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)

The Mediterranean brown trout is one of the most endangered freshwater 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% 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%

The Use of Ovarian Fluid as Natural Fertilization Medium for Cryopreserved Semen in Mediterranean Brown Trout: The Effects on Sperm Swimming Performance

In the context of the “Life—Nat.Sal.Mo” project, obtaining an effective semen cryopreservation protocol was an important milestone that allowed the implementation of the first European cryobank of native Mediterranean brown trout (S. cettii) inhabiting Molise rivers (Italy). The main use of our semen cryobank is represented by its practical application in artificial reproduction practices aimed at maximizing the genetic variability of the offspring and reducing the genetic introgression in the native trout populations in the project area. Thus, the choice of the most suitable activation/fertilization medium represents the last key step in the development of artificial reproduction protocols using cryopreserved semen. Therefore, the aim of the present study was to investigate the effect of ovarian fluid as a natural activation media of spermatozoa on the post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid. Our results suggest that the presence of ovarian fluid alone or in combination with D-532 in the artificial microenvironment of reproduction represents a key factor to increase the success of fertilization when the frozen semen of Mediterranean brown trout is used

Femoral Plaque Echogenicity and Cardiovascular Risk in Claudicants

OBJECTIVES: The present study was designed to verify whether the evaluation of femoral plaque echogenicity might be a useful tool for cardiovascular risk assessment in patients affected by lower extremity peripheral arterial disease. BACKGROUND: Lower extremity peripheral arterial disease is a common manifestation of atherosclerosis and is associated with a high risk of developing major cardiovascular events. Vulnerable atherosclerotic plaque plays a central role in the occurrence of acute ischemic events in different vascular territories. Furthermore, atherosclerosis is a systemic disease, and the presence of an unstable atherosclerotic plaque in a certain vascular district, characterized by low echogenicity at B-mode ultrasound, is associated to a greater prevalence of unstable plaques in other vascular beds. METHODS: Femoral plaque echogenicity of 246 claudicants with ankle/brachial index ≤0.90 was evaluated at B-mode ultrasound by visual analysis and by calculating the grayscale median (GSM) value. In these patients, the occurrence of myocardial infarction and stroke was prospectively assessed. RESULTS: Femoral GSM values and plaque types assessed by visual analysis were highly correlated by Spearman analysis (rho = 0.905, p < 0.001). During a median follow-up of 30 months, 32 patients (13%) had a major cardiovascular event. Compared with patients without events, those who experienced an event during the follow-up had a lower femoral plaque GSM value (42.9 ± 26.2 vs. 58.8 ± 19.3, p = 0.002) and a higher prevalence of hypoechoic femoral plaque at visual analysis (68.8% vs. 19.6%, p < 0.001). At Cox analysis, femoral GSM showed an inverse relationship with cardiovascular risk, even after adjustment for possible confounders (hazard ratio: 0.96, 95% confidence interval [CI]: 0.95 to 0.98, p < 0.001). Furthermore, patients with hypoechoic femoral plaques at visual analysis had a 7.24-fold increased cardiovascular risk compared with patients with hyperechoic plaques after adjustment for possible confounders (95% CI: 3.23 to 16.22, p < 0.001). CONCLUSIONS: This study demonstrates that the presence of hypoechoic atherosclerotic femoral plaques is associated with higher cardiovascular risk in lower extremity peripheral arterial disease patients

Biometrics measurements in red foxes (Vulpes vulpes) in the Picentini area. Part I: intersexual differences between young adult and adult subjects.

A series of biometrics measurements have been obtained in 16 Red foxes (Vulpes vulpes) duly hunted in the Picentini area, outside the protected area. Data recorded included the sex, bodyweight, and age class (young adults 2 y.o.) as determined on tooth wearing by an experienced wildlife operator. Occipito-coccygeal (OC), nasal-occipitalis (NO), tail and total lengths, wither height, ear and hind foot (HF) length, upper and lower canine length and width (UCL, LCL, UCW, LCW) and carnassial length and width (data not shown), were measured directly on the carcasses. Condilo-basal (CB), hard palate (HP), scapular, mandibular and upper dentition (UD) lengths, inter-carnassial (IC) and zygomatic (Z) width were measured on latero-lateral and ventro-dorsal radiograms. A Wilcoxon sum rank test was applied to compare variables not normally distributed. For normally distributed variables, homoscedasticity was investigated by using a Levene’s test, post hoc a Student’s t test or a Welch ANOVA was applied. Significance was set at P<0.05. Results are summarized in table 1

Zootechnical Brown Trout (Salmo trutta L. 1758) Ovarian Fluid Fails to Upregulate the Swimming Performances of Native Mediterranean Brown Trout (Salmo cettii Rafinesque, 1810) Sperm in the Biferno River

In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (Salmo cettii) is threatened by genetic introgression with strains of domestic brown trout (Salmo trutta) that were introduced for recreational purposes. The aim of our study was to test if native S. cettii females, rather than zootechnical S. trutta, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native S. cettii vs. zootechnical S. trutta female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical S. trutta fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past

Semen cryopreservation for the Mediterranean brown trout of the Biferno River (Molise-Italy): comparative study on the effects of basic extenders and cryoprotectants

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 &lt; 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 trou

Digital holographic microscopy for the evaluation of human sperm structure

The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization (IVF), and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI). In this paper, we have studied whether Digital Holographic (DH) microscopy may be useful to obtain quantitative data on human sperm head structure and compared this technique to high power digitally enhanced Nomarski microscope. The main advantage of DH is that a high resolution 3-D quantitative sample imaging may be obtained thorugh numerical refocusing at different object planes without any mechanical scanning. We show that DH can furnish useful information on the dimensions and structure of human spermatozoo, that cannot be revealed by conventional phase contrast microscopy. In fact, in this paper DH has been used to evaluate volume and indicate precise location of vacuoles, thus suggesting its use as an additional useful prognostic quantitative tool in assisted reproduction technology (ART)

Dimerization of GPCRs:Novel insight into the role of FLNA and SSAs regulating SST₂ and SST₅ homo- and hetero-dimer formation

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)-based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P &lt; 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P &lt; 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulatesSST2 and SST5 intracellular trafficking induced by octreotide and pasireotide. </p

The potential role of advanced glycation end products in food allergy pathogenesis

prevalence has dramatically increased in the last two decades. Among dietary factors, it has been hypothesized that advanced glycation endproducts(AGEs), present at high level in junk food, could be involved in FA pathogenesis. AGEs are a heterogeneous group of compounds deriving from sugars(sweets and beverages), autoclaved/processed foods, microwaved foods, more roasted/barbecued meat. To evaluate the AGEs levels in FA children compared with healthy controls and subjects with respiratory allergy. Methods: We evaluated paediatric patients with challenge-proven FA, children with respiratory allergy(RA) and age and sex-matched healthy controls. Subcutaneous AGEs levels were evaluated through the AGE reader. Food-frequency questionnaires were evaluated in all study subjects. In vitro studies were performed on human enterocytes(Caco-2 cells) stimulated with 200 mg/ml of BSA-AGE for 24and48 hours to evaluate effects on gut barrier function: mucin2(mucus production), transpithelial electrical resistance(TEER), ZO-1, occludin expression(intestinal permeability). The direct effects elicited on peripheral blood mononuclear cells (PBMCs) after the treatment with 200 mg/ml of BSA-AGE for 48hours, 4and 7days of treatment were also evaluated. RESULTS: 115 subjects were evaluated and subdivided into 3 groups: group 1 patients with FA (n=31); group 2 patients with RA (n=18), group 3 healthy controls (n=66). The consumption of food containing AGEs was higher in subjects with FA compared to RA children and healthy controls (p<0.05). FA and RA children presented significant higher subcutaneous AGEs levels compared to healthy controls (p<0.05). Linear regression analysis confirmed a significant positive correlation between subcutaneous levels of AGEs and consumption of food containing AGEs. Human enterocytes exposed to BSA-AGE treatment showed a reduction of TEER, of Muc2 and tight junction proteins (Occludin and ZO-1). Moreover, the treatment with BSA-AGE on human PBMCs stimulates pro-inflammatory cytokines TNF-α and Th2 cytokines(IL-5 and IL-13)production , but it was unable to modulate IL-10 production. Finally, after7days of treatment with BSAAGE, we found a low percentage of proliferating CD4+T. CONCLUSIONS: Current hypotheses and models of FA do not adequately explain the dramatic increase observed in the last years

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage