Effect of antioxidant supplementation on pig and horse gamete storage

According to recent studies, antioxidant supplementation on gamete processing and/or storage solutions improvesgamete quality parameters, after cooling or storage at sub zero temperature. The aim of the present study was to investigate the effects of antioxidant supplementation on pig and horse gamete storage. The first study aimed to determine the effects of resveratrol (RESV) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. RESV(2µM) was added during: IVM (A); 2 h post-warming incubation (B); vitrification/warming and 2 h post-warming incubation (C); all previous phases (D). The obtained data demonstrate that RESV supplementation in the various steps of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage. In the second work different concentrations of RESV (10, 20, 40, and 80µM) were added during liquid storage of stallion sperm for 24 hours at either 10°C or 4°C, under anaerobic conditions. Our findings demonstrate that RESV supplementation does not enhance sperm quality of stallion semen after 24 hours of storage. Moreover, the highest RESV concentrations tested (40 and 80µM) could damage sperm functional status, probably acting as pro-oxidant. Finally, in the third work other two antioxidants, ascorbic acid (AA) (100 µM) and glutathione (GSH) (5mM) were added on boar freezing and/or thawing solutions. In our study different sperm parameters were evaluated before freezing and at 30 and 240 minutes after thawing. Our results showed that GSH and AA significantly improved boar sperm cryotolerance, especially when supplemented together to both freezing and thawing media. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels.

Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

Marinetti ultimo mitografo

La ricognizione delle opere composte da Filippo Tommaso Marinetti tra il 1909 e il 1912 è sostenuta da una tesi paradossale: il futurismo di Marinetti non sarebbe un'espressione della modernità, bensì una reazione anti-moderna, che dietro a una superficiale ed entusiastica adesione ad alcune parole d'ordine della seconda rivoluzione industriale nasconderebbe un pessimismo di fondo nei confronti dell'uomo e della storia. In questo senso il futurismo diventa un emblema del ritardo culturale e del gattopardismo italiano, e anticipa l’analoga operazione svolta in politica da Mussolini: dietro un’adesione formale ad alcune istanze della modernità, la preservazione dello Status Quo. Marinetti è descritto come un corpo estraneo rispetto alla cultura scientifica del Novecento: un futurista senza futuro (rarissime in Marinetti sono le proiezioni fantascientifiche). Questo aspetto è particolarmente evidente nelle opere prodotte del triennio 1908-1911, che non solo sono molto diverse dalle opere futuriste successive, ma per alcuni aspetti rappresentano una vera e propria antitesi di ciò che diventerà il futurismo letterario a partire dal 1912, con la pubblicazione del Manifesto tecnico della letteratura futurista e l'invenzione delle parole in libertà. Nelle opere precedenti, a un sostanziale disinteresse per il progressismo tecnologico corrispondeva un'attenzione ossessiva per la corporeità e un ricorso continuo all'allegoria, con effetti particolarmente grotteschi (soprattutto nel romanzo Mafarka le futuriste) nei quali si rilevano tracce di una concezione del mondo di sapore ancora medioevo-rinascimentale. Questa componente regressiva del futurismo marinettiano viene platealmente abbandonata a partire dal 1912, con Zang Tumb Tumb, salvo riaffiorare ciclicamente, come una corrente sotterranea, in altre fasi della sua carriera: nel 1922, ad esempio, con la pubblicazione de Gli indomabili (un’altra opera allegorica, ricca di reminiscenze letterarie). Quella del 1912 è una vera e propria frattura, che nel primo capitolo è indagata sia da un punto di vista storico (attraverso la documentazione epistolare e giornalistica vengono portate alla luce le tensioni che portarono gran parte dei poeti futuristi ad abbandonare il movimento proprio in quell'anno) che da un punto di vista linguistico: sono sottolineate le differenze sostanziali tra la produzione parolibera e quella precedente, e si arrischia anche una spiegazione psicologica della brusca svolta impressa da Marinetti al suo movimento. Nel secondo capitolo viene proposta un'analisi formale e contenutistica della ‘funzione grottesca’ nelle opere di Marinetti. Nel terzo capitolo un'analisi comparata delle incarnazioni della macchine ritratte nelle opere di Marinetti ci svela che quasi sempre in questo autore la macchina è associata al pensiero della morte e a una pulsione masochistica (dominante, quest'ultima, ne Gli indomabili); il che porta ad arrischiare l'ipotesi che l'esperienza futurista, e in particolare il futurismo parolibero posteriore al 1912, sia la rielaborazione di un trauma. Esso può essere interpretato metaforicamente come lo choc del giovane Marinetti, balzato in pochi anni dalle sabbie d'Alessandria d'Egitto alle brume industriali di Milano, ma anche come una reale esperienza traumatica (l'incidente automobilistico del 1908, “mitologizzato” nel primo manifesto, ma che in realtà fu vissuto dall'autore come esperienza realmente perturbante).

Caratteristiche merceologiche, nutrizionali, sensoriali e di freschezza di Nasello (Merluccius merluccius), Lanzardo (Scomber japonicus) e Suro (Trachurus trachurus)

Three finfish species frequently caught in the waters of the Gulf of Manfredonia (Apulia, Italy) were studied in order to know how the flesh composition (proximate, fatty acid, macro- and micro- element contents) could be affected by the season effect. The species we examined were European hake (Merluccius merluccius), chub mackerel (Scomber japonicus) and horse mackerel (Trachurus trachurus), which were analysed at the raw state in three catch season and after cooking in two catch season. More precisely, European hake and chub mackerel caught during winter, summer and fall were analysed at the raw state. The composition of the flesh of grilled European hake and chub mackerel was study on fish caught in winter and fall. Horse mackerel of summer and winter catches were analysed both at the raw and grilled state. Furthermore, an overall sensory profile was outlined for each species in two catch season and the relevant spider web diagrams compared. On the whole, two hundred and eighty fish were analysed during this research project in order to obtain a nutritional profile of the three species. One hundred and fifty was the overall number of specimens used to create complete sensory profiles and compare them among the species. The three finfish species proved to be quite interesting for their proximate, fatty acids, macro- and micro-element contents. Nutritional and sensory changes occurred as seasons elapsed for chub and horse mackerel only. A high variability of flesh composition seemed to characterise these two species. European hake confirmed its mild sensory profile and good nutritional characteristics, which were not affected by any season effect.

Acqua: solvente elettivo in Organocatalisi e Biocatalisi

Water is a safe, harmless, and environmentally benign solvent. From an eco-sustainable chemistry perspective, the use of water instead of organic solvent is preferred to decrease environmental contamination. Moreover, water has unique physical and chemical properties, such as high dielectric constant and high cohesive energy density compared to most organic solvents. The different interactions between water and substrates, make water an interesting candidate as a solvent or co-solvent from an industrial and laboratory perspective. In this regard, organic reactions in aqueous media are of current interest. In addition, from practical and synthetic standpoints, a great advantage of using water is immediately evident, since it does not require any preliminary drying process. This thesis was found on this aspect of chemical research, with particular attention to the mechanisms which control organo and bio-catalysis outcome. The first part of the study was focused on the aldol reaction. In particular, for the first time it has been analyzed for the first time the stereoselectivity of the condensation reaction between 3-pyridincarbaldehyde and the cyclohexanone, catalyzed by morpholine and 4-tertbutyldimethylsiloxyproline, using water as sole solvent. This interest has resulted in countless works appeared in the literature concerning the use of proline derivatives as effective catalysts in organic aqueous environment. These studies showed good enantio and diastereoselectivities but they did not present an in depth study of the reaction mechanism. The analysis of the products diastereomeric ratios through the Eyring equation allowed to compare the activation parameters (ΔΔH≠ and ΔΔS≠) of the diastereomeric reaction paths, and to compare the different type of catalysis. While morpholine showed constant diasteromeric ratio at all temperatures, the O(TBS)-L-proline, showed a non-linear Eyring diagram, with two linear trends and the presence of an inversion temperature (Tinv) at 53 ° C, which denotes the presence of solvation effects by water. A pH-dependent study allowed to identify two different reaction mechanisms, and in the case of O(TBS)-L-proline, to ensure the formation of an enaminic species, as a keyelement in the stereoselective process. Moreover, it has been studied the possibility of using the 6- aminopenicillanic acid (6-APA) as amino acid-type catalyst for aldol condensation between cyclohexanone and aromatic aldehydes. A detailed analysis of the catalyst regarding its behavior in different organic solvents and pH, allowed to prove its potential as a candidate for green catalysis. Best results were obtained in neat conditions, where 6-APA proved to be an effective catalyst in terms of yields. The catalyst performance in terms of enantio- and diastereo-selectivity, was impaired by the competition between two different catalytic mechanisms: one via imine-enamine mechanism and one via a Bronsted-acid catalysis. The last part of the thesis was dedicated to the enzymatic catalysis, with particular attention to the use of an enzyme belonging to the class of alcohol dehydrogenase, the Horse Liver Alcohol Dehydrogenase (HLADH) which was selected and used in the enantioselective reduction of aldehydes to enantiopure arylpropylic alcohols. This enzyme has showed an excellent responsiveness to this type of aldehydes and a good tolerance toward organic solvents. Moreover, the fast keto-enolic equilibrium of this class of aldehydes that induce the stereocentre racemization, allows the dynamic-kinetic resolution (DKR) to give the enantiopure alcohol. By analyzing the different reaction parameters, especially the pH and the amount of enzyme, and adding a small percentage of organic solvent, it was possible to control all the parameters involved in the reaction. The excellent enatioselectivity of HLADH along with the DKR of arylpropionic aldehydes, allowed to obtain the corresponding alcohols in quantitative yields and with an optical purity ranging from 64% to >99%.

Detection and localization of GLUTs in spermatozoa from different domestic species

Sperm cells need hexoses as a substrate for their function, for both the maintenance of membrane homeostasis and the movement of the tail. These cells have a peculiar metabolism that has not yet been fully understood, but it is clear that they obtain energy from hexoses through glycolisis and/or oxidative phosphorylation. Spermatozoa are in contact with different external environments, beginning from the testicular and epididymal fluid, passing to the seminal plasma and finally to the female genital tract fluids; in addition, with the spread of reproductive biotechnologies, sperm cells are diluted and stored in various media, containing different energetic substrates. To utilize these energetic sources, sperm cells, as other eukaryotic cells, have a well-constructed protein system, that is mainly represented by the GLUT family proteins. These transporters have a membrane-spanning α-helix structure and work as an enzymatic pump that permit a fast gradient dependent passage of sugar molecules through the lipidic bilayer of sperm membrane. Many GLUTs have been studied in man, bull and rat spermatozoa; the presence of some GLUTs has been also demonstrated in boar and dog spermatozoa. The aims of the present study were - to determine the presence of GLUTs 1, 2, 3, 4 and 5 in boar, horse, dog and donkey spermatozoa and to describe their localization; - to study eventual changes in GLUTs location after capacitation and acrosome reaction in boar, stallion and dog spermatozoa; - to determine possible changes in GLUTs localization after capacitation induced by insulin and IGF stimulation in boar spermatozoa; - to evaluate changes in GLUTs localization after flow-cytometric sex sorting in boar sperm cells. GLUTs 1, 2, 3 and 5 presence and localization have been demonstrated in boar, stallion, dog and donkey spermatozoa by western blotting and immunofluorescence analysis; a relocation in GLUTs after capacitation has been observed only in dog sperm cells, while no changes have been observed in the other species examined. As for boar, the stimulation of the capacitation with insulin and IGF didn’t cause any change in GLUTs localization, as well as for the flow cytometric sorting procedure. In conclusion, this study confirms the presence of GLUTs 1, 2 ,3 and 5 in boar, dog, stallion and donkey spermatozoa, while GLUT 4 seems to be absent, as a confirmation of other studies. Only in dog sperm cells capacitating conditions induce a change in GLUTs distribution, even if the physiological role of these changes should be deepened.