Riccardo Zelli

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis

SummaryIn the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasmamembrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.

Power and pulsed Doppler evaluation of ovarian hemodynamic changes during diestrus in pregnant and nonpregnant bitches

The aim of the study was to further characterize the relationship between hemodynamic changes in the ovary and luteal function in pregnant and nonpregnant bitches. Fourteen German Shepherd bitches were monitored three times a week from the first day of cytological diestrus (D1) until parturition or the end of diestrus (progesterone <2 ng/mL) by color Doppler, pulsed wave spectral Doppler, and power Doppler (PD) ultrasonography. By means of PD the total number of color pixels were calculated. The Doppler parameters evaluated were: peak systolic velocity (PSV), end diastolic velocity (EDV), and both resistive and pulsatility indices. Blood samples were collected three times a week throughout the experiment to determine progesterone (P4) concentrations. The length of diestrus in pregnant versus nonpregnant group was significantly shorter (P < 0.01; 57 ± 1 vs. 63 ± 1, respectively). By means of pulsed wave spectral Doppler the waveform showed a typical pattern of a low-resistive vessel characterized by a rapid systolic peak followed by a slow telediastolic decrease with a relatively high end-diastolic velocity. Blood flow parameters did not differ between left and right ovary. In both groups PSV and EDV showed a gradual decrease with the progress of diestrus; however, the values of PSV and EDV were significantly higher (P < 0.05) in the pregnant group versus nonpregnant group from D31 to D61 and from D49 to D58 respectively. Moreover, a significantly decrease (P < 0.05) of PSV and EDV in the pregnant group was observed from D46 to D58 and from D49 to D55, respectively. The resistive and pulsatility indices showed an increase during diestrus and the values were significantly lower (P < 0.05) in the pregnant group from D49 to D61. By means of PD, the pixel number was significantly higher (P < 0.05) in the pregnant versus nonpregnant group from D40 to D61. In particular, a significant decrease (P < 0.05) in the pixel number in the pregnant group was observed from D46 to D61. The comparison of the P4 values with the ovarian pixel number in the pregnant and nonpregnant group showed a direct correlation (r = 0.792, N = 59 and r = 0.774, N = 59, respectively). In particular, the P4 values were higher (P < 0.05) in the pregnant than in the nonpregnant group from D37 to D52. In conclusion, significant physiological differences between pregnant and nonpregnant bitches in terms of P4 and ovarian blood supply are reported. In addition it was possible to define that blood flow pattern during diestrus in pregnant bitches is not always closely related with P4 production.

Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

Immunohistochemical localization of orexin A and orexin type 2 receptor-positive cells in the placenta of dogs

The aim of the present study was to examine the presence and distribution of cells that express immunopositivity for orexin A (OXA) and its type 2 receptor (OX2R) in the dog placenta toward the end of pregnancy using immunohistochemical techniques. In the placental fetal portion, a few OXA and OX2R-positive cells were seen scattered in the outermost coating layer of chorionic villi and in the trophoblastic protrusions. Closer to the maternal portion, immunopositive labeling for both peptides was visible in the glandular epithelia and that for OXA also in the endothelium of the capillaries. These observations allow us to hypothesize that the canine placenta may be not only a source of orexin A, but also its target, and that orexin A may play an important role in controlling the function of this important organ for normal fetal development.

Dysuria in a Sardinian Ram with Glans Penis Traumatic Injury

Signs of dysuria in rams are usually associated with urethral obstruction caused by calculi, especially in castrated males, while penile trauma is rarely reported as a cause of dysuria. In this paper, a case of a 4-year-old, 65 kg bodyweight, Sardinian intact ram referred to the Teaching Veterinary Hospital (OVUD) at the Department of Veterinary Medicine University of Perugia, Italy, with a 1-month history of dysuria is reported. At the moment of referral, the animal showed increased heart and respiratory rate, normal rectal temperature, urine dropping, straining and false kyphosis. Ultrasound investigation revealed heterogeneous hypoechoic tissue surrounded by a hyperechoic capsule in the apical end of the penis, normal testicular parenchyma, and distended urinary bladder. After sedation, the protruded glans penis was necrotic at inspection. Cystography showed no alterations in the urinary tract, while catheterization was unsuccessful; after the amputation of the urethral process, involuntary urine loss appeared. The ram was hospitalized and antimicrobial, anti-inflammatory and sustain therapy was administered for 5 days, associated with daily preputial flushing with iodine solution. Two injections of neostigmine metilsulphate, 1 mg SC, 24 hours apart, were administered to increase the bladder tone. The animal slowly improved both the general condition and the voluntary urine emission. After coming back to the farm, the ram observed a further period of sexual rest. To our knowledge, there are no reported cases of iatrogenic penile trauma and subsequent necrosis and strangury in small ruminants. Since necrosis of the glans penis was noticed, caution should be used in formulating prognosis concerning the future mating ability of the animal, based on the extension of the necrotic process.