Shape mode analysis exposes movement patterns in biology: flagella and flatworms as case studies

We illustrate shape mode analysis as a simple, yet powerful technique to concisely describe complex biological shapes and their dynamics. We characterize undulatory bending waves of beating flagella and reconstruct a limit cycle of flagellar oscillations, paying particular attention to the periodicity of angular data. As a second example, we analyze non-convex boundary outlines of gliding flatworms, which allows us to expose stereotypic body postures that can be related to two different locomotion mechanisms. Further, shape mode analysis based on principal component analysis allows to discriminate different flatworm species, despite large motion-associated shape variability. Thus, complex shape dynamics is characterized by a small number of shape scores that change in time. We present this method using descriptive examples, explaining abstract mathematics in a graphic way.Comment: 20 pages, 6 figures, accepted for publication in PLoS On

Counting the Number of Active Spermatozoa Movements Using Improvement Adaptive Background Learning Algorithm

The most important early stage in sperm infertility research is the detection of sperm objects. The success rate in separating sperm objects from semen fluid has an important role for further analysis. This research performed the detection and calculation of human spermatozoa. The detected sperm was the moving sperm in the video data. An improvement of Adaptive Background Learning was applied to detect the moving sperm. The purpose of this method is to improve the performance of Adaptive Background Learning algorithm in background subtraction process to detect and calculate moving sperm on the microscopic video of sperm fluid. This paper also compared several other background subtraction algorithms to conclude the appropriate background subtraction algorithm for sperm detection and sperm counting. The process done in this research was preprocessing using the Gaussian filter. The next was background subtraction process, followed by morphology operation. To test or validate the detection results of any background subtraction algorithm used, the foreground mask results from the morphological operation were compared to the ground truth of moving sperm image. For visualization purposes, every BLOB area (white object in binary image) on the foreground were given a bounding box to the original frame and the number of BLOB objects present in the foreground mask were counted. This shows that the system had been able to detect and calculate moving sperm. Based on the test results, Adaptive Background Learning method had a value of F-measure of 0.9205 and succeeded in extracting sperm shape close to the original form compared to other methods

Towards the improvement of sheep welfare: Exploring the use of qualitative behavioural assessment (QBA) for the monitoring and assessment of sheep

Challenges faced by sheep in Australia in terms of disease, injury and management may compromise not only health and productivity but also welfare. These challenges represent a growing concern for both producers and the public. Hence there is an obvious need for the development of measures to allow producers, who may have limited access to stock or are constrained by time and/or resource availability, to monitor their sheep. There is a clear benefit to producers being able to readily identify animals whose welfare might be compromised and thus are in need of further care. However, the assessment of animal welfare is challenging under commercial conditions and to date, few measures are available to help producers recognise animals in compromised welfare states. Qualitative behavioural assessment (QBA) is an approach that captures the expressive behaviour of an animal, through the integration and summary of details of behavioural events, posture, and movement. In this way, QBA represents a valuable tool that offers insight into the physical and physiological aspects of animal welfare, and when used in conjunction with other key measures helps to provide a more complete and comprehensive picture of an animal’s welfare state. Furthermore, QBA should be used together with other welfare measures, where it has been proposed to guide the interpretation of welfare data. As a welfare tool, QBA has been applied to assess the behavioural expression in numerous livestock species including pigs and cattle, however, this methodology is less well studied in sheep and more work is needed to validate QBA for practical application. The aim of the research described in this thesis was to investigate whether the QBA methodology could be applied to assess the welfare of sheep subject to various welfare issues relevant to the Australian sheep industry. To this end, over four experimental chapters, QBA was applied to video footage captured of sheep in various states of compromised welfare, including those suffering from common injury and diseases; lameness, inappetence, flystrike, and gastro-intestinal parasitism, and those experiencing pain caused by routine husbandry procedures (ear tagging, castration, mulesing, and tail docking). Moreover, in two experimental chapters (Chapters 4 & 6), video footage was captured of sheep in positive welfare states (reduced gastro-intestinal parasite burden, and habituation to human presence). This video footage was also analysed quantitatively and other welfare measures including those of health/disease status, physical condition and locomotive activity were collected for validation purposes in each study. Over four experimental chapters, it was demonstrated that observers, blind to experimental procedures and treatments, can reach a significant consensus in their interpretation and assessment of the behavioural expression of sheep, and that these assessments can relate meaningfully to the welfare state of the animal. In Chapter 3, observers were able to distinguish between flystruck and non-flystruck sheep using the QBA methodology, and the behavioural expression scores given to each sheep corresponded to the severity of strike and the condition of the wool. In Chapter 4, observers identified differences in the behavioural expression of sheep that related to the severity of gastro-intestinal parasitism (subclinical v. clinical). Moreover, it was discovered that the treatment of sheep to lessen gastro-intestinal parasite burden altered the behavioural expression of parasitised sheep. A significant consensus was also reached amongst observers in the assessment of lambs subject to routine husbandry procedures (ear tagging, castration, mulesing, and tail docking) in Chapter 5. Observers were able to distinguish lambs that were subject to these painful husbandry procedures and were administered either a placebo or analgesics (Tri-Solfen® and meloxicam), from the control lambs which were only restrained. Hence suggesting that the pain caused by these husbandry procedures alters the behavioural patterns and demeanour of lambs in a way that is identifiable to observers using the QBA methodology. Lastly, when observers viewed video footage of sheep traversing a walk-over-weigh (WoW) apparatus in Chapter 6, they were able to distinguish sheep that were either lame or habituated to the test apparatus and human presence, from the control animals. However, in this Chapter, observers were not able to distinguish between all treatment groups evaluated based on their behavioural expression, specifically differences in the demeanour of inappetent and control sheep was not evident, nor were observers able to distinguish between lame and habituated sheep. In summary, the research presented in this thesis indicates that assessments of behavioural expression can be used under most of those conditions investigated to distinguish sheep in poor welfare states due to injury or disease, from those that are healthy. Furthermore, it appears that observers can reliably identify differences in behavioural expression related to positive welfare states. This work has detailed the behavioural expression of sheep as perceived by observers and has led to a greater understanding of the behavioural expression of sheep in different welfare states. It appears that through the assessment of demeanour or body language, QBA offers both relevant and valid assessments which may help producers gain an insight into the welfare state of their sheep. It is suggested that when used in conjunction with other select behavioural measures, QBA may represent a valuable tool for producers to improve the welfare of sheep in their care

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.Fil: Buchelly Imbachí, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Pastore, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Greco, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Iniesta-Cuerda, M.. Universidad de Castilla-La Mancha; EspañaFil: Garde, J. J.. Universidad de Castilla-La Mancha; EspañaFil: Soler, A. J.. Universidad de Castilla-La Mancha; EspañaFil: Ballarin, Virginia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Bio-inspired Magnetic Systems: Controlled Swimming, Fluid Pumps, and Collective Behaviour

This thesis details the original experimental investigations of magnetically actuated and controlled microscopic systems enabling a range of actions at low Reynolds number. From millimetre-robots and self-propelled swimmers to microfluidic and lab-on-a-chip technology applications. The main theme throughout the thesis is that the systems reply on the interactions between magnetic and elastic components. Scientists often take inspiration from nature for many aspects of science. Millimetre to micrometre machines are no exception to this. Nature demonstrates how soft materials can be used to deform in a manner to create actuation at the microscale in biological environments. Nature also shows the effectiveness of using beating tails known as flagella and the apparent enhancements in flow speeds of collective motion. To begin with, a swimmer comprised of two ferromagnetic particles coupled together with an elastic link (the two-ferromagnetic particle swimmer), was fabricated. The system was created to mimic the swimming mechanism seen by eukaryotic cells, in which these cells rely on morphological changes which allows them to propel resulting in approximate speeds of up to 2 body lengths per second. The aim of this system was to create a net motion and control the direction of propagation by manipulating the external magnetic field parameters. It was shown that the direction of swimming has a dependence on both the frequency and amplitude of the applied external magnetic field. A key factor discovered was that the influence of a small bias field, in this case, the Earth’s magnetic field (100 orders of magnitude smaller than the external magnetic field) resulted in robust control over the speed (resulting in typical swimming speeds of 4 body lengths per second) and direction of propulsion. Following this work, swimmers with a hard ferromagnetic head attached to an elastic tail (the torque driven ferromagnetic swimmer) were investigated. These systems were created to be analogous to the beating flagella of many natural microscopic swimmers, two examples would be sperm cells and chlamydomonas cells. These biological cells have typical speeds of 10s of body lengths per second. The main focus of this investigation was to understand how the tail length affects the swimming performance. An important observation was that there is an obvious length tail (5.7 times the head length) at which the swimming speed is maximised (approximately 13 body lengths per second). The experimental results were compared to a theoretical model based on three beads, one of which having a fixed magnetic moment and the other two non-magnetic, connected via elastic filaments. The model shows sufficient complexity to break time symmetry and create a net motion, giving good agreement with experiment. Portable point-of-care systems have the potential to revolutionise medical diagnostics. Such systems require active pumps with low power (USB powered devices) external triggers. Due to the wireless and localisation of magnetic fields could possibly allow these portable point-of-care devices to come to life. The main focus of this investigation was to create fluid pump systems comprising from the previously investigated two-ferromagnetic particle swimmer and the torque driven ferromagnetic swimmer. Building on the fact that if a system can generate a net motion it would also be able to create a net flow. Utilising the geometry of the systems, it has been demonstrated that a swimmer-based system can become a fluid pump by restricting the translational motion. The flow structure generated by a pinned swimmer in different scenarios, such as unrestricted flow around it as well as flow generated in straight, cross-shaped, Y-shaped and circular channels were investigated. This investigation demonstrated the feasibility of incorporating the device into a channel and its capability of acting as a pump, valve and flow splitter. As well as a single pump system, networks of the previously mentioned pump systems were fabricated and experimentally investigated. The purpose of this investigation was to utilise the behaviour of the collective motion. Such networks could also be attached to the walls or top of the channel to create a less invasive system compared to pump based within the channel system. The final investigation involved creating collective motion systems which could mimic the beating of cilia - known as a metachronal wave. Two methods were used to create an analogous behaviour. The first was using arrays of identical magnetic rotors, which under the influence of an external magnetic field created two main rotational patterns. The rotational patterns were shown to be controllable producing useful flow fields at low Reynolds numbers. The second system relied on the magnetic components having different fixed magnetisation to create a phase lag between oscillations. The magnetic components were investigated within a channel and the separation between the components was shown to be a key parameter for controlling the induced flow. In both cases, a simple model was produced to help understand the behaviour. Finally, a selection of preliminary investigations into possible applications were conducted experimentally. These investigations included, measuring the effective surface viscosity of lipid monolayers, created cell growth microchannels, as well as systems which could be used for blood plasma separation. The properties of lipid monolayers vary with the surface density, resulting on distinct phase transitions. Slight differences in the molecular lattice are often accompanied by significant changes in the surface viscosity and elasticity. The idea was to use a swimmer as a reporter of the monolayer viscosity, resulting in a less invasive method compared to current techniques to monitor monolayer viscosity, for example torsion pendulums and channel viscometers. The reported effective surface viscosity closely matched the typical Langmuir trough measurements (with a systematic shift of approximately 17 Ų/molecule). The blood plasma separation preliminary work shows the previously investigated two-ferromagnetic particle swimmer mixing a typical volume (100 μm) blood sample with a buffer solution in 21 seconds. The system was also able to create locations with a high population of red blood cells. This resulted in a separation between the blood plasma and red blood cells. Two other preliminary results of future investigations were presented; the collective motion of free swimmers, and the fabrication of ribbon-like structures with fixed magnetic moment patterns.European CommissionEngineering and Physical Sciences Research Council (EPSRC

Understanding the interface that enables the interaction between two dopamine receptor subtypes known as D1 and D2 implicated in depression

The dopamine receptor subtypes known as DI and D2 have been shown to form a heteromer complex, which is thought to lead to the disruption of a multitude of signaling pathways within the brain. As a result, this has been proposed to lead to diseased states such as Alzheimer\u27s and depression. In previous studies, it has been shown that the use of synthetic peptides corresponding to the surface interface of the interactions (specifically in the third intracellular loop of D2 interface) within the complex have served to compete against the formation of the heteromer. Through in vitro biochemical techniques such as SDS-Page and Western blotting, we observed the degree of dissociation of the Dl-D2 complex through a variety of small peptides. These peptides consist of peptide 1, 2, 3 and 4 with the sequences corresponding to Ac-EAARRAQE, AcEERRAQ, Ac-ARRA, and Ac-AARRAQ, respectively. Peptide 1 was found to be the most effective in preventing complex formation

SOMATIC AND GENETIC EFFECTS OF LOW SAR 2.45 GHz MICROWAVE RADIATION ON WISTAR RATS

The somatic and genetic effects of 2.45 GHz Microwave radiation (MWR) on male and female Sprague Dawley rats were studied. Two hundred rats were used for this study. They were grouped into control and exposed according to the parameters under consideration. The animals were exposed to various levels of specific absorption rate (SARs) using the microwave generator, model ER660E, Serial No MX704CCR from Toshiba UK Ltd. All animals were kept in healthy, and radiation free environment with water and feed provided ad libitum. The study is divided into three parts; firstly, the effects of MWR on the anxiolytic and exploratory behaviour of the animals. MWR produced dose-dependent significant decrease in the cumulative time spent in the open arms of maze in the first twelve days after exposure and gradually increased towards the control value before the end of two weeks. It also affects the exploratory behavoiur of the animals, for the first two weeks after exposure but by the third week there was no significant difference between the control and the exposed animals. The modifications in the behaviour were not sex dependent as there were no significant differences between male and female rats. Secondly, the effects of MWR on the vital organs of male and female most especially, male reproductive functions were studied. The sperm count, gross motility and sperm morphology were determined using standard methods. The results showed that 2.45 GHz MWR reduced the sperm concentration, gross motility while increasing the percentage abnormal sperm cells. There was a decrease in reproductive organ weights and increased dead sperm cells as large numbers of the sperm cells membranes took up the eosin-nigrosin vital stain in the exposed group. Lastly, the genotoxic effects of MWR were studied using Chromosomal aberrations, single cell gel electrophoresis (comet assay) and direct amplification of length polymorphisms (DALP). There was a significant increase in the frequency of chromosomal aberrations between control and xvi xvii exposed groups. The exposure of vital mammalian cells to 2.45 GHz radiation altered the band patterns of the exposed animals, especially in the range 40 – 120 bp. Starting with appearance of additional peaks compared with the control which was not seen in their tail DNA before exposure to MW and these were confirmed through the densitometric gel analysis. There was a statistically significant difference in the Olive moment and % DNA in tail of the exposed animals compared with control (p < 0.05). Thus, these findings support the hypothesis that exposure to 2.45 GHz MWR at studied SARs may have potential somatic and genetic effects