Motility Characteristics Research Articles

Sulforaphane inclusion in a freezing medium augments post-thaw motility, functional and biochemical features, and fertility potential of buffalo (Bubalus bubalis) spermatozoa

Sulforaphane is a natural and highly effective antioxidant safeguarding the reproductive system, and alleviate oxidative stress. This study was designed in order to elaborate L-sulforaphane effect on semen quality, biochemical parameters, and fertility of buffalo (Bubalus bubalis) spermatozoa. Semen was collected from five buffalo bull with artificial vagina (42 °C) three times and evaluated for volume, consistency (color), motility, and sperm concentration. After critical examination, semen was diluted (50 × 106 spermatozoa per ml, 37 °C) in extenders with (2 μM, 5 μM, 10 μM, and, 20 μM) or without (control) sulforaphane, cooled (from 37 to 4 °C), equilibrated (4 °C), filled (straws, 4 °C), and cryopreserved (LN2, −196 °C). Data analysis exhibited that sulforaphane addition in extender augments total motility (%, 10 μM, and 20 μM than control), progressive motility (%), and rapid velocity (%, 20 μM than control), and velocity parameters (average path velocity, μm/s, straight line velocity, μm/s and curved linear velocity, μm/s, 20 μM than control, and 2 μM). Moreover, sulforaphane augments functional features (membrane functionality, mitochondrial potential, and acrosome integrity) of buffalo sperm (20 μM than control). Sulforaphane preserves biochemical features of seminal plasma of buffalo i.e., Calcium (μM), and total antioxidant capacity (μM/L), followed by reduction in lactate dehydrogenase (IU/L), reactive oxygen species (104 RLU/20 min/ 25 million), and lipid peroxidation (μM/ml) in 20 μM than control. Lastly, sulforaphane augments fertility rate of buffalo sperm at 20 μM than control, and 2 μM. Conclusively the existing study revealed that adding L-sulforaphane (20 μM) in a freezing medium augments motilities, kinematics, functional parameters, and fertility rate of buffalo spermatozoa. Correspondingly, sperm favorable biochemical features were also augmented with sulforaphane followed by reduction in oxidative stress parameters. Further studies are highly recommended to define the particular mechanism of action of sulforaphane in augmenting buffalo post-thawed semen quality, and in vitro fertility potential.

Determining the Fertility Characteristics of Iraqi Local Chickens by Studying the Variance in the Characteristics of Semen

The Iraqi local chicken is national wealth, so it is necessary to work on increasing knowledge of its fertilization capabilities. The current research aims to identify the local breeds that have fertility characteristics by studying the variance in semen quality and comparing it with some characteristics of the semen of other local breeds. Replicates of white breeds W, Black B, and striped BW were adopted at 60 weeks of age. Semen values according to the characteristics of colour, the volume of semen, number and concentration of sperm, percentage of abnormalities, live and dead sperm, and individual and collective motility. Semen colour for all breeds was pearly white, and there had been no big differences in the characteristics of semen size, sperm count, concentration, individual and collective motility, and percentage of dead sperm. While, the white and striped breeds significantly outperformed the black breed (P≤0.05) in the proportion of stay sperms. Black breed significantly outperformed the rest of the study breeds in the percentage of sperm abnormalities, as it turned out that the study breeds had good fertility characteristics compared to other local breeds.

Eukaryotic Initiation Factor 5A2 localizes to actively translating ribosomes to promote cancer cell protrusions and invasive capacity

BackgroundEukaryotic Initiation Factor 5A (eIF-5A), an essential translation factor, is post-translationally activated by the polyamine spermidine. Two human genes encode eIF-5A, being eIF5-A1 constitutively expressed whereas eIF5-A2 is frequently found overexpressed in human tumours. The contribution of both isoforms with regard to cellular proliferation and invasion in non-small cell lung cancer remains to be characterized.MethodsWe have evaluated the use of eIF-5A2 gene as prognosis marker in lung adenocarcinoma (LUAD) patients and validated in immunocompromised mice. We have used cell migration and cell proliferation assays in LUAD lines after silencing each eIF-5A isoform to monitor their contribution to both phenotypes. Cytoskeleton alterations were analysed in the same cells by rhodamine-phalloidin staining and fluorescence microscopy. Polysome profiles were used to monitor the effect of eIF-5A2 overexpression on translation. Western blotting was used to study the levels of eIF-5A2 client proteins involved in migration upon TGFB1 stimulation. Finally, we have co-localized eIF-5A2 with puromycin to visualize the subcellular pattern of actively translating ribosomes.ResultsWe describe the differential functions of both eIF-5A isoforms, to show that eIF5-A2 properties on cell proliferation and migration are coincident with its features as a poor prognosis marker. Silencing of eIF-5A2 leads to more dramatic consequences of cellular proliferation and migration compared to eIF-5A1. Overexpression of eIF-5A2 leads to enhanced global translation. We also show that TGFβ signalling enhances the expression and activity of eIF-5A2 which promotes the translation of polyproline rich proteins involved in cytoskeleton and motility features as it is the case of Fibronectin, SNAI1, Ezrin and FHOD1. With the use of puromycin labelling we have co-localized active ribosomes with eIF-5A2 not only in cytosol but also in areas of cellular protrusion. We have shown the bulk invasive capacity of cells overexpressing eIF-5A2 in mice.ConclusionsWe propose the existence of a coordinated temporal and positional interaction between TFGB and eIF-5A2 pathways to promote cell migration in NSCLC. We suggest that the co-localization of actively translating ribosomes with hypusinated eIF-5A2 facilitates the translation of key proteins not only in the cytosol but also in areas of cellular protrusion.5zFBY1s4Yn-gzgeYzNdWy3Video

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

Abstract P3-08-06: Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model

Abstract Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions (&amp;lt;~0.001mm2). This allowed for computation of the total tumor area including average area of each cluster. In addition, the area moment, which describes the basic directional growth pattern of the tumor cells, was quantified by multiplying a cluster distance from the center of mass by its area. Conclusions: A key novel finding from structured illumination imaging data was the observation of LVI occurring early, similar with the clinical presentation in IBC patients. This model was able to effectively track tumor cluster migration. This approach of short-term longitudinal imaging time frame in studying transient or dynamic events of diffuse, collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility and vessel characteristics is an innovation that can be used to investigate other cancer cell types exhibiting LVI. Funding in part by DoD W81XWH-17-1-0297; W81XWH201053 (GRD); ACS Mission Boost grant MBG-20-141-01 (GRD) and NIH grant P30-CA014236 (Imaging Core/GMP) Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Effect of BNT162b2 mRNA COVID-19 vaccine on sperm morphokinetics and DNA integrity

Objective: To assess whether the coronavirus disease 2019 (COVID-19) mRNA vaccine affects sperm morphokinetics using a computer-assisted semen analyzer and other semen parameters using a sperm chromatin structure assay. Methods: Healthy male volunteers in two Japanese clinics between May 2021 and December 2021 were prospectively analyzed. Participants donated sperm twice, two days apart, in the following phases: before vaccination, 2 weeks after the first vaccine dose, and 2, 4, and 12 weeks after the second dose. Basic sperm parameters, sperm motility characteristics, and the percentage of DNA-damaged sperm were compared among the different phases. Results: Ninety-six semen samples from ten volunteers, who were vaccinated with the BNT162b2 mRNA vaccine, were evaluated. There were no significant differences between any phases in basic semen findings and parameters of the sperm chromatin structure assays. Regarding sperm motion characteristics, the average linear velocity, beat-cross frequency, and sperm motility index significantly decreased after the second vaccine dose (P=0.018, P=0.003, and P=0.027, respectively), with no significant differences between any two phases by post-hoc pairwise comparisons. Conclusions: After COVID-19 mRNA vaccination, while sperm motion characteristics might fluctuate, no apparent deterioration of basic sperm parameters or sperm DNA integrity was observed. Given the adverse effects of COVID-19 on sperm, our findings suggest that there might be no reason to refrain from vaccination for healthy individuals.

Interpreting T-cell search "strategies" in the light of evolution under constraints.

Two decades of in vivo imaging have revealed how diverse T-cell motion patterns can be. Such recordings have sparked the notion of search "strategies": T cells may have evolved ways to search for antigen efficiently depending on the task at hand. Mathematical models have indeed confirmed that several observed T-cell migration patterns resemble a theoretical optimum; for example, frequent turning, stop-and-go motion, or alternating short and long motile runs have all been interpreted as deliberately tuned behaviours, optimising the cell's chance of finding antigen. But the same behaviours could also arise simply because T cells cannot follow a straight, regular path through the tight spaces they navigate. Even if T cells do follow a theoretically optimal pattern, the question remains: which parts of that pattern have truly been evolved for search, and which merely reflect constraints from the cell's migration machinery and surroundings? We here employ an approach from the field of evolutionary biology to examine how cells might evolve search strategies under realistic constraints. Using a cellular Potts model (CPM), where motion arises from intracellular dynamics interacting with cell shape and a constraining environment, we simulate evolutionary optimization of a simple task: explore as much area as possible. We find that our simulated cells indeed evolve their motility patterns. But the evolved behaviors are not shaped solely by what is functionally optimal; importantly, they also reflect mechanistic constraints. Cells in our model evolve several motility characteristics previously attributed to search optimisation-even though these features are not beneficial for the task given here. Our results stress that search patterns may evolve for other reasons than being "optimal". In part, they may be the inevitable side effects of interactions between cell shape, intracellular dynamics, and the diverse environments T cells face in vivo.

Comprehensive insights into the metabolism characteristics of small RNA Qrr4 in Vibrio alginolyticus.

Vibrio alginolyticus is an important foodborne pathogen that can infect both humans and marine animals and cause massive economic losses in aquaculture. Small noncoding RNAs (sRNAs) are emerging posttranscriptional regulators that affect bacterial physiology and pathological processes. In the present work, a new cell density-dependent sRNA, Qrr4, was characterized in V. alginolyticus based on a previously reported RNA-seq analysis and bioinformatics approach. The effects of Qrr4 actions on the physiology, virulence, and metabolism of V. alginolyticus were comprehensively investigated based on molecular biology and metabolomics approaches. The results showed that qrr4 deletion markedly inhibited growth, motility and extracellular protease activities. Additionally, nontargeted metabolism and lipidomics analyses revealed that qrr4 deletion induced significant disturbance of multiple metabolic pathways. The key metabolic remodelling that occurred in response to qrr4 deletion was found to involve phospholipid, nucleotide, carbohydrate and amino acid metabolic pathways, providing novel clues about a potential mechanism via which mutation of qrr4 could interfere with cellular energy homeostasis, modulate membrane phospholipid composition and inhibit nucleic acid and protein syntheses to regulate the motility, growth and virulence characteristics of V. alginolyticus. Overall, this study provides a comprehensive understanding of the regulatory roles of the new cell density-dependent sRNA Qrr4 in V. alginolyticus. KEY POINTS: • A novel cell density-dependent sRNA, Qrr4, was cloned in V. alginolyticus. •Qrr4 regulated growth and virulence factors of V. alginolyticus. • Phospholipid, nucleotide and energy metabolisms were modulated obviously by Qrr4.

Tau acts as a selective barrier along microtubules to regulate multi-motor cargo transport.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. Tau is a neuronal MAP that stabilizes microtubules within the axon. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. To dissect tau's role in regulating transport, we isolate endocytic organelles from cells and reconstitute their motility along tau-decorated microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility characteristics and response to tau. Here, we found that early phagosomes (EPs) undergo unidirectional retrograde transport while late phagosomes (LPs) move bidirectionally. Correspondingly, different numbers and combinations of kinesins-1, -2, -3, and dynein are bound to EPs and LPs. Previous studies showed that tau preferentially inhibits kinesin motors, which biases the transport of LPs towards the microtubule minus-end. Recently, we showed that tau strongly inhibits long-range, dynein-mediated EP motility. Dynein is the dominant motor in EP transport, and tau strongly impedes forces generated by teams of multiple dynein motors and accelerates dynein unbinding under load. These results are surprising, as previous single-molecule studies showed that dynein is less sensitive to tau compared to kinesin. Our results show that specific cargoes differentially respond to tau, where dynein-complexes on EPs are more sensitive to tau inhibition than those on LPs, suggesting that pathological changes in tau associated with Alzheimer's disease and other tauopathies will also have disparate impact on cargoes. We will next compare the effects of pathogenic tau mutations on the transport of different cargoes in iPSC-derived neurons with the aim of identifying the pathways most disrupted in neurodegenerative disease.

Morphodynamic and motility feature-based deep learning classification for subtypes of cancer-associated fibroblasts.

The cancer-associated fibroblasts (CAFs) have risen to prominence as key players in cancer progression. It is now widely appreciated that CAFs are composed of heterogeneous populations, either pro-tumorigenic or anti-tumorigenic. In addition, a growing body of evidence supports the multifaceted nature of CAFs. Therefore, researchers in the field have been trying to identify subtypes of CAFs with molecular markers. However, those markers cannot accurately classify the subpopulations and can be co-expressed in different subtypes. In order to utilize CAFs as a target for cancer treatment, issues with subtypes of CAFs must be resolved such that specific pro-tumorigenic subtypes can be suppressed or reprogrammed to anti-tumorigenic ones. The morphology and the motile characteristics of CAFs result from gene expression combinations. Thus, those characteristics can be holistic readouts of CAFs. Unlike biomolecular analysis, a fixed cell-based end-point assay, morphology or motility features of cells can be traced with live-cell imaging. Those features can provide information on the dynamic changes of CAFs. Here, in order to comprehensively identify subtypes CAFs, we adopt a deep learning-based cell classification strategy. Using multiple unsupervised and supervised machine learning algorithms, we extract the morphodynamic and motility features of cells from label-free live-cell imaging data of CAFs. To this end, we established in vitro breast CAFs, which were fibroblasts cocultured with two different breast cancer cell lines with different aggressiveness. As a result, we show that the morphodynamic and motility features can successfully classify heterogeneous subpopulations of cells.

Controlling Collision-Induced Aggregations in a Swarm of Micro Bristle Robots

Systematically designing local interaction rules to achieve collective behaviors in robot swarms is a challenging endeavor, especially in micro robots, where size restrictions imply severe sensing, communication, and computation limitations. In such robot swarms, performing useful functions is often preconditioned on the formation of high-density aggregations which can facilitate collective signaling and information sharing. In this article, we present a systematic approach to control aggregation behaviors by leveraging the physical interactions in a swarm of 300 3-mm vibration-driven micro bristle robots that we designed and fabricated. We demonstrate the ability to control the degree of aggregation by varying the motility characteristics of the robots through global vibration frequency and amplitude inputs, after comprehensive characterization, modeling, and simulation of the locomotion dynamics and robot interactions. To quantify the degree of aggregation, we also introduce a new metric, the motility-induced phase separation index index, which unlike many existing methods does not require a scenario-specific tuning of parameters. Our investigations reveal how physics-driven interaction mechanisms can be exploited to achieve desired behaviors in minimally equipped robot swarms and highlight the specific ways in which hardware and software developments aid in the achievement of collision-induced aggregations.

Gastric dysmotility and gastrointestinal symptoms in myalgic encephalomyelitis/chronic fatigue syndrome

Background Gastrointestinal symptoms are common in ME/CFS, but there is a knowledge gap in the literature concerning gastrointestinal motility features and detailed symptom description. Objective In this study, we aimed to characterize gastric motility and gastric symptoms in response to a liquid meal. Methods We included 20 patients with ME/CFS with abdominal complaints who were recruited to a double-blind randomized placebo-controlled trial of Rituximab. The patients of this sub study were examined with an ultrasound drink test, and gastrointestinal symptoms were evaluated using the Rome III questionnaire and Irritable Bowel Syndrome Symptom Severity Scale (IBS-SSS) questionnaire. Results We found that patients commonly reported fullness/bloating (75%), abdominal pain (45%) and nausea (35%). Ultrasound measurements revealed lower proximal measurements of the stomach after a meal (p < 0.01) and larger fasting antral area (p = 0.019) compared to healthy controls. The patients had a stronger symptomatic response to the liquid meal compared to healthy controls regarding epigastric pain, discomfort and nausea (p < 0.05). Ninety percent of the patients reported bowel movement frequencies within the normal range but scored high on bowel habit dissatisfaction and life disruption. Conclusion The patients presented with fullness/bloating, nausea and epigastric pain, showed signs of impaired gastric accommodation and visceral hypersensitivity, showing that the gastrointestinal symptoms of ME/CFS patients are similar to functional dyspepsia. Key summary Gastrointestinal symptoms are common in ME/CFS, but there is a knowledge gap in the literature concerning gastrointestinal motility features and detailed symptom description. • In this study, patients with ME/CFS had signs of impaired gastric accommodation after a liquid meal. • Out of 20 patients, 15 patients reported fullness/bloating, 9 reported abdominal pain, and 7 reported nausea. The patients showed signs of visceral hypersensitivity on a drink test. • Our findings suggest that patients with ME/CFS share many similarities with patients with Functional Dyspepsia. The findings were not typical for Irritable Bowel Syndrome.

Detection of sperm cells by single-stage and two-stage deep object detectors

Today, infertility is a common health concern that affects approximately 15%–20% of the world population. The evaluation of male patients with infertility includes diverse examination and laboratory tests in comparison to female patients. In the evaluation of male infertility, sperm specimens are examined in terms of morphometry, concentration, and motility. Detection of sperm is the critical step to determine the concentration and motility parameters. In this study, a fusion approach of deep learning-based object detection techniques is utilized in the sperm detection problem for obtaining more accurate and consistent concentration and motility characteristics of sperm specimens. First, 12 sperm specimen videos acquired from different infertile patients were recorded. Then, regions in the video frames were labeled as sperm and non-sperm by the experts. Two different scenarios have been tested over the labeled dataset. Differently arranged train and test data ratios were also utilized for each scenario. In the first scenario, deep learning-based object detection algorithms were individually performed for the sperm detection of patient-oriented videos in terms of different train/test split ratios. Videos with the lowest mAP (mean Average Precision) values resulted in the first scenario were selected for the second scenario as target videos. The rest of the videos were used for model training without any patient-oriented manner. In the second scenario, the detection performance for more challenging videos is aimed to increase by using different videos instead of using the small part of the same video in the model training. Additionally, a fusion idea of the utilized deep learning-based techniques was proposed and performed over these low mAP resulted videos to increase the detection performance. The results are compared regarding the general mAP, class-wise APs, and training time. In the first scenario, YOLOv5 achieved the best results, while the proposed fusion approach achieved the best mAP scores in the second scenario.

Sedimentation Rate of Dunaliella salina in Dark Conditions

Microalgae are a source of carbohydrates, proteins and lipids. Thus, they can be considered as raw material to transition from current fossil fuel-based refineries to biorefineries. Microalgae harvesting is considered a major challenge in biomass production. There are several harvesting techniques, but the majority of them are either expensive or not effective. The harvesting method that we propose is sedimentation-induced by light blockage, taking advantage of the motility characteristics of certain microalgae. In this research, the halophilic microalgae Dunaliella salina was selected. Experiments were conducted under light and dark conditions to compare the sedimentation rates. Sedimentation behavior was measured by collecting data on the optical density and cell count under both light and dark conditions. The results showed that, under light conditions, the cell count in the middle of the flask decreased from 1 × 106 cell/mL to 5 × 104 cell/mL after 50 days. Under dark conditions sedimentation took less than 10 days for complete settlement. Leaving Dunaliella salina under dark conditions may constitute a promising harvest method as this provides a high recovery rate and requires low energy.

Evaluation of the in vitro cryopreservative performance of Juniper berry oil (Juniperus communis) on frozen-thawed bull semen

This study aimed to reveal the effects of juniper (Juniperus communis) berry oil (JB) when added to the Tris extender, on bull (Holstein) sperm motility characteristics, abnormal sperm ratio, DNA fragmentation and some oxidative stress markers. The pooled ejaculate was divided into five equal parts as control (C) and Tris diluent containing 25, 50, 100, 200 μg/ml JB. Diluted semen was incubated at 4 °C for 4 h and then frozen in liquid nitrogen (−196 °C). After freezing-thawing, motility characteristics were determined using a sperm analyzer system. The results showed that the 25 μg/ml dose of JB had an improving effect on overall sperm motility (P &lt; 0.05). The total abnormal spermatozoa ratio was not significantly different between the groups (P &gt; 0.05). The JB25 and JB50 groups had higher DNA integrity than the other groups (P &lt; 0.05). This finding was supported by low malondialdehyde levels (P &lt; 0.05). Especially the 25 μg/ml dose of JB was found to have a remedial effect on DNA integrity, as well as increasing progressive and total motility rates. This study has revealed new information on the effects of JB on bull semen cryopreservation. Juniper berry oil may be used as an antioxidant in bull semen freezing.

Reconstitution of Organelle Transport Along Microtubules In Vitro.

In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.

Tribulus terrestris ekstraktının ilavesiyle gökkuşağı alabalığının (Oncorhynchus mykiss) sperm motilitesinin arttırılması

Tribulus terrestris (TT) is a famous traditional plant of family Zygophyllaceae and widely distributes around the world. TT has potential to elevate the testosterone, testosterone precursor and luteinizing hormone levels. In this study, trials were conducted to assessment the impact of Tribulus terrestris addition on sperm motility of rainbow trout (Oncorhynchus mykiss) for the first time. In the trial, we used to different concentrations [0 µg L-1 (Control), 200 µg L-1, 400 µg L-1, 600 µg L-1, 800 µg L-1 and 1000 µg L-1] of T. terrestris extract. Sperm motility characteristics and longevity were determined. In addition, protodioscin content of T. terrestris extract was assessed. The present study revealed that the presence of T. terrestris caused to increase in sperm motility. The increases in duration (49.00±3.61 s) and motility rate (91.67±2.89%) at 400 µg L-1 were statistically significant (p&amp;lt;0.05). Overall, addition of T. terrestris to activation solution can increase the sperm motility of rainbow trout.

LACTATIONAL EXPOSURE OF METHOXYCHLOR IMPAIRS SPERM QUALITY IN BALB/C MICE

Numerous pesticides derived from human activity are now disseminated in the environment that have been identified as an estrogenic activity that mainly affects the male reproductive system. The objective study was to determine the dose of methoxychlor (MXC) pesticides administered to the mother during the lactation period that would affect the sperm quality of the offspring in mice. Twenty-four BALB/c strains were divided into control and treatment groups. MXC was administered once daily by intraperitoneal to female mice for days 1 to 21 of lactation periods, with 0.14 mg/g, 0.28 mg/g, and 0,42 mg/g doses of MXC. Male litters were killed at postnatal day (PND) 60, and the cauda epididymis was used to observe sperm motility and morphology characteristics. Parameters observed were the number of spermatozoa abnormal and the percentage of progressive spermatozoa. ANOVA showed that the effect of MXC was significantly different in the case of the number of abnormal sperm and the rate of progressive sperm parameters. Duncan's Multiple Range Test (DMRT) showed that D3 (0.42 mg/g) gave the highest effect of MXC on increasing abnormal spermatozoa morphology and decreasing the percentage of motile sperm. These results indicate that exposure to MXC, which showed a physiological response in the lactation period, reduces the sperm quality of offspring

Santolina pinnata Viv. Exerts Promising Antitumor Activity against Breast Cancer Cells and Anti-Inflammatory Effects in LPS-Stimulated RAW 264.7 Cells.

Cancer is one of the largest causes of mortality in the world, and due to its incidence, the discovery of novel anticancer drugs is of great importance. Many successful anticancer drugs used in clinical practices are derived from natural products. The genus Santolina is a group of species distributed in the Mediterranean area and used in traditional medicine for their biological properties. The aim of this work was to investigate, for the first time, the multi-target biological potential of Italian Santolina pinnata in relation to their chemical profile, by which an interesting natural source of valuable phytochemicals endowed with anticancer and anti-inflammatory features could be assessed. n-Hexane (EHSP) and methanol (EMSP) extracts were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography (UHPLC), respectively. Anti-proliferative activity was analyzed on MCF-7 and MDA-MB-231 breast cancer cells, as well as on non-tumorigenic MCF-10A cells, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptotic death was assessed by comet assay. Cell motility and invasive features were examined in highly invasive MDA-MB-231 by wound-healing scratches, while, in both breast cancer cell lines, by gel-zymography experiments. The anti-inflammatory potential was analyzed by nitric oxide (NO) production and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) staining experiments in bacterial lipopolysaccharides (LPS) which stimulated RAW 264.7 cells. EHSP and EMSP extracts exhibited anticancer activity against breast cancer cells, promoting apoptotic death, as well as decreasing cell migration and invasive behaviours. The highest activity (IC50 of 15.91 μg/mL) was detected against MDA-MB-231 cells, a highly invasive breast cancer cell line. Both extracts were also able to promote anti-inflammatory effects (IC50 values ranging from 27.5 to 61.14 μg/mL), as well as to reduce NO levels by inducing inhibitory effects on NF-κB nuclear translocation in LPS-stimulated RAW 264.7 cells. The different biological behaviours found between the extracts could be related to their different chemical compositions. Herein, the multi-target biological potential of S. pinnata in inducing antitumor and anti-inflammatory effects was comprehensively demonstrated. These findings will provide important stepping-stones for further investigations and may lead to the development of highly effective S. pinnata extract-based treatments for breast cancer and inflammatory processes.

L-carnitine improves quality parameters and epigenetic patterns of buck’s frozen-thawed semen

Buck sperm cryopreservation is an effective method to distribute qualified sperm for reproductive purposes, but this procedure reduces sperm quality. The current study was conducted to investigate the effect of L-carnitine (LC) on the quality and epigenetic patterns of buck’s post-thawed semen. Semen samples were collected from five male goats twice a week and diluted in extenders supplemented with 0 (LC-0), 1 (LC-1), 5 (LC-5) and 10 (LC-10) mM LC. Samples were cryopreserved according to standard protocol. After thawing, motility characteristics, lipid peroxidation, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, epigenetic modifications, viability, apoptotic-like changes and DNA fragmentation were assessed. Samples supplemented with 5 mM LC showed greater (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, DNA methylation, viability, and lower (P ≤ 0.05) apoptotic-like changes. Lipid peroxidation was lower (P ≤ 0.05) in LC-5 and LC-10 compared to the control group. Addition of LC to the cryopreservation extender had no effect (P > 0.05) on velocity parameters, abnormal morphology, histone modifications, or DNA fragmentation. In conclusion, supplementing the cryopreservation extender with 5 mM LC significantly preserves the quality of buck sperm after the cryopreservation process.