Serum concentration dynamic of energy homeostasis hormones, leptin, insulin, thyroid hormones, and cortisol throughout canine pregnancy and lactation.

Pregnancy and lactation represent critical periods of canine reproductive life and different hormones are required to maintain homeostasis and the correct energy requirements. The aim of this study was to evaluate leptin, insulin, triiodotyronine (T3), thyroxin (T4) and cortisol serum concentrations to better describe the canine maternal energy metabolism. Twelve Bloodhound bitches that had at least one litter and were considered to be healthy submitted blood samples every 15 days starting from day 0 (ovulation) throughout pregnancy up to the end of lactation (day 120). We found that leptin concentrations increased from day 0 to reach maximum value at day 45 (P < 0.001), decreased at day 60 (P < 0.05) and rose again at day 105 and day 120 (P < 0.05 and P < 0.01 respectively). Insulin concentrations significantly increased during pregnancy peaking at day 60 (P < 0.05). After parturition dropped and reached progressively lower values similar to those at day 0 at the end of lactation. During pregnancy Body Weight was in low correlation with leptin (r = 0.32, P < 0.01) and there was no correlation with insulin (r = 0.13, P > 0.1); moreover, no correlation was found during lactation. T3 concentrations showed a U-shaped trend with a significant reduction at day 75 (P < 0.05). T4 concentrations fluctuated without any significant change (ranging from 27.0 ± 1.6 nmol/l to 32.1 ± 1.9 nmol/l). Cortisol concentrations ranged within the reference values (minimal value at day 30 (49.8 ± 6.3 nm/L) and maximal value at day 45 (72.5 ± 16.1 nm/L)). Our study states that canine pregnancy and lactation evoke many hormonal changes necessary to mother and fetuses. In particular, this is the first report regarding the pattern of leptin during whole pregnancy and lactation. The present work lays the groundwork for further studies on endocrine homeostatic mechanisms using the dog as a model for human medicine.

Addition of different concentrations of prostasome-like vesicles at neutral or slightly alkaline pH decreases canine sperm motility.

Prostasome-like vesicles (PV) are components of the canine prostatic fluid during ejaculation and despite some enzymatic activities have been identified recently, their functions in the reproductive events in this species are still poorly understood. In this study, we evaluated at neutral or slightly alkaline pH, the effects of different concentrations of purified PV on sperm cell motility characteristics and the impact on the short- and long-term preservation of preserved semen maintained at room temperature. Two different experiments were performed. In the first experiment, purified PV were added at increasing concentration (1.25, 2.5, 5, 10, and 20 µL equivalent to 1, 2, 4, 8, and 16 µg of protein, respectively) to aliquots of 100 µL of preserved semen maintained at 22 °C at the following time points: 0, 30, 60, 120, 240, and 480 min. Computer-assisted sperm motility characteristics and pH were assessed three times at each time points, for each sample and for every concentrations. In the second experiment, the purified PV were added with the same methods as described above but only at time 0. Sperm motility characteristics and pH were assessed over the time. This study showed how the addition of purified PV to preserved semen affects negatively (p < 0.05) at neutral and alkaline pH, both total and progressive motility in a concentration depending manner. Furthermore, prostasome addition was demonstrated to change the quality of sperm movement which may represent a mechanism facilitating sperm cells attachment to the uterine epithelium and facilitating energy preservation before fertilization.

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.

Evaluation of testicular artery blood flow by Doppler ultrasonography as a predictor of spermatogenesis in the dog.

The present study examines the relationship between reproductive and seminal characteristic with blood flow supply of the testis in male dogs by means of Doppler ultrasonography. Five male dogs were subject to a clinical examination, blood sampling, B-Mode and Doppler ultrasonography of the testis, semen evaluation at collections and after storage at 4°C for 24, 48 and 72 h in three replicates 30 days apart. Peak Systolic Velocity was positively correlated with testicular volume and negatively with live sperm at collections and after 24h. A negative correlation existed between Resistive Index and Pulsatility Index with total and progressive motility at collections and also after 24, 48 and 72 h. Resistive Index and Pulsatility Index were also negatively correlated with the percentage of membrane intact sperms with curled tails at collections while the latter was positively correlated with End Diastolic Velocity. Moreover, positive linear correlations of weight with bilateral testicular volume and total sperm concentration and also between age and abnormal sperm at collections and after 24h were established. The results suggest that the Resistive Index and Pulsatility Index seem to be potential markers of seminal quality in dogs.

Power and pulsed Doppler evaluation of prostatic artery blood flow in normal and benign prostatic hyperplasia-affected dogs.

This study was carried out with the objectives to test the differences in the haemodynamic characteristics of the prostatic artery in normal and benign prostatic hyperplasia (BPH)-affected dogs using Doppler ultrasonography. In sixteen male German shepherd dogs, prostatic volume was detected and prostatic biopsy was performed. The prostatic artery blood flow parameters determined were as follows: peak systolic velocity (PSV), end diastolic velocity (EDV), Resistive Index (RI) and Pulsatility Index. The power Doppler mode was used for colour flow mapping of the prostatic artery. In PW mode at marginal locations, the waveforms recorded showed a continuous pattern typical of the high-resistance vessels, while in subcapsular locations, the waveforms recorded were continuous characteristic of the low-resistance vessel. Peak systolic velocity and EDV in both locations were significantly higher in BPH group (p < 0.001) than normal group (p < 0.05). Pixel number in BPH group in both locations was significantly higher (p < 0.05) compared to the normal group. This study shows that Doppler ultrasonography represents a valid and non-invasive method for the characterization of the blood flow in the prostatic artery in dogs affected with BPH. Moreover, statistically significant differences of blood flow velocities in prostatic artery in normal and BPH-affected dogs were detected. The RI was not able to differentiate normal dogs from dogs affected by BPH and therefore is not a parameter usable for diagnostic purposes, while Power Doppler could represent an additional diagnostic tool.

Clinical efficacy of the GnRH agonist (deslorelin) in dogs affected by benign prostatic hyperplasia and evaluation of prostatic blood flow by Doppler ultrasound.

In six German Shepherds dogs, GnRH agonist implants (Deslorelin) were inserted subcutaneously one month after histological confirmation of benign prostatic hyperplasia (BPH). Prostatic volume (PV), characteristics of ejaculate, serum testosterone concentrations and Doppler parameters of prostatic and subcapsular arteries were detected at different time intervals, for 6 month. The prostatic volume showed a significantly reduction starting at day 37. The decrease in sperm concentration, motility and increase in morphological abnormal sperm were observed from day 22 to day 37, when it was no longer possible to obtain the ejaculate. The values of peak systolic velocity and end-diastolic velocity in prostatic and subcapsular arteries showed from day 11 a gradual decrease, significant at day 22 until day 37 and reaching the lowest values at day 52 until the end of observation. The power Doppler pixel intensity of both arteries showed a gradual decrease from day 5 until day 52. In particular, a significant decrease was observed for both arteries from day 11. Testosterone serum concentration decreased to undetectable levels by day 11 until the end of the observations. All these Doppler parameters and testosterone values were positively correlated with the prostatic volume. Furthermore, testosterone values were positively correlated with peak systolic velocity, end diastolic velocity and pixel numbers. The use of implants containing GnRH analogues, even in asymptomatic subjects, is effective for the control of BPH and the application of Doppler exam of prostatic blood flow represent an non-invasive tool for monitoring the response of medical treatment.

Ultrastructural and enzymatic activity of membranous vesicles isolated from canine seminal plasma.

The objectives of this study were to verify the presence of membranous vesicles (MV) in canine seminal plasma by mean of transmission electron microscopy (TEM), to describe the ultrastructural characteristics and to identify some enzymatic activity associated with them. Semen samples, collected by digital manipulation from dogs with proven fertility, were pooled and used for membrane vesicles preparation according to conventional procedures. TEM observations showed the existence of vesicular membranous structures of more or less spherical shape with different sizes. These vesicles were surrounded by a single-, double- or multiple-layered laminar membranes. The mean vesicle diameter was 117.6 ± 86.9 nm ranging from 24.4 to 716.6 nm. Enzyme activity determinations showed the presence of adenosine deaminase, 5'-nucleotidase, ADPase, ATPase, dipeptilpeptidase IV, alkaline phosphatase, total acid phosphatase and prostatic acid phosphatase, while the aminopeptidase activity was absent. In conclusion, results of this study, compatible with results from other mammals, showed for the first time the presence of MV, their ultrastructural and enzymatic characteristics in dog seminal plasma.

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.

Doppler evaluation of maternal and fetal vessels during normal gestation in the bitch.

The aim of this work is to evaluate the haemodynamic characteristics of maternal and fetal vessels during normal pregnancy in bitches, using Colour and Pulsed wave Doppler ultrasonography, in order to obtain more information about maternal and fetal circulation. The blood waveforms of the uteroplacental arteries, aorta, caudal cava vein and umbilical cord of the fetuses were recorded weekly in 16 pregnant bitches. Also, the measurements of Peak Systolic, End Diastolic Velocities, Resistance and Pulsatility Indices were carried out. Uteroplacental blood flow was biphasic while the ones of the umbilical artery and aorta were first systolic and then diastolic. The cava showed a typical waveform of venous vessels. During gestation the EDV and PSV of fetal vessels increased (alpha<0.05) while the PI and RI of all vessels examined decreased (alpha<0.05) except for the IP of the Aorta. The Doppler ultrasonography was used to study the characteristics of maternal and fetal vessel flow and their progressive changes during pregnancy. This study can be considered a further contribution in diagnosing and monitoring high-risk pregnancies in Veterinary Medicine.

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

In 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 plasma membrane, 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.

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development.

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.

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.

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.