were activated in 0.3% NaCl at a dilution ratio of 1:400. To evaluate the post thawing semen motility the straws were thawed in a 40°C water bath [17] and then activated using a solution with 1% NaHCO 3 at a dilution ratio of 1:30 in order to reach a sperm concentration of 100 × 10 6 sperm/mL. After an aliquot of 3 µL was loaded onto a 20 micron Leja slide (Leja Standard Count, Nieuw Vennep The Netherlands) and the following sperm motility parameters were…”

“…In this regard, six aliquots of equal volume from each sperm sample (n = 10 male), were split into 1.5 ml cryovials and stored on ice for 1, 2, 3, 4, 5 and 6 hours respectively, until freezing (6 intervals × 10 males = 60 total aliquots). After each storage time interval, an aliquot of fresh semen was subjected to the sperm motility evaluation by a Computer-Assisted Sperm Analysis (CASA) system, whilst the other one was frozen using the cryopreservation procedure optimized in our previous work [17]. Brie y, each semen aliquot was diluted with freezing extender in order to reach the concentration of 0.15 M of glucose, 7.5% of methanol and a sperm concentration of 3.0 × 10 9 sperm/mL.…”

“…In this regard, the creation of the rst European semen cryobank results as a "tank" of genetic variability for S. cettii to be used for arti cial breeding activities. In order to obtain an effective freezing protocol aimed at the implementation of the rst European sperm cryobank for the native Mediterranean brown trout, a series of systematic studies were performed [14][15][16][17].…”