Environmental temperature and relative humidity differently affect the sperm characteristics in Brown Swiss and Belgian Blue bulls.

In a temperature-increasing scenario, due to global warming, the individual thermic resilience of the male assumes a crucial role in the reproductive efficiency of a male since the thermic stress, such as the inability of the male to reduce body or regional temperature on a physiological level, impairs testicular function. In this study, the effect of the environmental conditions on the fresh semen quality, in terms of volume, concentration, total sperm in the ejaculate, total motility, normal morphology, membrane integrity, and discarding rate, were compared longitudinally in Belgian Blue (BB) and Brown Swiss (BS) bulls. The environmental conditions, summarized in the mean temperature-humidity index (THI), were calculated on the day of collection, as well as 7 days (epididymal maturation), 35 days (late spermatogenesis), and 70 days (early spermatogenesis) before the collection, to reflect spermatogenesis time. Our findings showed that limited seasonal effects were present in the semen quality of BS bulls. On the other hand, in BB bulls lower semen quality was found between July and November, with a different timing depending on the seminal parameter. This effect of the season on BB semen parameters appears to be related to the THI. The data presented in this study shows that the temperature and humidity, summarized in THI, could affect the semen quality of the bull on breed basis, given that volume, concentration, total sperm in the ejaculate, total motility, membrane integrity, and sperm normal morphology were significantly reduced by an increasing THI in the Belgian Blue bulls, but not in Brown Swiss bulls.

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.

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates.

BACKGROUND: Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation, was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars, but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality. RESULTS: Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample. CONCLUSIONS: These data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.

The effects of antibiotic additions to extenders on fresh and frozen-thawed bull semen.

Reproduction in dairy cows is based around the use of cryopreserved semen. Antibiotics are utilized to control bacterial contamination and growth in cryopreserved bull semen. The antibiotic resistance of some bacteria required the evaluation of new antibiotic combinations with a high level of antibacterial effectiveness and a negligible effect on spermatozoa. In this research, we studied the effect of the fluorinate carboxyquinolone ofloxacin and the combination of ceftiofur/tylosin on bull spermatozoa and in-field bacterial growth. In Experiment 1, the toxicity of different levels of ofloxacin and ceftiofur/tylosin was tested by the incubation of bull spermatozoa and the evaluation of sperm kinetic parameters, membranes and acrosome integrity after dilution, and at 60 and 120 min after incubation. The data reported in this study reveals that both antibiotic combinations, at all concentrations, seem to have a negligible effect on spermatozoa with respect to all of the parameters examined (p>0.05). Furthermore, progressive motility was significantly higher for sperm diluted with both antibiotic combinations compared with samples without antibiotics (p<0.01). In Experiment 2, the ability of ofloxacin or ceftiofur/tylosin to control bacterial growth during bovine semen cryopreservation was compared with the combination of gentamicin/tylosin/spectinomycin/lincomycin. A significant reduction in progressive motility was found in cooled semen with respect to all of the antibiotic treatments (p<0.05). However, the membrane integrity was found to significantly rise in frozen samples with, compared to samples without, antibiotics (p<0.05). In a bull, gentamicin, tylosin, spectinomycin, and lincomycin failed to control bacterial growth in the cryopreserved sample, while no such growth was found in samples extended with ceftiofur/tylosin or ofloxacin. In conclusion, both ceftiofur/tylosin and ofloxacin can be safely added to bull seminal extenders, and both can protect insemination doses from bacteria that are resistant to other antibiotic combinations.

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.

Successful use of a gonadotropin-releasing hormone (GnRH) analog for the treatment of tertiary hypogonadism (GnRH deficiency) in a 5-year-old Belgian Blue bull.

A bull was referred for a progressive oligoasthenotheratozoospermia that resulted in a unsuitable seminal quality for the cryopreservation. Breeding soundness evaluation results suggested gonadal dysfunction. Because of the lack of normal ranges for these hormones in the bull, in this study, the hypogonadism and the site of the dysfunction (hypothalamus) were diagnosed by the gonadotropin-releasing hormone (GnRH) stimulation test. The evaluation of pituitary and testicular responsiveness by a GnRH stimulating test revealed a responsiveness of the pituitary and testis, thus a secondary hypogonadism (hypothalamic hypogonadism) was postulated and a therapeutic approach based on the subcutaneous administration of GnRH analog was attempted. An increase in semen volume, concentration and sperm characteristics were detected 9 weeks after the start of the treatment, corroborating the hypothalamic origin of the disease and the useful of the GnRH therapy.

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.