Pulse wave Doppler ultrasound of testicular arteries and their relationship with semen characteristics in healthy bulls.

BACKGROUND: Semen evaluation is used to estimate the testicular function. In bulls, the spermatozoa present in the ejaculate are the result of a process that begun more than 2 mo earlier, bequeathing a delayed depiction of the actual function of the testis. Since testis vascularization might be critical for the gonad function, selected pulse wave Doppler ultrasound parameters were assessed in this study, for instance the peak systolic velocity, the end diastolic velocity and the resistive index of the testicular artery along the spermatic cord, the marginal portion of the testicular artery and the intratesticular branches of the testicular artery both in healthy adult and young bulls. Correlations between these parameters and characteristics of semen that was collected numerous times, before and after the Doppler ultrasound examination. RESULTS: The peak systolic velocity and the end diastolic velocity measured in the testicular artery along the spermatic cord (supratesticular artery - SA) were variable among the bulls and within individual bulls, likely due to the convoluted course of the vessel. The resistive index was found highly repeatable in the same bull. A reduction in the resistive index was found between the supratesticular artery and the marginal portion of the testicular artery (P < 0.01), and between the marginal portion of the testicular artery and the intratesticular branches of the testicular artery (P < 0.05). No differences were recorded for the pulse wave Doppler ultrasound parameters in young bulls compared with adults. A significant correlation was found between the resistive index of the marginal portion of the testicular artery and total sperm in the ejaculate (r = 0.516, P < 0.05), the immature sperm (r = 0.462, P < 0.05), the teratoid sperm (r = 0.375, P < 0.05), and the "Dag defect" sperm (r = 0.389, P < 0.05). Similarly, the resistive index of the intratesticular branches of the testicular artery were found correlated with the total sperm number in the ejaculate (r = 0.568, P < 0.05), the immature sperm (r = 0.523, P < 0.05), the teratoid sperm (r = 0.418, P < 0.05), and the "Dag defect" sperm (r = 0.341, P < 0.05). CONCLUSIONS: The data presented in this study suggest that the resistive index, measured at the marginal portion of the testicular artery, could be an easy-to-perform parameter to evaluate the spermatogenesis quality in young bulls and normal adults.

Factors affecting staining to discriminate between bull sperm with greater and lesser mitochondrial membrane potential.

The sperm mitochondrial membrane potential (MMP) is usually evaluated using the JC-1 dye. This study aimed to verify the effect of incubation temperature (25 °C or 38 °C), incubation time (10, 30, and 45 min), JC-1 stain concentration (0.2 µM, 2 µM, 8 µM, 12 µM), and the presence of glycerol (6.6% compared with 0%), on the capacity of the stain to discriminate between sperm with high mitochondrial membrane potential (hMMP) and low mitochondrial membrane potential (lMMP) in fresh and frozen bull sample by both flow cytometry and epifluorescence microscopy. The temperature (38 °C for 10 min) and the dye concentration (8 µM and 12 µM) resulted in a greater proportion of hMMP (P < .05). The incubation for 45 min at 38 °C resulted in a significant reduction of hMMP in samples stained with JC-1 dye at 8 µM and 12 µM (P < .01). A longer incubation time (45 min) and greater dye concentration (8 µM and 12 µM) resulted in an increased proportion of hMMP sperm in cryopreserved samples. Fresh sperm incubated with glycerol had a hMMP (P < .05). Data for the present study indicate that the optimal incubation temperature was 38 °C, with an incubation time differing between fresh (10-30 min) and cryopreserved sperm (at least 45 min). Furthermore, the JC-1 dye concentration used that could reliably detect the proportion of hMMP sperm was 2 µM in fresh samples, and at least 8 µM in cryopreserved sperm.

Kinematic study on the effect of pH on bull sperm function.

Since the mammalian spermatozoa became capable of motion, during the epididymal transit, the spermatozoon swims in a liquid medium and it is completely dependent on the environmental conditions. Some reports have suggested an influence of pH on sperm kinetic characteristics, but no study has objectively described how motility changes in a different environmental pH. In this study, we evaluated the effect of different environmental pHs (5.5, 6, 6.5, 7, 7.5, 8, and 8.5) on kinetic parameters, sperm viability, mitochondrial activity, and sperm morphology of bull semen immediately and 1h after dilution. The results showed higher values for sperm motility characteristics, viability, and mitochondrial activity at pH 7 and 7.5. Values of pH lower than 6.5 and higher than 8 resulted in suboptimal motility, with a decrease in most parameters. At pH 8 and 8.5, a discrepancy between viability and total and progressive motility was found, with a significant amount of spermatozoa that were live but immotile. This reduction seemed related to a decrease in mitochondrial activity, possibly due to the increase in pH. The flow cytometric evaluation of sperm viability assessed by calcein AM was very consistent with the amount of spermatozoa with membrane integrity, evaluated in fluorescence by propidium iodide/SYBR-14 stain. Thus, the calcein AM stain could be used as viability stain instead the classic propidium iodide/SYBR-14 stain because this could allow the addiction of other functional stains without a overlapping of the fluorescent signal in the flow cytometer.

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa.

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 x 10(6) sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 x 10(6) sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 x 10(6) sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.

Dynamics of infection and immunity in a dairy cattle population undergoing an eradication programme for Infectious Bovine Rhinotracheitis (IBR).

Several countries within the European Union (EU) have successfully eradicated Infectious Bovine Rhinotracheitis (IBR), while others (e.g. Germany) are making efforts to achieve IBR-free status. EU member states IBR eradication programmes must meet Community legislation requirements that ban breeding farms from purchasing positive animals, from using whole-virus IBR vaccines, and from inseminating cows with semen from positive bulls. A follow-up study from 2002 to 2005 was carried out in the province of Trento (Italy), where a compulsory programme for IBR eradication was started in 1998. IBR outbreaks (identified on the basis of seroconversion of sentinel animals) were concentrated in larger positive herds. A higher incidence was recorded between 2003 and 2004. An association between markedly high temperatures in the summer of 2003 and virus reactivation has been suggested but is yet to be confirmed. The practice of driving cattle to common alpine pastures for the summer season did not play a significant epidemiological role in IBR transmission. Premising that only seronegative animals are allowed to enter dairy farms, animal movement increases the infection risk to a moderate extent. The long-term persistence of IBR antibodies was more pronounced in animals positive for antibodies to the glycoprotein E (gE). Scattered seroconversions, occurring mostly in positive herds, require careful interpretation in order to avoid overestimating the incidence of the infection at herd level.