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The aim of this study was to evaluate the effect of different cool storage time intervals between collection and semen freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice for 1 to 6 hours, until freezing. Fresh and post-thawing sperm motility were evaluated by Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency was recorded, whilst the amplitude of lateral displacement of the spermatozoon head underwent a significant increase. Velocity parameters (VCL, VAP and VSL) were not affected by the cold storage time, whilst the duration of sperm movement was significantly higher at 1h compared to the other times tested. Freezing procedure overall decreased almost all post-thaw sperm motility parameters, however no significant differences was observed over time, both in term of fast and linear movement. Cool storage time did not significantly affect the percentage of post-thaw eyed embryos. Our results showed that Mediterranean brown trout semen can be stored on ice even up to 6 hours before freezing, without decreasing its post-thawing quality and fertilizing ability.

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.

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 &times, 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 &deg, C/5 s or 10 &deg, 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 &times, 105, 4.5 &times, 105 and 3 &times, 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 &deg, C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 &times, 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.

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p &lt, 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.