Franco Martelli

Membrane integrity evaluation in rabbit spermatozoa

OBJECTIVES The behaviour of head and tail sperm membrane subjected to different vitality tests was evaluated, searching for possible correlations with sperm motility. STUDY DESIGN On fresh semen from seven rabbits we performed: the Eosin Exclusion Test (EET) and, with different incubation times (T(0), T(5), T(30)), a Hypoosmotic Swelling Test (HOS-test) and a combined Hypoosmotic and Eosin Test (HE-test). Kinetic parameters were determined by using the CASA system CELL-TRAK. Statistical analysis was performed by one way ANOVA and Pearsons correlation coefficient. RESULTS The HOS-test revealed significant differences between percentages of curled spermatozoa at T(0) (67.86+/-4.28) versus T(5) (72.86+/-3.28) and versus T(30) (75.14+/-6.5). Among four types of spermatozoa obtained with the HE-test, percentage of Type 1 sperm (HOS+/Eosine) at T(0) (32.3%+/-6.65), was lower than motile sperm (66.86%+/-9.72) but positively correlated. CONCLUSIONS The HE-test is a rapid instrument for predicting sperm motility, selecting spermatozoa with a high degree of membrane resistance both in the tail and the head compartment.

AN ANALYSIS OF THE EFFECTS OF GALANIN ON GASTRIC-ACID SECRETION AND PLASMA-LEVELS OF GASTRIN IN THE DOG

The effects of galanin on gastric acid secretion and plasma levels of gastrin were studied in conscious dogs chronically fitted with gastric fistulas. Continuous i.v. infusion of galanin (2 micrograms.kg-1.h-1) for 2 h did not affect unstimulated total acid output or plasma levels of gastrin. In contrast, simultaneous i.v. infusion of galanin (1-2 micrograms.kg-1.h-1) inhibited the bombesin-stimulated output of acid whereas the effects of bombesin on gastrin output were not significantly modified. Galanin (2-4 micrograms.kg-1 i.v.) also depressed the secretory response to 2-deoxy-D-glucose without significantly affecting plasma gastrin levels. Galanin (2-4 micrograms.kg-1 i.v.) did not depress bethanechol-stimulated gastric acid output or inhibit histamine-stimulated gastric acid secretion. These findings indicate that glanin inhibits the bombesin- and 2-deoxy-D-glucose-stimulated secretion of gastric acid in conscious dogs by an action which is probably exerted at the level of the cholinergic nerve terminals.

Coenzyme Q10, exercise lactate and CTG trinucleotide expansion in myotonic dystrophy

Steinerts myotonic dystrophy (DM) is a genetic autosomal dominant disease and the most frequent muscular dystrophy in adulthood. Although causative mutation is recognized as a CTG trinucleotide expansion on 19q13.3, pathogenic mechanisms of multisystem involvement of DM are still under debate. It has been suggested that mitochondrial abnormalities can occur in this disease and deficiency of coenzyme Q 10 (CoQ10) has been considered one possible cause for this. The aim of this investigation was to evaluate, in 35 DM patients, CoQ10 blood levels and relate them to the degree of CTG expansion as well as to the amount of lactate production in exercising muscle as indicator of mitochondrial dysfunction. CoQ10 concentrations appeared significantly reduced with respect to normal controls: 0.85 +/- 0.25 vs. 1.58 +/- 0.28 microg/ml (p < 0.05). Mean values of blood lactate were significantly higher in DM patients than controls (p < 0.05) both in resting conditions (2.9 +/- 0.55 vs. 1.44 +/- 1.11 mmol/L) and at the exercise peak (6.77 +/- 1.79 vs. 4.90 +/- 0.59 mmol/L), while exercise lactate threshold was anticipated (30-50% vs. 60-70% of the predicted normal maximal power output, p < 0.05). Statistical analysis showed that serum CoQ10 levels were significantly (p < 0.05) inversely correlated with both CTG expansion degree and lactate values at exercise lactate threshold level. Our data indicates the occurrence of reduced CoQ10 levels in DM, possibly related to disease pathogenic mechanisms associated with abnormal CTG trinucleotide amplification.

Behaviour of mean erythrocyte volume during submaximal treadmill exercise in the horse

Abstract: The aim of this work was to study the role of spleen contraction in the modification of circulating erythrocyte mean volume during treadmill exercise in the horse. Red blood cells count (RBC), haematocrit (Hct), haemoglobin (Hgb), mean cellular volume (MCV), mean cellular haemoglobin (MCH), mean cellular haemoglobin concentration (MCHC) and red cell distribution width (RDW) were evaluated in five Haflinger mares performing a submaximal incremental test on the treadmill. Blood was collected every minute during the trial without stopping the treadmill. Significant changes of all measured parameters, except for MCH, have been observed. Hct values increased significantly from the 2nd minute from the start of the test. MCV and MCHC values were significantly different from their basal values from the 6th minute of exercise, with MCV being increased and MCHC decreased, suggesting cellular swelling. Hct modifications during brief submaximal exercise have been shown to be mainly and directly related to mobilization of the pool of red blood cells stored in the spleen. However, the evidence in this study of different times of onset of changes between Hct and erythrocyte indices (MCV and MCHC) suggests that exercise-induced alterations in red cell indices are not related to spleen emptying.

AFM characterization of rabbit spermatozoa

Atomic force microscopy (AFM) has been applied for determining the topological and structural features of rabbit spermatozoa. Fresh ejaculated spermatozoa were adsorbed passively onto a silicon slide or by motility from suspension onto a poly(L‐lysine)‐coated glass coverslip and then imaged in air and in buffer saline, respectively. AFM images clearly highlighted many details of spermatozoa head, neck, and tail. Distinct features were observed in the plasmatic membrane of spermatozoa. In particular, head topography easily recognized the acrosome, equatorial segment, equatorial subsegment, and postacrosome regions. Moreover, AFM images revealed the presence of double belt of invaginations around the spermatozoa head, at the boundary between equatorial subsegment and postacrosome regions. All together, the collected AFM images clearly defined a detailed map of spermatozoa morphology while giving some hints on the internal structure. Microsc. Res. Tech., 2008.

Application of a constant blood withdrawal method in equine exercise physiology studies

The aim of the present study was to test a constant blood withdrawal method (CBWM) to collect blood samples from horses during treadmill exercise. CBWM was performed in 4 Standardbreds and 5 Haflinger horses. A peristaltic pump was used to control blood aspiration from an i.v. catheter via an extension line. Blood was collected using an automatic fractions collector, with a constant delay time between the drawing of blood and sample collection. Blood withdrawal using CBWM was made during a treadmill standardised exercise test (SET). A blood flow of 12 m/min was used and samples collected every 60 s during the entire period of exercise. The volume of blood collected in each sample tube was 12.1+/-0.2 ml, with a delay time of mean +/- s.d. 25.3+/-0.8 s. Plasma lactate kinetics based on measurement of lactate in each fraction showed an exponential increase during the first 13 min of exercise (10.5 min of SET and 2.5 min recovery). The peak plasma lactate concentration was observed between 2.5 and 5.5 min after the end of SET. CBWM permits the kinetics of lactate and other blood-borne variables to be studied over time. This method could be a valuable aid for use in studying equine exercise physiology.

GABA-A-mediated gastrin release induced by baclofen in the isolated vascularly perfused rat stomach.

In order to investigate the role of peripheral GABA-B receptors, the effects of the putative GABA-B agonist baclofen on immunoreactive gastrin release from an isolated vascularly perfused rat stomach preparation were examined. The vascular infusion of baclofen at graded concentrations induced a dose-dependent increase in gastrin release; this was unaffected by the GABA-B antagonist delta-aminovaleric acid, but was fully prevented by the selective GABA-A antagonist bicuculline as well as by atropine or tetrodotoxin. These results suggest that the stimulant effects of baclofen are mediated by nervous cholinergic structures, associated with GABA-A receptors, and indicate that this GABA-B agonist must be regarded as a partial agonist of peripheral GABA-A receptors.

Assessment of trans, trans-muconic acid in human seminal plasma.

Summary. Trans, trans‐muconic acid (tt‐MA) is one of the most important metabolites of benzene, a pollutant ubiquitously distributed in ambient air and classified in 1982 as a group I carcinogen. For its sensitivity and specificity, tt‐MA excreted in urine is considered a good biological marker of benzene exposure. In this study, seminal tt‐MA levels in occupationally nonexposed subjects (n = 32) have been determined. The seminal fluid of normozoospermic subjects contained an average tt‐MA concentration (170 ± 100 ng ml−1) significantly lower than that of teratozoospermic (310 ± 180 ng ml−1; P < 0.01), oligozoospermic (400 ± 180 ng ml−1; P < 0.001), and oligoasthenozoospermic (430 ± 230 ng ml−1; P < 0.01) subjects. A negative correlation existed between tt‐MA levels and sperm concentration (r = − 0.62; P < 0.001), percentage of normal spermatozoa (r = − 0.41; P < 0.05), and percentage of vital spermatozoa (r = − 0.89; P < 0.001). Average tt‐MA levels detected in seminal plasma were higher in smokers (350 ± 160 ng ml−1) than in nonsmokers (280 ± 210 ng ml−1). These results show that seminal plasma tt‐MA content could be an important biological indicator for evaluating the negative effects of benzene on spermatogenesis.

Potential involvement of ubiquinone in myotonic dystrophy pathophysiology: new diagnostic approaches for new rationale therapeutics

Abstract An impairment of mitochondrial function may contribute to the pathophysiology of myotonic dystrophy (MyD). Coenzyme Q10 (CoQ10) deficiency has been previously observed, even if in an restricted sample of patients. The aim of this investigation was to obtain more information about coenzyme Q10 and its relationships to the aerobic metabolism in a group of MyD patients. Serum CoQ10 appeared significantly reduced with respect to normal controls: 0.93±0.22 vs. 1.58±0.28 μg/ml (p<0.05). Moreover, the results demonstrated an inverse tendency between CoQ10 levels and the CTG expansion degree. Basal blood lactate levels were significantly higher than controls (p<0.05). A borderline inverse correlation between CoQ10 and lactate, corresponding to lactate threshold, was found. These data suggest a possible role of CoQ10 in the pathogenesis of MyD, which may be mediated by mechanisms of cellular damage common to the oxidative pathway. Therapeutic strategies may be devised by virtue of this rationale.

A SOURCE OF FALSE-NEGATIVE RESULTS IN CLENBUTEROL ANALYSIS IN TISSUES OF VEAL CALVES

The possibility of false-negative results in clenbuterol analysis was investigated in bovine tissues. An extraction procedure currently in use was adapted to process 100 specimens of different tissues each time. Its efficiency and accuracy were investigated radiometrically by means of a series of different molar concentration of the tritiated drug. In samples not submitted to extensive delipidation, unreliability of the analysis was evident. The measurement of tissue clenbuterol content, by a competitive ELISA, gave results numerically similar to those existing in literature, but with an accuracy high enough to minimize the frequency of false-negative results.