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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.

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.

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 120min 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.

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.

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.

In this study, the effect of the chamber used for the automated analysis of sperm motility by a computer-assisted semen analysis system on sperm kinematics was evaluated, and the cause of this effect was also verified. Twenty-three bull semen batches were thawed, and semen was diluted, aliquoted and analysed with six different chambers, (three capillary-loaded chambers and three droplet (DR)-loaded chambers). For each chamber type, each sample was analysed in quadruplicate, and the reliability of the analysis was tested using an intraclass correlation coefficient (ICC). Furthermore, sperm membrane integrity (MI) was evaluated, for each sample and chamber, in 12 randomly selected central and 12 edge fields. The ICC analysis showed that some parameters could have a significant variability related to the chamber. High stability of results was detected in Leja 4-chamber slide. Furthermore, as previously reported in other studies, capillary-loaded chambers seemed to affect the total and progressive motility and sperm velocities (average path velocity, straight line velocity and curvilinear velocity). These findings were corroborated by the evaluation of sperm MI that was significantly higher in the DR-loaded chambers. This study confirms that the chamber used for the objective kinetic evaluation of bull-thawed spermatozoa significantly affects the result. These differences could be present also in other species, even if the specific effect on the sperm kinematics should be verified. The Makler chamber seemed to give reliable results with negligible effects on sperm kinematics.

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.