Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa.

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.

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors.

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 106 mL-1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 µM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 µM (CCCP), uncoupling agent; antimycin A 1 µg/mL (ANTI), complex III inhibitor; oligomycin 5 µM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O2•- production and H2O2 intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

Effects of Intra-Uterine Fluid Accumulation after Artificial Insemination on Luteal Function in Mares.

After breeding or artificial insemination, especially with frozen/thawed semen, mares often develop a persistent uterine inflammation, which is diagnosed by intra-uterine fluid accumulation. Here, we explored whether intra-uterine fluid accumulation affects corpus luteum function and tested the hypothesis that intra-uterine fluid accumulation after artificial insemination alters blood flow in the corpus luteum and plasma progesterone concentrations. A total of 40 Standardbred mares were artificially inseminated with frozen-thawed semen 30 to 36 h after induction of ovulation, and cases with or without intra-uterine fluid accumulation were detected by ultrasound 12 h after insemination. Luteal blood flow was measured by Power Doppler ultrasonography 3 and 6 days after ovulation, progesterone concentration was measured in peripheral plasma by ELISA 6 days after ovulation, and pregnancy was diagnosed by ultrasonography 14 days after ovulation. Luteal blood flow increased between 3 and 6 days after ovulation, but blood flow did not differ significantly between cases with (n = 28) and without (n = 25) intra-uterine fluid accumulation after insemination. Surprisingly, progesterone concentrations were higher in cases of intra-uterine fluid accumulation than cases without (9.3 ± 1.1 vs. 6.6 ± 0.5 ng/mL, p = 0.048). Pregnancy was less likely in cases with intra-uterine fluid accumulation than in cases without (10/28 vs. 17/25, p = 0.019), and there was a negative correlation between the severity of intra-uterine fluid accumulation and per cycle pregnancy rate. These data suggest that although intra-uterine fluid accumulation increases the secretion of progesterone, pregnancy is more dependent on uterine health than ovarian function.

Effect of platelet lysate on uterine response of mares susceptible to persistent mating-induced endometritis.

Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24 h pre- or 4 h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine response in mares susceptible to PMIE. A total of 14 mares susceptible to PMIE (based on presence of fluid or inflammatory cells 24 h after AI) underwent an untreated (Ctr) cycle followed by a treated (PL) cycle. From each mare, 100 mL of citrated whole blood was obtained for PRP production by centrifugation. The resultant PRP was brought to a final volume of 10 mL with platelet poor plasma and frozen at -80 °C to obtain PL. On untreated cycles, mares were inseminated with frozen-thawed semen 36 h after ovulation induction. On treated cycles, PL was thawed, infused into the uterus 12 h after ovulation induction, and AIs were performed 24 h later. The number of neutrophils in uterine cytology (score 1(normal)-3(severe inflammation)) evaluated by optical microscopy, uterine fluid accumulation (height x width) and uterine edema (score 0-3) observed in ultrasonography, were analysed. Pregnancy was evaluated by ultrasonography 14 days after ovulation. A significant decrease (P < 0.05) was observed on cytology score (PL 1.3 ± 0.1 vs Ctr 2.0 ± 0.1), fluid accumulation (PL 79.5 ± 30.1 mm2 vs Ctr 342.7 ± 52.9 mm2) and edema score (PL 1.8 ± 0.2 vs Ctr 2.3 ± 0.2) in treated mares. Pregnancy rate in PL-treated cycles (3/12) and control cycles (2/14), were not significantly different (P > 0.05). According to the results, we conclude that treatment with PL in mares classified as susceptible to PMIE appears to reduce the inflammatory response after breeding, based on clinical signs of uterine edema, IUF accumulation and PMNs migration.

Comparison between chlorhexidine and povidone-iodine solutions for flushing the preputial cavity on penile mucosa and semen bacterial counts in beef bulls.

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.

Impact of glyphosate and its formulation Roundup® on stallion spermatozoa.

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.

Use of specific mitochondrial complex inhibitors to investigate mitochondrial involvement on horse sperm motility and ROS production.

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.

Pharmacokinetics of oxytetracycline long-acting on plasma and semen of beef bulls.

The objectives of this investigation were to evaluate the pharmacokinetic parameters of oxytetracycline long-acting in plasma and seminal plasma after a single administration through either subcutaneous or intramuscular route at 10 mg/kg or 20 mg/kg dose. Four Simmental bulls, healthy and satisfactory potential breeders, were used. The route of administration either subcutaneous or intramuscular did not affect the mean values for 10 mg/kg dose in plasma (1,470 ng/mL vs. 1,330 ng/mL; P = 0.82) or seminal plasma (5,710 ng/mL vs. 5,390 ng/mL; P = 0.88), or for 20 mg/kg dose in plasma (2,540 ng/mL vs. 2,590 ng/mL; P = 0.96) or seminal plasma (25,600 ng/mL vs. 19,400 ng/mL; P = 0.58), respectively. Comparison between the 10 mg/kg and 20 mg/kg doses showed a difference in terms of mean plasma levels (1400 ng/mL vs. 2570 ng/mL; P = 0.07) and mean seminal plasma levels (6,480 ng/mL vs. 26,200 ng/mL; P = 0.004), respectively. After the dose of 10 mg/kg, plasma Cmax was 2,841 ng/mL at 12 h (Tmax) with a half-life of 20.1 h; seminal plasma Cmax was 11,515 ng/mL at 24 h (Tmax) with a half-life of 23.7 h. After the dose of 20 mg/kg, plasma Cmax was 5,269 ng/mL at 12 h (Tmax) with a half-life of 18.1 h; seminal plasma Cmax was 55,040 ng/mL at 24 h (Tmax) with a half-life of 15.7 h. Oxytetracycline long-acting may be an appropriate antibiotic, owing to its pharmacokinetic properties, that could be used for treating bulls' genital infections when its usage is indicated.

Assessment of an open-access CASA software for bovine and buffalo sperm motility analysis.

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.

Pharmacokinetics of tulathromycin on plasma and semen of beef bulls.

The objective of this investigation was to evaluate the pharmacokinetic parameters of tulathromycin in plasma and semen of beef bulls after administering a single sc dose at two different sites in the neck. Four Simmental bulls with excellent temperament received a comprehensive physical exam that included breeding soundness examination. In addition, blood was collected and analyzed for CBC and chemical panel in order to rule out any subclinical liver or kidney disease. All bulls were diagnosed as healthy and satisfactory potential breeders. The mean plasma levels of tulathromycin for the two neck sites of sc administration were not different between posterior aspect of the ear where it attaches to the head (RP; regio parotidea; 77.9 ± 43.3 ng/mL; X ± SD) and to the middle of the neck (RC; regio collis lateralis; 73.7 ± 39.7 ng/mL; P = 0.84). The mean seminal plasma levels of tulathromycin after administration in the RP was 608 ± 374 ng/mL and for RC was 867 ± 599 ng/mL without differences between both sites (P = 0.29). The mean level of tulathromycin in plasma was 75.8 ± 40.2 ng/mL, which was lower than mean seminal plasma levels of 781 ± 482 ng/mL (P = 0.001). The plasma peak tulathromycin concentration (Cmax) was 160 ± 27 ng/mL at 21 ± 6 h (Tmax) post-administration. The seminal plasma Cmax was 1539 ± 44.4 ng/mL at 33.00 ± 18.00 h (Tmax) post-administration. The Cmax between plasma and seminal plasma were different (P = 0.008) without any differences in Tmax between plasma and seminal plasma (P = 0.35). The terminal half-life for plasma tulathromycin (81.4 ± 27.6 h) showed a tendency to be shorter than in seminal plasma (114.7 ± 21.7; P = 0.10). The plasma area under the curve concentration time from the first to the last sample (AUC0-last) was 15,440 ± 1717 ng/mL/h, which was significatively smaller compared with 171,071 ± 58,556 ng/mL/h for seminal plasma AUC0-last (P = 0.01). The plasma means residence time from the first to the last sample (MRT0-last) was 89.3 ± 5.1 h and it was shorter than for seminal plasma of 96.6 ± 5.0 h (P = 0.05). From the present investigation, it was concluded that tulathromycin is a suitable antibiotic based in its pharmacokinetic properties that could be used for treatment of bull genital infections when its application is indicated.