Cryobank of Mediterranean Brown Trout Semen: Evaluation of the Use of Frozen Semen up to Six Hours Post-Collection

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

Strategies to improve the postharvest management of flat oyster (Ostrea edulis) from aquaculture using the short-term storage and package in an innovative closed-circuit system

This study aimed to improve postharvest management of flat oysters reared in a longline system in the mid Adriatic Sea, using short-term storage and package in an innovative closed-circuit system. For the trial, 870 oysters were employed, divided into three experimental groups (A, B, and C), N = 270 oysters each group, whereas the remaining 60 oysters were used for the 2 controls. Each group differed in relation to the time spent in the depuration tank and the time of packaging: group A was packed and immediately transferred to the cell; group B was depurated in a tank for 48 h, then packed and transferred to the cell; group C was depurated in a tank for 48 h and then packed, depurated for another 24 h and transferred to a cell. Samples of each group were sampled at different times of permanence in cell (t0) up until 12 days (t12) for biomorphometric, sensorial, nutritional, and microbiological analysis. Although the nutritional and sensorial quality of the oysters was more pronounced in group A, B and C groups also showed good results. In these two groups, thanks to the use of the modern water recirculation system the quality and safety of oysters was improved by reducing the presence of sludge and eliminating fecal contaminants completely than A treatment and seawater control. These results were also confirmed by the tank control, where a more extended depuration period positively influenced the same parameters emphasizing the importance of the adequate depuration processes in oyster productio

Risk of Salmonella transmission via cryopreserved semen in turkey flocks

To investigate the possibility to carry pathogen bacteria in turkey flocks via cryopreserved semen, research was carried out 1) to investigate the microbial contamination of fresh and frozen thawed turkey semen and 2) to evaluate the effect of the freezing-thawing process on the survival of 3 serovars of Salmonella spp. experimentally inoculated in turkey semen. Five pools of semen diluted 4-fold were cooled, added with 8% of dimethylacetamide as a cryoprotectant, and aliquots of 80 μL were directly plunged into liquid nitrogen to form frozen pellets. Mesophilic viable counts, total and fecal coliforms, Enterobacteriaceae, enterococci, Campylobacter spp., and Salmonella spp. were investigated on fresh and thawed samples. Further, 5 pools of diluted semen were each divided into 3 subsamples, inoculated with 7.8 ± 0.2 log cfu·mL ―1 of Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup, respectively, and cryopreserved before to assess the postthaw viability of Salmonella spp. strains. Fresh semen was highly contaminated by all of the saprophytic bacteria investigated and the cryopreservation process reduced the amount of mesophilic viable count and total coliforms (P < 0.05) and fecal coliforms, Enterobacteriaceae, and enterococci (P < 0.01) by about 1 log cfu·mL ―1 . Conversely, neither Campylobacter spp. nor Salmonella spp. were found as endogenous bacteria in semen. In the inoculated semen, both Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup colonies were recovered postthaw, showing a significant reduction of 2.03 ± 0.28, 3.08 ± 0.22, and 2.72 ± 0.23 log cfu·mL ―1 , respectively, compared with the fresh semen (P < 0.001). In conclusion, the cryopreservation process allowed us to obtain a low reduction of microbial count both in endogenous saprophytic bacteria and artificially inoculated Salmonella spp. strains; therefore, the possibility of Samonella spp. transmission to flocks through the use of infected cryopreserved semen does exist

Validation of the Turkey semen cryopreservation by evaluating the effect of two diluents and the inseminating doses

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 7 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 7 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis\u2014CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 7 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates

Finding an effective freezing protocol for Turkey semen: Benefits of ficoll as non-permeant cryoprotectant and 1:4 as dilution rate

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

Dynamics of black hole-neutron star binaries in young star clusters

Young star clusters are likely the most common birthplace of massive stars across cosmic time and influence the formation of compact binaries in several ways. Here, we simulate the formation of black hole-neutron star binaries (BHNSs) in young star clusters, by means of the binary population synthesis code MOBSE interfaced with the N-body code NBODY6++GPU. BHNSs formed in young star clusters (dynamical BHNSs) are significantly more massive than BHNSs formed from isolated binaries (isolated BHNSs): ~40 per cent of the dynamical BHNS mergers have a total mass of &gt; 15 M0, while only ~0.01 per cent of the isolated BHNS mergers have mass in excess of this value. Hence, our models strongly support a dynamical formation scenario for GW190814, given its total mass of ~26 M0, if this event is a BHNS merger. All our dynamical BHNSs are ejected from their parent star cluster before they reach coalescence. Thus, a significant fraction of BHNS mergers occurring in the field might have originated in a young star cluster. The mass spectrum of BHNS mergers from gravitational-wave detections will provide a clue to differentiate between dynamical and isolated formation of BHNSs

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated

New insights on binary black hole formation channels after GWTC-2: young star clusters versus isolated binaries

With the recent release of the second gravitational-wave transient catalogue (GWTC-2), which introduced dozens of new detections, we are at a turning point of gravitational wave astronomy, as we are now able to directly infer constraints on the astrophysical population of compact objects. Here, we tackle the burning issue of understanding the origin of binary black hole (BBH) mergers. To this effect, we make use of state-of-the-art population synthesis and N-body simulations, to represent two distinct formation channels: BBHs formed in the field (isolated channel) and in young star clusters (dynamical channel). We then use a Bayesian hierarchical approach to infer the distribution of the mixing fraction $f$, with $f=0$ ($f=1$) in the pure dynamical (isolated) channel. %that controls the proportion of isolated and dynamical BBHs. We explore the effects of additional hyper-parameters of the model, such as the spread in metallicity $\sigma_{\text{Z}}$ and the parameter $\sigma_{\text{sp}}$, describing the distribution of spin magnitudes. We find that the dynamical model is slightly favoured with a median value of $f=0.26$, when $\sigma_{\text{sp}}=0.1$ and $\sigma_{\text{Z}}=0.4$. Models with higher spin magnitudes tend to strongly favour dynamically formed BBHs ($f\le{}0.1$ if $\sigma_{\text{sp}}=0.3$). Furthermore, we show that hyper-parameters controlling the rates of the model, such as $\sigma_{\rm Z}$, have a large impact on the inference of the mixing fraction, which rises from $0.18$ to $0.43$ when we increase $\sigma_{\text{Z}}$ from 0.2 to 0.6, for a fixed value of $\sigma_{\text{sp}}=0.1$. Finally, our current set of observations is better described by a combination of both formation channels, as a pure dynamical scenario is excluded at the $99\%$ credible interval, except when the spin magnitude is high.Comment: 13 pages, 10 figures, 2 tables, published in MNRA

Guanosine-mediated anxiolytic-like effect: Interplay with adenosine a1 and a2a receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A1R (DPCPX) or A2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [3H]GUO binding became saturable at 100–300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [3H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [3H]GUO binding, but the sum of the two effects was able to displace [3H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A1R and A2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs

Italian semen cryobank of autochthonous chicken and turkey breeds: a tool for preserving genetic biodiversity

The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations.HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in&nbsp;vivo conserved