Maria Tsantarliotou

Potential of long-term dietary administration of rosemary in improving the antioxidant status of rat tissues following carbon tetrachloride intoxication

In this study, 24 Wistar rats were allocated to 4 groups of 6 animals each. Groups 1 and 2 were fed a basal diet, while groups 3 and 4 were fed the basal diet supplemented further with ground rosemary at 1% level. Following 6-weeks feeding, groups 2 and 4 were injected 1 ml CCl(4)/kg bw and after six hours all animals were sacrificed. Results showed that feeding rosemary before CCl(4) treatment resulted in decline (P<0.05) of the increased aspartate transaminase, alanine transaminase and alkaline phosphatase activities and increase (P<0.05) of the reduced cholesterol and triacylglycerols in serum. It also decreased (P<0.05) lipid peroxidation and increased (P<0.05) the reduced hydroxyl anion radical and hydrogen peroxide scavenging activities in serum, liver, kidney and heart tissues. In addition, it increased (P<0.05) the reduced ABTS radical cation and the superoxide anion scavenging activities in all tissues except in heart and in kidney and heart tissues, respectively. These results suggest that dietary rosemary has the potential to become a promising functional food component.

The effect of melatonin implants on blood testosterone and acrosin activity in spermatozoa of the ram

In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non‐breeding season). Total acrosin activity in spermatozoa was increased between days 35–56 (autumn) and days 49–70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non‐breeding season (after oestrus induction).

Dexamethasone reduces acrosin activity of ram spermatozoa

The aim of this study was to investigate the effect of dexamethasone on acrosin activity of spermatozoa in Chios rams during autumn (breeding season for sheep in Greece), in correlation with possible changes in blood testosterone. Dexamethasone was administered in four equal consecutive intramuscular injections, one every four hours (total dose: 3 mg kg−1). Total acrosin activity was determined in semen samples collected 48 h before and on the 4th and 7th day and thereafter once every week until the 77th day after dexamethasone administration. Blood samples for testosterone radioimmunoassays were collected 24 h before, during dexamethasone administration and on the 4th, 7th, 14th and 21st day after administration. Total acrosin activity in spermatozoa was reduced between days 7–28 after dexamethasone administration. Dexamethasone also induced a reduction in mean value and basal level of blood testosterone and inhibited its episodic secretion between 1 and 4 days after administration. As the reduction of acrosin activity appeared relatively soon after dexamethasone administration (7th day), it is likely that the increased amount of dexamethasone did not influence the synthesis of proacrosin in the late spermatids. As glucocorticoid receptors exist in the epididymis and accessory glands in various species, dexamethasone may have a direct influence on the synthesis and/or release of acrosin inhibitors in epididymal fluid or seminal plasma. These changes in acrosin activity in ovine spermatozoa mediated by dexamethasone may be of importance regarding the role of stress in the reduction of sperm fertilizing ability.

Effect of α-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa

The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe.

CCl4 induces tissue-type plasminogen activator in rat brain; protective effects of oregano, rosemary or vitamin E

The high metabolic rate and relatively low antioxidant defenses of the lipid-rich brain tissue render it highly susceptible to reactive oxygen species (ROS) and oxidative stress, whereas the implication of ROS in the pathogenesis of several diseases in the central nervous system is well-established. The plasminogen activator (PA) system is a key modulator of extracellular proteolysis, extracellular matrix remodeling and neuronal cell signaling and has been implicated in the pathogenesis of these diseases. This study evaluates the role of tissue-type PA (t-PA) in oxidative stress and the protective role of dietary antioxidants in the rat brain. We used the CCl4 experimental model of ROS-induced lipid peroxidation and evaluated the antioxidant effect of oregano, rosemary or vitamin E. CCl4-treated Wistar rats exhibited elevated brain t-PA activity, which was decreased upon long-term administration of oregano, rosemary or vitamin E. PA inhibitor-1 (PAI-1) activity was also slightly elevated by CCl4, but this increase was not affected by the antioxidants. We hypothesize that the CCl4-induced t-PA activity indicates extracellular proteolytic activity that may be linked to neuronal cell death and brain damage. Vitamin E or antioxidants present in oregano or rosemary are effective in inhibiting t-PA elevation and can be considered as a potential protection against neuronal damage.

Simultaneous Determination of Retinoic Acid, Retinol, and Retinyl Palmitate in Ram Plasma by Liquid Chromatography

Abstract A liquid chromatographic method is described for the simultaneous determination of retinoic acid, retinol, and retinyl palmitate in ram plasma. Samples of 0.2 mL are extracted with 2‐propanol‐dichloromethane, the extracts are centrifuged, the supernatants are collected, evaporated, reconstituted in mobile phase, and analyzed on a C8 column using two consecutive isocratic elutions with methanol tetrahydrofuran‐acetate buffer. Detection is performed at 350 and 325 nm using wavelength change during the run. The method exhibits analytical characteristics well within acceptable limits. Overall recoveries were 73.7% for retinoic acid, 90.2% for retinol, and 87.7% for retinyl palmitate. Precision values, expressed as % relative standard deviation, were in the range of 1.16–6.18%, while limits of detection were in the range of 0.3–10.0 ng/mL for all analytes.

Crocetin administration ameliorates endotoxin-induced disseminated intravascular coagulation in rabbits.

Disseminated intravascular coagulation (DIC), a life-threatening secondary complication in several diseases, is characterized by large amounts of thrombin that lead to fibrin deposition and microthrombus formation throughout the microcirculation. Recent in-vitro studies suggest that crocin, crocetin or safranal, carotenoid constituents of the spice Crocus sativus L. (saffron), have antithrombotic properties, especially anti-Xa activity. The present study was designed to evaluate the effect of crocetin on thrombosis procedure using a rabbit model of bacterial endotoxin-induced DIC. Crocetin administration (3 mg/kg), 30 min before the beginning of endotoxin infusion, improved DIC-related haemostatic indices such as platelet blood counts (P ⩽ 0.05), blood plasma fibrinogen and protein C concentration (P ⩽ 0.05). In addition, it ameliorated DIC-associated disease and fibrin deposition in the glomeruli (P ⩽ 0.05). These results indicate that crocetin reveals a preventive antithrombotic role in vivo and prescribe further investigation on the possibility of developing crocetin-based DIC treatment modalities.

Seasonal Variation of Plasminogen Activator Activity in Spermatozoa and Seminal Plasma of Boar, Buck, Bull and Stallion

Plasminogen activators (PA) are proteolytic enzymes present in the spermatozoa and seminal plasma of various species. They play a role in the binding of the spermatozoon and its penetration through the layers surrounding the oocyte. Plasminogen activator activity (PAA) is modulated by hormones that have a seasonal variation, such as testosterone and melatonin. The present study investigates the seasonal variation of PA activity in sperm extracts and seminal plasma of four farm animal species: boar, buck, bull and stallion. Semen samples were collected every second week during a 12-month period and PAA was determined. With respect to sperm enzyme activity, the boar showed a peak from late January until the beginning of April, whereas the activity in the bull was at the highest levels from April until October and gradually declined during autumn and winter period. Plasminogen activator activity of stallion spermatozoa peaked during March and April, and remained low throughout the rest of the year, whereas in the buck sperm, PAA increased from late October until the end of January. No biologically significant variation was detected regarding the seminal PAA activity in any of the species studied. While seasonality of reproduction is typically studied from the female perspective, the present data provide compelling information about a factor that may affect the reproductive ability of the male.

The effect of melatonin on bovine in vitro embryo development

Abstract The aim of the present study was to evaluate the effect of melatonin supplementation during in vitro maturation on fertilization and embryo development in cattle. Bovine cumulus-oocyte-complexes (COC), recovered from abattoir ovaries, were matured in vitro in the absence (control) and in the presence of 10 µM, 100 µM and 1 mM of melatonin. Matured oocytes were fertilized in vitro with frozen-thawed sperm and cultured up to the blastocyst stage. The results of this work demonstrated that melatonin enrichment of the in vitro maturation (IVM) medium does not affect both cleavage (71.0, 72.8, 72.5 and 72.7 % in the control group and in the groups supplemented with 10 µM, 100 µM and 1 mM of melatonin respectively) and blastocyst rates (41.3, 33.8, 39.4 and 38.3 % respectively) in cattle.

Gossypol-induced inhibition of plasminogen activator activity in human and ovine acrosomal extract

The effect of gossypol—a polycyclic compound isolated from cotton seeds—on the plasminogen activator activity of man and ram acrosomal extracts was explored in vitro. The action of gossypol on the plasminogen activator activity was investigated by a spectrophotometric method using the chromogenic substrate S‐2251. Gossypol, a known antispermatogenic agent, was found to effectively inhibit human and ovine acrosomal plasminogen activator activity. The inhibition was dose‐dependent. Plasminogen activator activity from man and ram extracts was completely inhibited by 350 μmol l−1 and 300 μmol l−1 of gossypol, respectively. In additional experiments, low, non‐spermicidal concentrations of gossypol (2.5–40 μmol l−1) were found to significantly inhibit plasmin activity in a dose‐dependent manner. The results suggest that inhibition of both acrosomal plasminogen activator and plasmin activity is a possible mechanism by which gossypol exerts its antifertility effect, since the plasminogen activator/plasmin system plays a role in the whole process of ovum fertilization.