This study was designed to determine: (i) the in vitro effects of different freezing rates on post-thaw semen quality of Mediterranean brown trout (Salmo trutta macrostigma) from the Biferno river; and (ii) the in vivo fertilization and hatching percentage of freezing rate giving rise to the best post-thaw semen quality. Pooled semen samples were diluted 1:3 (v:v) in a freezing extender composed of 300 mM glucose, 10% egg yolk and 10% dimethyl sulfoxide (DMSO). The extended semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen surface (1, 5 or 10 cm) for 10 min to give three different freezing rates. Semen samples were thawed at 30°C for 10 s. The variables assessed after thawing were sperm motility, duration of motility and viability. Our results clearly indicate a significant effect of freezing rate on post-thaw semen quality. Semen frozen 5 cm above the liquid nitrogen surface showed the best quality after freezing/thawing. Based on these in vitro data, 2 groups of 200 eggs were fertilized with fresh semen or semen frozen 5 cm above the liquid nitrogen surface. Fertilization and hatching rates recorded for eggs fertilized with frozen semen were significantly lower (25.4% and 22.5%, respectively) than the ones obtained using fresh semen (87.8% and 75.5%, respectively). An effective freezing protocol will allow for the creation of a sperm cryobank to recover the original population of Mediterranean brown trout in the Biferno river.

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 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.

This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P < 0.05). These freezing protocols were selected for fertilization trials in vivo . Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trout.

Over the past 20 years, the introduction of new molecular techniques has given a new impetus to genetic and genomic studies of fishes. The main traits selected in the aquaculture sector conform to the polygenic model, and, thus far, effective breeding programmes based on genome-wide association studies (GWAS) and marker-assisted selection (MAS) have been applied to simple traits (e.g. disease resistance and sexual maturation of salmonids) and known Quantitative Trait Loci (QTLs). Genomic selection uses the genomic relationships between candidate loci and SNPs distributed over the entire genome and in tight linkage disequilibrium (LD) with genes that encode the traits. SNP (low and high density) arrays are used for genotyping thousands of genetic markers (single nucleotide polymorphisms, SNPs). The genomic expected breeding value (GEBV) of selection candidates is usually calculated by means of the GBLUP or ssGBLUP (single step) methods. In recent years, in several aquaculture breeding programmes, the genomic selection method has been applied to different fish and crustacean species. While routine implementation of genomic selection is now largely carried out in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), it is expected that, in the near future, this method will progressively spread to other fish species. However, genomic selection is an expensive method, so it will be relevant mostly for traits of high economic value. In several studies (using different salmonid species), the accuracy of the GEBVs varied from 0.10 to 0.80 for different traits (e.g. growth rate and disease resistance) compared to traditional breeding methods based on geneology. Genomic selection applied to aquaculture species has the potential to improve selection programmes substantially and to change ongoing fish breeding systems. In the long term, the ability to use low-pass genome sequencing methods, low-cost genotyping and novel phenotyping techniques will allow genomic selection to be applied to thousands of animals directly at the farm level.

This research aimed to provide a characterization of fresh semen in wild and threatened Mediterranean brown trout (Salmo cettii), inhabiting the Biferno River basin and to study the variance of sperm parameters in a reproductive system with high male competition. The evaluated sperm quality parameters were: volume, density, viability, motility and sperm movement duration. High variability in sperm traits were found probably in response to the heterogeneity of individual condition, social status, spawning activity and migration costs. Duration of motility was positively correlated (P<0.05) with age class of breeders and motility. Stronger positive correlations (P<0.001) were observed between motility and viability and between sperm counts and viable sperms. This study reported the first data about semen quality of an Italian wild population of native trout. These results are useful for planning efficient artificial fertilization protocols in restoration programs involving supportive breeding together with other innovative conservation strategies as gamete cryopreservation.