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The growing and widespread use of glyphosate-based herbicides (GBHs) has raised an intense public debate about the impact of environmental contamination on animal and human health, including male fertility. The aim of this study was to deepen the impact of glyphosate (Gly) and GBHs on mammalian sperm investigating the effect of in vitro exposure of stallion spermatozoa to Gly and to its commercial formulation Roundup® (R). Spermatozoa were incubated at 37 °C with different Gly or R concentrations (from 0.5 to 720 μg/mL Gly or R at the same Gly-equivalent concentrations). After 1 h of incubation motility, viability, acrosome integrity, mitochondrial activity and ROS production were assessed. Gly, at all the concentrations tested, did not induce any detrimental impact on the sperm quality parameters evaluated. Conversely, R starting from 360 μg/mL (Gly-equivalent dose) significantly (P < 0.05) decreased total and progressive motility, viability, acrosome integrity, mitochondrial activity and the percentage of live spermatozoa with intact mitochondria not producing ROS. Our results indicate that the commercial formulation R is more toxic than its active molecule Gly and that the negative impact on stallion sperm motility might be likely due to a detrimental effect mainly at membrane and mitochondrial level and, at least in part, to redox unbalance. Moreover, based on the data obtained, it can be hypothesized a species-specificity in sperm sensitivity to Gly and GBHs as horse spermatozoa were negatively influenced at higher concentrations of R compared to those reported in literature to be toxic for human and swine male germ cells.

Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies.

This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 μM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy Open Access Funding provided by Universita degli Studi di Bologna within the CRUI-CARE Agreement

The aim of this study was to verify the reliability of an open access CASA software (BGM) to evaluate the sperm motility of cattle and buffalo, comparing motility and kinematic parameters to those of a commercial one (HTM). Thirty frozen-thawed samples for each species were analyzed with both HTM and BGM, after 1 h of incubation at 37 °C. Sperm viability and mitochondrial membrane potential (MMP) were evaluated through flow cytometric analysis. Agreement of all motility variables between the two systems was assessed. Correlation analysis was performed to identify relationships between motion parameters and sperm viability and MMP. Bland Altman analysis showed good agreement between methods for all motility parameters except for curvilinear velocity (VCL) in cattle, and for average path (VAP), VCL and (amplitude of lateral head displacement) ALH in buffalo, that showed a proportional bias (P > 0.05). In both systems, positive correlation between both viability and high MMP and total and progressive motility of cattle spermatozoa were found; viability and the sperm with high MMP were positive correlated only with VAP, straight-line (VSL), VCL and ALH evaluated with HTM system. Different results were found for buffalo sperm motility parameters, since viability had positive correlations and mitochondrial activity negative ones. Results suggested that motility assessment performed by these two systems are comparable. The discrepancy of VCL, VAP, and ALH could be due to the difference in the algorithms between software. The open-access CASA plug-in is a reliable alternative to the expensive commercial CASA system for sperm motility assessment in cattle and buffalo.

The objectives of the present study were to estimate the number of colonies forming units (CFU) from penile mucosa and semen, the effect of two antiseptic solutions used to flush the preputial cavity to reduce the bacterial counts from those sites, and compare them. Six clinically healthy bulls between 15 and 16 mo old declared satisfactory potential breeders were used. A prospective, randomized, and controlled cross-over design was performed, in which each bull was first sampled from the penile mucosa and semen without treatment (control group) and 24 h later, after antiseptic preputial flushing (treated group). In the treated group, the preputial area was cleaned, the preputial hair was cut, urination was stimulated, prepuce area was scrubbed twice, and the preputial cavity was flushed with either 1% of povidone-iodine solution (POI; 500 mL) or 0.05% chlorhexidine digluconate (CHG; 500 mL), maintained for 10 min. Then, the preputial cavity was emptied and flushed with 500 mL of sterile saline solution. Next, the accessory sexual glands were massaged per rectum. Finally, protrusion, erection, and ejaculation were obtained by electroejaculation, and samples from penile mucosa and semen were collected for microbiological culture. The number of CFU was determined for each sample by enumerate total aerobic bacteria using Standard Plate Surface Count cultured for 48 h. In the first replicate, half of the bulls were treated with CHG, and the other half were treated with POI. After 58.8 ± 5.3 days (x ± SD) of wash-out period, the treatments were reverted, and the same protocols were applied again. In the control group, the median number of CFUs from the penile mucosa was 750,000 (range from 60,000 to 1,800,000) and the median number of CFUs in semen was 8,000,000 (700,000-45,000,000). The CFU in semen was higher than the penile mucosa (P = 0.005). Both antiseptic solutions reduced the median number of CFUs on the penile mucosa to 915 (P = 0.002) and in semen to 1,680 (P = 0.002). The antiseptic effect on the penile mucosa was higher for CHG solution (490) than for POI solution (6,650; P = 0.05). The antiseptic effect on semen of CHG was also greater (200) than for the POI solution (31,000; P = 0.05). It can be concluded that the median number of CFU was higher in semen compared with penile mucosa, and flushing the preputial cavity either with 0.05% CHG or 1% POI maintained for 10 min reduced the number of CFUs from penile mucosa and semen. The level of antiseptic activity was higher for CHG than for POI.

Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1 h and 3 h at 37 °C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.8 μM, ANTI), the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine (5 μM, CCCP), ATP synthase inhibitor oligomycin (5 μM, OLIGO), and 2 μL vehicle DMSO (control, CTL). Samples were analyzed for sperm motility and for mitochondrial membrane potential (MMP), mitochondrial integrity, mitochondrial O2•– production, and cytoplasmic H2O2. A multivariate analysis was performed on the data. CCCP caused a pronounced MMP reduction at both time points while ROT and ANTI showed the same effect at 3 h. All treatments at 3 h incubation significantly reduced the percentage of sperm with early changes in membrane permeability with active mitochondria. The H2O2 production of live cells was low at 1 h incubation in all treatments; after 3 h a slight decrease in the percentage of low-H2O2 producing cells was recorded. All treatments, except DMM, induced a significant decline in sperm motility and kinematics and modified the pattern of sperm subpopulations. The effect of DMM was evident only after 3 h, increasing the percentage of slow sperm subpopulation. In conclusion, the disruption of mitochondrial integrity induces an increase of mitochondrial ROS production that could be detrimental for cell function and survivior.

Context While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6(3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. Aims The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. Methods Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6(3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. Key results DiOC6(3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6(3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. Conclusions JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6(3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. Implications Both DiOC6(3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.