Artificial Mucus Research Articles

Experimental Model of Atelectasis in Newborn Piglets

There are few studies using animal models in chest physical therapy. However, there are no models to assess these effects in newborns. This study aimed to develop a model of obstructive atelectasis induced by artificial mucus injection in the lungs of newborn piglets, for the study of neonatal physiotherapy. Thirteen newborn piglets received artificial mucus injection via the endotracheal tube. X-rays and blood gas analysis confirmed the atelectasis. The model showed consistent results between oxygenation parameters and radiological findings. Ten (76.9%) of the 13 piglets responded to the intervention. This did not significantly differ from the expected percentage of 50% by the binomial test (95% CI 46.2-95%, P = .09). Our model of atelectasis in newborn piglets is both feasible and appropriate to evaluate the impact of physical therapies on atelectasis in newborns.

Non-steroidal anti-inflammatory drug for pulmonary administration: Design and investigation of ketoprofen lysinate fine dry powders

Pulmonary inflammation is an important therapeutic target in cystic fibrosis (CF) patients, aiming to limit and delay the lung damage. The purpose of the present research was to produce respirable engineered particles of ketoprofen lysinate, a non-steroidal anti-inflammatory drug able to fight lung inflammatory status by direct administration to the site of action. Micronized drug powders containing leucine as dispersibility enhancer were prepared by co-spray drying the active compound and the excipient from water or hydro-alcoholic feeds. Microparticles were fully characterized in terms of process yield, particle size distribution, morphology and drug content. The ability of the drug to reach the deepest airways after aerosolization of spray-dried formulations was evaluated by Andersen cascade impactor, using the monodose DPI as device. In order to investigate the behaviour of the drug once in contact with lung fluid, an artificial CF mucus was prepared. Drug permeation properties were evaluated interposing the mucus layer between the drug and a synthetic membrane mounted in Franz-type diffusion cells. Finally, the effect of the engineered particles on vitality of human airway epithelial cells of patients homozygous for ΔF 508 CF (CuFi1) was studied and compared to that of raw active compound. Results indicated that powders engineering changed the diameter and shape of the particles, making them suitable for inhalation. The mucus layer in the donor compartment of vertical diffusion cells slowed down drug dissolution and permeation, leucine having no influence. Cell proliferation studies evidenced that the spray drying process together with the addition of leucine reduced the cytotoxic effect of ketoprofen lysine salt as raw material, making the ketoprofen lysinate DPI a very promising product for the inflammation control in CF patients.

The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation

The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and “apoptotic-like” changes).

Nasal administration of liquid crystal precursor mucoadhesive vehicle as an alternative antiretroviral therapy

The purpose of this study was to develop a mucoadhesive stimuli-sensitive drug delivery system for nasal administration of zidovudine (AZT). The system was prepared by formulating a low viscosity precursor of a liquid crystal phase, taking advantage of its lyotropic phase behavior. Flow rheology measurements showed that the formulation composed of PPG-5-CETETH-20, oleic acid and water (55, 30, 15% w/w), denominated P, has Newtonian flow behavior. Polarized light microscopy (PLM) revealed that formulation P is isotropic, whereas its 1:1 (w/w) dilution with artificial nasal mucus (ANM) changed the system to an anisotropic lamellar phase (PD). Oscillatory frequency sweep analysis showed that PD has a high storage modulus (G′) at nasal temperatures. Measurement of the mucoadhesive force against excised porcine nasal mucosa or a mucin disk proved that the transition to the lamellar phase tripled the work of mucoadhesion. Ex vivo permeation studies across porcine nasal mucosa exhibited an 18-fold rise in the permeability of AZT from the formulation. The Weibull mathematical model suggested that the AZT is released by Fickian diffusion mechanisms. Hence, the physicochemical characterization, combined with ex vivo studies, revealed that the PPG-5-CETETH-20, oleic acid, and water formulation could form a mucoadhesive matrix in contact with nasal mucus that promoted nasal absorption of the AZT. For an in vivo assessment, the plasma concentrations of AZT in rats were determined by HPLC method following intravenous and intranasal administration of AZT-loaded P formulation (PA) and AZT solution, respectively, at a dose of 8mg/kg. The intranasal administration of PA resulted in a fast absorption process (Tmax=6.7min). Therefore, a liquid crystal precursor formulation administered by the nasal route might represent a promising novel tool for the systemic delivery of AZT and other antiretroviral drugs. In the present study, the uptake of AZT absorption in the nasal mucosa was demonstrated, providing new foundations for clinical trials in patients with AIDS.

The effect of the in vitro supplementation of exogenous long-chain fatty acids on bovine sperm cell function

This study aimed to investigate the effects of long-chain fatty acids (α-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linoleic acid (LA), oleic acid (OA) and palmitic acid (PA)) at concentrations of 10-100 µM, on extended bull spermatozoa stored in vitro for up to 7 days. Progressive linear motion (PLM), viability (Experiments 1-3), ability to penetrate artificial mucus (Experiment 1), reactive oxygen species (ROS; Experiment 2) and superoxide production (Experiment 3) were assessed. Spermatozoa maintained the ability to penetrate artificial mucus up to Day 4, irrespective of treatment. In Experiments 2 and 3, DHA and EPA had detrimental effects on PLM and viability. PA preserved PLM and viability at levels greater than the control (P<0.05), whilst keeping ROS levels to a minimum, particularly on Days 1 and 3 (P<0.01) when ROS generation peaked in other treatments. In contrast, superoxide production peaked on Day 0 (Experiment 3) and declined thereafter with no significant effect of fatty acid. This study supports the notion that superoxide dominates on Day 0, whereas its breakdown products, hydrogen peroxide and the hydroxyl radical as assessed by CM-H2DCFDA, contribute to ROS generation on subsequent days.

Gentamicin and leucine inhalable powder: What about antipseudomonal activity and permeation through cystic fibrosis mucus?

The aim of this study was to evaluate the permeation properties of gentamicin (G) in a novel dry powder form for inhalation through an artificial mucus model. Moreover, since respiratory infections sustained by Pseudomonas are a major cause of sickness and death in CF patients, the susceptibility of P. aeruginosa to engineered G powders was investigated. Micronized G and G/leucine (85:15) formulations were produced by co-spray-drying, using process parameters and conditions previously set. Powders were characterized in terms of yield, drug content and aerodynamic profiles, analyzed by Andersen Cascade Impactor. Different mucus models were prepared, showing composition and viscosity similar to those of the native CF mucus. To investigate the impact on drug permeation, Franz-type vertical diffusion cells were used; the powders were applied directly on a synthetic membrane with or without the interposition of the artificial mucus layer. In buffer, gentamicin showed a diffusion controlled release; the presence of leucine reduced powder wettability and, consequently, the permeation rate. Otherwise, mucus delayed drug permeation from both G and G/leucine formulations, with a faint influence of the aminoacid. Antimicrobial tests revealed that G/leu engineered particles are able to preserve the antipseudomonal activity, even in presence of the mucus.

Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.

Symmetrical-Waveform High-Frequency Oscillation Increases Artificial Mucus Flow Without Changing Basal Mucus Transport in In Vitro Ovine Trachea

The efficacy of symmetrical-waveform high-frequency oscillating (HFO) air flow for airway secretion clearance is controversial and debated in the literature. We conducted in vitro experiments with ovine tracheae to investigate the effects of symmetrical-waveform HFO on tracheal transport of artificial mucus. We mounted each trachea as an intact tube, with a 15(o) head-down tilt, infused artificial mucus (10 mL over one hour) at the caudal end of the trachea, and measured mucus-transport velocity as the time between the beginning of infusion and the first appearance of artificial mucus over 2 near-infrared sensors at the rostral end of the trachea and by measuring the amount of mucus emerging. In a second series of experiments we opened each trachea flat and with video microscopy we measured the transport velocity of plaques over the endogenous mucus sheet. In the intact-trachea preparation, HFO at 20 Hz and 50 cm H(2)O increased mucus-transport velocity from 5.8 mm/min to 7.8 mm/min. HFO led to nearly half the artificial mucus being cleared during the infusion period. In the opened-trachea experiments the mean control transport velocity was 8.7 mm/min, and HFO, at 14 Hz or 20 Hz (and 50 cm H(2)O), did not significantly alter that velocity. Symmetrical-waveform HFO increases mucus-transport velocity and mucus clearance when a thick layer of mucus is present. This may be important when considering the mechanisms of mucus clearance and using HFO for secretion clearance.

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.

Mannitol‐Guided delivery of ciprofloxacin in artificial cystic fibrosis mucus model

Abnormal airway mucus presents a significant challenge for inhalational drug delivery. Recognizing the thick and tenacious airway mucus seen in the cystic fibrosis (CF) patients as a critical barrier to effective drug delivery, both into the mucus layer itself as well as across that layer to the underlying airway epithelium, we hypothesize that mannitol or NaCl can form inhalable drug carriers, improve drug penetration into the mucus, and ultimately enhance the drug's therapeutic effect. The objective of this study is to test whether mannitol and NaCl particles, as inhalable drug carriers, improve drug delivery into and enhance a drug's activity within a mucus-like material. Microparticles containing Ciprofloxacin (Cipro), an active ingredient, and either mannitol or NaCl were produced by spray-drying. Cipro encapsulated in mannitol particles (Cipro-mannitol) was significantly more effective at killing Pseudomonas aeruginosa (P. aeruginosa) grown in artificial mucus (AM) than Cipro encapsulated in either NaCl particles (Cipro-NaCl) or in hydrophobic particles consisting of dipalmitoylphosphatidylcholine (DPPC), albumin, and lactose (Cipro-DAL). The relatively high antibacterial effectiveness of Cipro-mannitol was not due to the effect of mannitol on bacteria or on Cipro. Rather, the unique performance of the mannitol-based particles in AM is attributable to its ability to increase local water content in the AM and enhance drug penetration into it. Mannitol is a promising excipient for inhalable microparticles that facilitate the drug delivery into the CF mucus.

Effect of artificial mucus properties on the characteristics of airborne bioaerosol droplets generated during simulated coughing

The effect of viscoelastic properties and surface tension of artificial mucus simulant samples on the size distribution and volume concentration of bioaerosol droplets generated during simulated coughing was investigated through in vitro experiments. The mucus simulant samples had viscoelastic properties in a similar range as those of real human airway mucus. The mucus simulant gels were prepared by mixing various proportions of 0.5–1.7% locust bean gum solution and 0.1 M sodium tetraborate (XLB) solution. Surface tension of one set of samples was varied by adding different amounts of SDS (sodium dodecyl sulfate) surfactant while the measurement of surface tension was performed using ADSA (axisymmetric drop shape analysis) method. The viscoelastic properties of the samples were measured using a Bohlin Gemini 200 HR (Malvern, UK) nano-rheometer with peltier plate assembly. An artificial cough machine was used to simulate human cough, generating aerosol droplets in a model trachea attached to the front of the cough machine. The size distribution and volume concentration of the droplets generated through simulated cough were measured using a laser diffraction particle sizer (SprayTec, Malvern, USA). The surface tension was found to have negligible effect on the characteristic of generated droplets within the range of this investigation. The experimental results showed a decrease in particle size as the samples changed from a viscous fluid type to a viscoelastic to an elastic solid type sample. The volume concentration also changed significantly as the viscoelasticity of the samples was varied.

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10 6/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly ( P < 0.001) up to 72 h after semen collection.

Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls

The performance of frozen-thawed spermatozoa from 10 Holstein bulls in a range of in vitro diagnostic tests and the relationship with adjusted in vivo fertility data was determined. The tests included an assessment of motility (subjective and computer-assisted), morphology, concentration, viability, acrosomal and chromatin integrity conducted immediately post-thaw and after swim-up, in conjunction with membrane status (CTC staining) and migration in an artificial cervical mucus. Adjusted in vivo fertility correlated with subjectively assessed post-thaw motility ( r = 0.672, p = 0.033), post-thaw straight-line velocity ( r = 0.636, p = 0.048), post-thaw sperm morphology ( r = −0.762, p = 0.010), post-thaw sperm viability ( r = 0.635, p = 0.048), the concentration of spermatozoa after swim-up ( r = 0.649, p = 0.042), sperm morphology after swim-up ( r = −0.687, p = 0.028), the number of spermatozoa migrating 10 mm into artificial cervical mucus ( r = 0.632, p = 0.050) and the distance migrated by the vanguard spermatozoon in artificial mucus ( r = 0.701, p = 0.024). A stepwise regression analysis identified tests which, when combined, produced models with a strong correlation ( R 2 > 0.9) to fertility.

Delivery of photosensitisers and light through mucus: Investigations into the potential use of photodynamic therapy for treatment of Pseudomonas aeruginosa cystic fibrosis pulmonary infection

Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In such patients chronic Pseudomonas aeruginosa infection is virtually impossible to eradicate using antibiotic therapy. Photodynamic antimicrobial chemotherapy (PACT) could be one potential alternative antimicrobial method. As photosensitisers could be delivered to the lungs of CF patients via inhalation, the current in vitro study investigated the potential use of PACT in the treatment of P. aeruginosa CF pulmonary infection. Delivery of red light (635 nm) and two photosensitisers (toluidine blue O (TBO) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP)) across artificial CF mucus was successfully achieved. Artificial CF mucus reduced the measured fluence of incident light in an almost exponential manner with increasing depth. The presence of dissolved photosensitisers also reduced light fluence. TMP diffused more efficiently across artificial CF mucus than TBO. However, receiver compartment concentrations of both drugs after 6 h were of the same order as those required to achieve high rates of kill (>99%) of P. aeruginosa isolates growing both planktonically and in biofilms. TMP required significantly higher concentrations (2.5 mg ml(-1)) than TBO to achieve high rates of kill (>99%) of P. aeruginosa isolates growing planktonically. Higher concentrations (5.0 mg ml(-1)) of both photosensitisers were required to achieve high rates of kill (>99%) of P. aeruginosa isolates growing in biofilms. When photosensitisers were prepared in artificial mucus, higher concentrations were required to achieve reasonably high kill rates (>80%) of P. aeruginosa (PAO1) growing both planktonically and in biofilm.

Static friction of porous bioceramic β-TCP on intestinal mucus films

The static friction behavior between a porous bioceramic material and an intestinal mucus film was investigated in order to develop a new intestine robotic endoscope. Here, the friction couple is porous β-tricalcium phosphate (β-TCP) and an artificial intestine mucus film. The effect of pore size of the β-TCP material on the friction behavior is investigated. The results illustrated that in this friction system there is a relatively small normal force upon the intestinal mucus film of the intestine wall during locomotion. The maximum static friction force in this friction couple varies with the pore size of the porous β-TCP material.

In vitro characteristics of fresh and frozen - thawed ram spermatozoa after sex sorting and re-freezing

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.

Endoscopic scoring of mucus quantity and quality: observer and horse variance and relationship to inflammation, mucus viscoelasticity and volume

Endoscopic scoring of airway mucus quantity and quality has not been critically assessed. To evaluate mucus scores for 1) observer- and horse-related variance and 2) association with inflammation, mucus viscoelasticity and measured volume. Variance of scoring within and between observers and over time within horses were determined for airway mucus accumulation, apparent viscosity, localisation and colour, and correlations of mucus accumulation scores with neutrophil ratios in secretions. The relationship of accumulation score to measured volumes of 'artificial mucus' was investigated. Correlations of mucus accumulation, apparent viscosity and colour scores with measured viscoelasticity were tested. Viscoelasticity was compared between tracheal secretion samples collected ventrally and dorsally. Mucus accumulation scoring showed excellent interobserver agreement and moderate horse-related variance, was related to measured volumes of 'artificial mucus', and correlated well with neutrophilic airway inflammation. Scores of mucus viscosity, colour and localisation showed high observer-related variance. Mucus accumulation, apparent viscosity and colour scores did not correlate with measured tracheal mucus viscoelasticity, but dorsally-localised mucus showed 2-fold higher measured viscoelasticity than ventrally-localised samples. Mucus accumulation scores are a reproducible measure of mucus volumes in the trachea. Endoscopic scoring of mucus accumulation is a reliable clinical and research tool. In contrast, apparent viscosity, localisation and colour scores should be interpreted with caution.

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 °C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 °C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples ( P<0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5±3.39%; 1.4±0.16 FPM versus 48.6±4.02%, 2.6±0.16 FPM; P<0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0±19.50 spermatozoa) and distance (56.3±5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5±2.77%) and FPM rating (2.8±0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0±3.67 spermatozoa, 21.6±3.05 mm, 12.2±1.31% and 1.2±0.10 FPM, respectively; P<0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0±1.2 mm; FT-GRADIENT: 57.2±0.76 mm; FT-CONTROL: 51.7±0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.

Experimental study of the effects of surface mucus viscosity on the glottic cycle

SummaryNumerous clinical findings indicate that viscosity of laryngeal mucosa is a crucial factor in glottal perfomance. Experience using experimental test benches has shown the importance of humidifying air stream used to induce vibration in excised larynges. Nevertheless, there is a lack of knowledge particularly regarding the physicochemical properties of laryngeal mucus. The purpose of this study was to research vocal fold vibration in excised larynges using artificial mucus of precisely known viscosity. Eight freshly harvested porcine larynges were examined. Parameters measured were Fo and vocal fold contact time. Measurements were performed under three conditions: basal (no fluid application on vocal cord surface), after application of a fluid of 60cP viscosity (Visc60), and after application of a fluid of 100cP viscosity (Visc 100). Electroglottographic measurements were performed at two different times for each condition: 1 s after airflow onset (T1) and 6 seconds after airflow onset (T2). Statistical analysis consisted of comparing data obtained under each condition at T1 and T2. The results showed a significant decrease in Fo after application of Visc60 and Visc100 fluids and a decrease in Fo at T2. Closure time was significantly higher under Visc60 conditions and under Visc100 conditions than under basal conditions. Application of artificial mucus to the mucosa of the vocal folds lowered vibratory frequency and prolonged the contact phase. Our interpretation of this data is that the presence of mucus on the surface of the vocal folds generated superficial tension and caused adhesion, which is a source of nonlinearity in vocal vibration.

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n=3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep® (AH) and stored at 5, 15 or 21°C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37°C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n=3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15°C for 24h or 5°C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15°C and daily for samples stored at 5°C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5°C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21°C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5°C.