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Cooled and frozen equine semen shows a reduction in fertility, compared to fresh one. In this study, cooled and frozen-thawed equine spermatozoa were compared and analyzed for plasma and acrosomal membrane integrity and mitochondrial membrane potential, combining three fluorescent probes: H258, CTC, JC-1 with a micro-spectrofluorimetric analysis (Quanticell equipped with a digital system for color images acquisition). Total and progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were measured by CASA system. We employed an innovative approach to study the reproductive potential of the male gamete subjected to cooling protocols for semen storage. In fact, we evaluated the modifications of equine sperm physiology induced by temperature during cooling and freezing treatments looking at the modifications of different functional sperm characteristics by a simultaneous analysis of different sperm markers with the aim of selecting those that are the most efficient signs for sperm fertility. We identified the mitochondrial membrane potential because it provides useful information on equine sperm quality strictly correlated with fertility. We consider it a useful marker for sperm fertility to be used as a guide to select high-quality semen to be employed in equine breeding farmers.

This work aimed to survey the slaughtering-linked plasma modifications of some stress-related hormones in horses subject to butcher standardized procedures. The blood samples of 12 males horses, ageing from 3 to 5 years, have been collected before slaughtering, in lairage, and after stunning by captive bolt gun, during exsanguination. The plasma levels of beta-endorphin, cortisol, epinephrine and norepinephrine have been assayed by EIA. The results indicate that beta-endorphin did not significantly increase after stunning, while cortisol (P

The presence of the μ-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (Nglycosidase F and O-glycosidase) and chemical (ßelimination) treatments. Immunoblotting analysis showed that the μ -opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by Oglycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the μ -opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galß(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the μ-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the μ-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after Nglycosidase F treatment, whereas it was preserved in the apical region after ß-elimination. Most non-ciliated cells expressed the μ-opioid receptor with both N- and Olinked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and ßelimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the μ -opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the μ-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the μ-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.

Artemisia annua L. è una pianta aromatica erbacea annuale appartenente alla famiglia delle Asteracee, endemica della Cina, ma ormai naturalizzata in Europa e in America. E' oggi coltivata in diversi paesi asiatici per l'estrazione di un composto chiamato artemisisina che risulta molto efficace nel combattere la malaria. L'artemisisina chimicamente è un sesquiterpene lattone caratterizzato da un ponte endoperossidico, al quale viene riconosciuta l'attività antimalarica. Lo scopo delle nostre ricerche è quello di mettere a punto colture in vitro di Artemisia annua L. per la produzione di artemisisina. Sono stati indotti calli da porzioni di foglie incubate al buio in vari mezzi di coltura, tutti con base minerale MS. I risultati preliminari finora ottenuti indicano che il sistema in vitro deve essere migliorato, e per questo pensiamo di utilizzare alcuni precursori della biosintesi del metabolita o di manipolare i parametri fisici e chimici per aumentarne la biosintesi.

The opioid receptor antagonist, naloxone, has been shown to have beneficial effects in the kidney and to be implicated in renal salt and water balance. In the present study the signal transduction pathways utilized by naloxone were studied in an epithelial cell line model of the cortical collecting duct, A6 cells. We found that naloxone has a dual effect depending on the concentration used: at a low concentration (10(-6) M) it antagonized the beta-endorphin-dependent increase in cytoplasmic calcium [Ca(2+)](i), while at higher concentrations (10(-5) M) it increased [Ca(2+)](i) and intracellular inositol phosphate levels. While naloxone-induced increases in [Ca(2+)](i) occurred in the absence of external calcium, it was significantly stimulated by increasing the external calcium concentration, suggesting that naloxone increases [Ca(2+)](i) via both calcium release and calcium influx. In polarized A6 cell monolayers naloxone inhibited the activity of the Na(+)/H(+) exchanger (NHE) only when added to the basolateral cell surface. This inhibition of the NHE was prevented by pretreatment of the cells with either the intracellular calcium chelator, BAPTA or with the protein kinase C inhibitor, calphostin C. These findings demonstrate that naloxone induces a rapid increase in intracellular calcium which inhibits the NHE via the calcium-dependent protein kinase C regulatory pathway.