Evaluation on the Morphology and Membrane Integrity of Immotile Human Sperm

Objective To determine the membrane integrity in the head and tail regions of individual spermatozoon, and observe sperm morphology for samples with totally immotile sperm. Methods Ten infertile men with immotile sperm were enrolled into this study （group A）. The membrane integrity in the head and tail regions of individual spermatozoon of immotile sperm was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test （HOS-EY test）. Sperm morphology was observed by light, scanning and transmission electron microscopy. Ten semen samples from normospermic donors were used as the control （group B）. Results The percentage of sperm with intact both head and tail membranes in group A was significantly lower than that in group B （P〈0.01）, whereas the value of sperm with defective head membrane but intact tail membrane in group A was significantly higher than that in group B （P〈0.01）. Abnormal sperm morphology in group A had a high incidence, and immotile sperm with viability and normal morphology could be observed in some cases. Most sperm had multiple ultrastructural defects. Conclussion Some immotile sperm had intact tail membrane but defective head membrane. Immotile sperm with viability and normal morphology could exist in some cases though abnormal sperm were in a great proportion. Carefully evaluating immotile sperm membrane integrity and morphology should benefit the treatment of patients with immotile sperm.

Portable fluorescence instrument for detecting membrane integrity in membrane bioreactor (MBR)

This study proposed the design, fabrication, and assembly of membrane integrity detection instruments in membrane bioreactors (MBR) based on fluorescence spectroscopy. Based on the PARAFAC model, we found that the peak at 280/335 nm strengthened after membrane breakage. The peak at 340/430 nm reflected the sludge concentration in the MBR and reduced the influence of internal filtration effects on detection. Therefore, we determined that the dual-LED light source excitation detection system can detect tryptophan-like substances at 280 nm (T-peak) and humic acid at 340 nm (C-peak). T-peak was identified as the core index indicating membrane integrity. Moreover, the C-peak is the reference indicator factor for a sensitive response to changes in the sludge concentration. The portable fluorescence instrument exhibited high sensitivity and good feedback accuracy compared to particle counting and turbidity detection, where the log reduction value was greater than 3.5. This overcomes the disadvantage of false alarms in particle counters and is not affected by the position of the pump system. This portable instrument provides a flexible and highly sensitive method for the assessment of industrial membrane integrity.

Changes on membrane integrity and ultrastructure of human sperm after freeze-drying

Objective To observe changes on membrane integrity and ultrastructure of human sperm after freeze-drying.Methods Semen samples from both normospermic donors（group A, n=15） and infertile men with abnormal sperm parameters（group B, n=15） were enrolled into this study. These samples were freeze-dried by using a freeze-drying method. The membrane integrity in the head and tail regions of individual spermatozoon was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test. Sperm ultrastructure in groups A（n=3） and B（n=3） was observed by scanning electron microscopy（SEM）.Results After freeze-drying, all spermatozoa were types I（damaged both head and tail membranes） and III（damaged head membrane and intact tail membrane） membrane integrity in groups A and B. Type III of group B had lower value than that of group A（P〈0.01）. Under SEM, intact freeze-dried spermatozoa including abnormal morphology and normal-looking morphology were observed in both groups A and B. A few freezedried sperm heads had unsmooth or fuzzy surface. Isolated sperm heads, bent tails,broken sperm tails or fragmentary tails were more frequently seen in group B than those in group A.Conclusion Freeze-dried human spermatozoa could have intact structural components. However, freeze-drying resulted in severe damage on membrane integrity and ultrastructure of sperm. Samples from infertile men would have less resistance to freeze-drying.

Single Layer Centrifugation with Androcoll-E^(TM) improved progressive motility and percentage of live spermatozoa with intact acrosome of chilled stallion semen but did not have an effect on DNA integrity

A significant number of stallions produce low quality ejaculates with high sensibility to chilling. Single Layer Centrifugation (SLC) with Andro-coll-ETM has been presented as an efficient method of selecting good quality spermatozoa. In the current study, changes in sperm quality (motility, viability, acrosome integrity and DNA damage) occurring during storage at 5?C for a maximum of 72 h, were investigated. For that, one ejaculate from 12 stallions was split in two aliquots: control and SLC-selected. Both aliquots were chilled and stored at 5?C and spermatozoa were evaluated for motility, viability and acrosome integrity at 24, 48 and 72 h post collection. DNA damage was evaluated at 48 h post collection using the comet assay. In the SLC-selected aliquots, there was a significant improvement in terms of progressive motility (0 h: P = 0.005;24 h: P 0.05). SLC with Androcoll-ETM improved semen quality prolonging sperm longevity of chilled semen (P = 0.012). This positive effect was more evident in ejaculates most sensitive to chilling that had a sharp decrease in motility in the first 24 h of refrigeration and for all ejaculates at 72 h post-chilling. Therefore, this method reveals to be a useful technique for selecting spermatozoa and maintain sperm quality during storage.

Calcitriol induces post-thawed bovine sperm capacitation

Background:Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity.In vivo,this process is directly associated with high calcium levels in sperm cytoplasm.Calcitriol,the vitamin D hypercalcemic metabolite,is related to human sperm motility,capacitation,and acrosome reaction.This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation.Methods:One million freezethawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min.Untreated cells(negative control)and treated ones with calcitriol or heparin(100μg/mL,positive capacitation control)were evaluated for motility,viability,and functional parameters.Menadione(70μM,30 min)treatment was included as a reactive oxygen species(ROS)positive sperm agent.Results:The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability,vigor,and capacitation than their positive and negative controls.The percentage of sperm with intact plasma and acrosome membranes,mitochondrial membrane potential(ΔΨm),and phosphatidylserine externalization was similar in all the conditions evaluated,while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control.Conclusion:Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility,viability,and vigor without altering key biological parameters such as redox status,ΔΨm,and cell death.

Effect of low-density lipoproteins, spermatozoa concentration and glycerol on functional and motility parameters of bull spermatozoa during storage at 4℃

An extender has been developed with low-density lipoproteins （LDLs） that eliminates the microbial risks associated with the use of whole egg yolk. The objective of this study was to assess the effects of substituting egg yolk with LDLs for use as an extender in sperm preservation at 4 ℃, as well as on spermatozoa motility, plasma membrane and acrosome integrity, at two different concentrations （80×10^6 and 240× 10^6 sperm per ml） for 8 days and to evaluate glycerol toxicity in both extenders. A total of 12 ejaculates were collected from three bulls. Spermatozoa motility was examined using computer-assisted semen analysis. Plasma membrane integrity was determined using the hypo-osmotic swelling test and acrosome integrity with the fluorescein isothiocyanate-Pisum sativum agglutinin test. The semen was subsequently divided into four aliquots and diluted with Tris-egg yolk-glycerol （TEG）, Tris-egg yolk without glycerol （TE）, LDL with glycerol （LDL＋） and LDL without glycerol （LDL-）, at 80×10^6 and 240 ×10^6sperm per ml. This study showed that the LDL＋ and LDL- extenders were more effective at preserving spermatozoa motility, plasma membrane integrity and acrosome integrity than TEG and TE （P〈0.05） during 8 days of incubation. After 3 days of incubation, a toxicity of glycerol was observed in TEG, whereas no significant difference was observed between LDL＋ and LDL-. We can therefore conclude that the LDL extender can be used to refrigerate semen at 4 ~C instead of TEG and TE at 80×10^6and 240×10^6 sperm per ml for elite bulls. This finding can be used to define a policy for the storage of high-quality bull semen.

In-vitro effect of Peganum harmala total alkaloids on spermatozoa quality and oxidative stress of epididymal ram semen

Objective:To determine the in-vitro effect of the total alkaloid extract of Peganum(P.)harmala seeds on ram epididymal sperm.Methods:Semen was divided into six groups according to the following concentrations of the P.harmala total alkaloids:1,5,10,50,and 100μg/mL,and the control group.The samples were incubated at ambient temperature(21℃-24℃)for 24 h,and analyzed in terms of motility,membrane integrity,and oxidative status.Results:The sperm kinematic parameters,i.e.straight-line velocity,curvilinear velocity,average path velocity,were significantly higher when treated with P.harmala at concentrations ranging from 1 to 10μg/mL compared to the control group(P<0.05).In addtion,the highest amplitude of the lateral head displacement value was found in the groups treated with concentrations 1 and 5μg/mL of P.harmala compared to the control group(P<0.05).Total and progressive motilities showed that the extracts at 1,5,and 10μg/mL exhibited a high percentage after 24 h of incubation.The effect of P.harmala extracts on the membrane integrity of ram epididymal sperm was concentration-dependent and significantly different compared to the control group(P<0.05).Non-significantly lower lipid peroxidation levels were observed after 24 h of incubation of ram epididymal sperm treated with concentrations 1,5,and 10μg/mL of P.harmala extracts compared to the control group(P>0.05).Conclusions:Low concentrations(1-10μg/mL)of P.harmala extracts stimulate sperm motility,preserve membrane integrity and protect ram spermatozoa from lipid peroxidation.

Stability of fluorochrome based assays to measure subcellular sperm functions

Aim： To evaluate the long-term stability of the fluorescence signals of new fluorescence-based semen analysis assays for clinical application. Methods： Semen samples from 87 unselected infertile patients were used to perform the following assays： （i） detection of active caspase-3 （n = 17）; （ii） integrity of the mitochondrial membrane potential （MMP） （n = 17）; （iii） externalization of phosphatidylserine （EPS） （n = 16）; and （iv） detection of intact acrosomes via CD46 （n = 37）. After the assays, 4% paraformaldehyde was added to all aliquots. The fluorescence intensity of each sample was evaluated by flow cytometry on days 0, 3, 7, 10 and 14. Results： Differences of up to ± 5% positive spermatozoa from the value measured at day 0 were estimated as acceptable deviation. The Caspase-3 FLICA^TM showed mean differences 〈 5% at day 3, 7 and 10. At day 14 the mean difference was 7.6%. In contrast, the disrupted MMP and the EPS detection showed differences 〉 5% at day 3. The CD46-FITC labeling displayed absolute differences 〈 5% CD46-positive spermatozoa at days 3, 7, 10 and 14. Conclusion： Although immediate analysis of the fluorescence signals is recommended, it is possible to evaluate caspase-3 activation up to 10 days and CD46 up to 14 days after staining of sperm. The FACS evaluation of MMP and EPS detection should be conducted on the same day.

Design of Subharmonic Mixers above 100 GHz

This paper presents the design and simulation of several fixed-tuned sub-harmonic mixers cover frequencies from 110 GH to 130 GHz, 215 GH to 235 GHz, 310 GH to 350 GHz, and 400 GH to 440 GHz. Among them, 120 GHz, 225 GHz, 330 GHz subharmonic mixers are designed with flip-chipped planar schottky diode mounted onto a suspended quartz-based substrate, the 225 GHz and 425 GHz subharmonic mixers are GaAs membrane integrated, and the 115 GHz subharmonic mixer has been fabricated and tested already.

Fate of microplastics in a coastal wastewater treatment plant: Microfibers could partially break through the integrated membrane system

Rare information on the fate of microplastics in the integrated membrane system (IMS) system in full-scale wastewater treatment plant was available. The fate of microplastics in IMS in a coastal reclaimed water plant was investigated. The removal rate of microplastics in the IMS system reached 93.2% after membrane bioreactor (MBR) treatment while that further increased to 98.0% after the reverse osmosis (RO) membrane process. The flux of microplastics in MBR effluent was reduced from 1.5 × 10^(13) MPs/d to 10.2 × 10^(11) MPs/d while that of the RO treatment decreased to 2.7 × 10^(11) MPs/d. Small scale fiber plastics (< 200 μm) could break through RO system according to the size distribution analysis. The application of the IMS system in the reclaimed water plant could prevent most of the microplastics from being discharged in the coastal water. These findings suggested that the IMS system was more efficient than conventional activated sludge system (CAS) for the removal of microplastics, while the discharge of small scale fiber plastics through the IMS system should also not be neglected because small scale fiber plastics (< 200 μm) could break through IMS system equipped with the RO system.

Effect on Life Span and Membrane Protein in Red Blood Cells by Integrated Medicine Therapy on Chronic Aplastic Anemia

Objective: To explore the mechanism ofintegrated traditional Chinese and Westernmedicine (TCM--WM ) therapy on chronicaplastic anemia (CAA). Methods: The RBClife span of 30 normal human subjects and 30patients with CAA were measured by sir labelled technique before and after TCM--WMtherapy. The morphology and distribution ofRBC membrane protein granules were observed by freeze fracture etching and transmission electron microscope. Results: The halflife of erythrocytes (RBC TI/2)was shortenedin CAA cases and there was a significant difference compared to healthy control (P <0. 01). After therapy, the RBC life span prolonged and approached the normal level. Before treatment, there existed abnormal in morphology, decrease in amount and uneven indistribution of protein granules in protoplasmicface (PF) and extracellular face (EF) of RBCmembrane. After treatment, the protein granules of RBC membrane was improved and approached to control. Conclusions: The morphology, amount, quality and distribution ofRBC membrane protein granule were closelyrelated to its life span. The therapeutic effectof TCM--WM was better than that of WMalone and it had a function both in stabilizingmembrane protein and extending the RBC lifespan.

Influence of in vitro capacitation time on structural and functional human sperm parameters

A cascade of dramatic physiological events is linked to the sperm acrosome reaction and binding to the oocyte's zona pellucida during human sperm capacitation.However,structural and functional sperm changes during capacitation currently remain poorly defined.Here,we performed a multibiomarker approach based on the utilization of sperm concentration,motility,viability,morphology,acrosome reaction,tyrosine phosphorylation,DNA fragmentation,and lectin-binding sites to analyze the impact caused by swim-up selection times(uncapacitated,1 h capacitated,and 4 h capacitated)on sperm function and structure in normozoospermic samples.We found that a 4 h swim-up capacitation increased sperm quality,because a large number of cells with normal morphology and lower DNA fragmentation rates were recovered.Furthermore,the long-term capacitation induced a higher percentage of cells with tyrosine phosphorylation of the principal piece as well as a redistribution of lectin-binding sites.Overall,the multivariate biomarkers analyzed showed a less variable distribution on spermatozoa recovered after 4 h capacitation than that with the shorter capacitation time.These findings stress the importance of capacitation time as a relevant factor in sperm quality with potential biological reproductive implications both for basic research and in assisted reproduction techniques.