The Department of Biological Sciences has an expanding research base, which, in addition to providing leading researchers of national and international standing in these areas, most importantly underpins the delivery of teaching. Research in Biological Sciences at Chester can be divided into three broad groups of expertise, namely Animal Behaviour and Conservation, Food Nutrition and Health, and Stress and Disease.

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  • Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice.

    Gouveia, Kelly; Hurst, Jane L; orcid: 0000-0002-3728-9624; email: jane.hurst@liverpool.ac.uk (2019-12-30)
    Handling can stimulate stress and anxiety in laboratory animals that negatively impacts welfare and introduces a confounding factor in many areas of research. Picking up mice by the tail is a major source of handling stress that results in strong aversion to the handler, while mice familiarised with being picked up in a tunnel or cupped on the open hand show low stress and anxiety, and actively seek interaction with their handlers. Here we investigate the duration and frequency of handling required for effective familiarisation with these non-aversive handling methods, and test whether this is sufficient to prevent aversion and anxiety when animals then experience immobilisation and a mild procedure (subcutaneous injection). Very brief handling (2 s) was sufficient to familiarise mice with tunnel handling, even when experienced only during cage cleaning. Brief but more frequent handling was needed for familiarisation with cup handling, while pick up by tail induced strong aversion even when handling was brief and infrequent. Experience of repeated immobilisation and subcutaneous injection did not reverse the positive effects of tunnel handling. Our findings demonstrate that replacing tail with tunnel handling during routine cage cleaning and procedures provides a major refinement with little if any cost for familiarisation.
  • Changes in selective biomarkers after transurethral resection of the bladder tumour (TURBT), and their association with Non-muscle invasive bladder cancer (NMIBC) recurrence and progression

    Ella-Twongiis, Peter (University of ChesterUniversity of Chester, 2019-10-01)
    Introduction Bladder Cancer (BC) is the 10th most common cancer in the UK, with about 10,000 new cases annually. It affects more men than women (ratio 3:1). Major risk factors include tobacco, chemical carcinogens, schistosomiasis infection and age. About 75-85% of BC are non-muscle invasive (NMIBC), which is associated with high recurrence and progression rates (50-60% within 7-10 years). Currently, diagnosis, treatment and management of BC is via clinical procedures such as transurethral resection of the bladder tumour (TURBT) and endoscopy. Concerning laboratory investigations, there are no routine biomarkers currently available for identifying BC patients at increased risk of developing recurrence and progression. By monitoring changes in selective biomarkers post-TURBT, any sustained changes may be a predictor of cancer recurrence or progression. The main-focus of this research study was to evaluate changes in selective novel biomarkers and their association with recurrence and progression in BC. Materials & Methods In this research, 40 patients (n=40) scheduled for TURBT at the Wrexham Maelor Hospital, North Wales were recruited after written informed consent. Ethical approval for the project was granted via IRAS (REC4: 14/WA/0033). Venous blood samples were taken at baseline (pre-operative) and following TURBT surgery at 1, 3 and 6 months post-operatively. Bladder tumour samples were also taken during TURBT according to standard procedure. Selective biomarkers to assess inflammation, angiogenesis and tumour growth, were measured using commercially available ELISA and BioPlex multiplex assay kits. Tissue immunoreactivity of novel biomarkers were also assessed in BC tissues using immunohistochemistry, with clinical outcome measures being recorded for all patients. Results Significant increases in serum Cluster of differentiation 31 (CD31) (p=0.003) and Stem Cell Factor (SCF) (p=0.032) concentration, as well as trends of increasing concentration of serum basic Fibroblast Growth Factor (bFGF) (p=0.14), Vascular Endothelial Growth Factor Receptor-1 and 2 (VEGFR-1) (p=0.15), VEGFR-2 (p=0.15) and Follistatin (p=0.40) were observed in BC patients up to 6 months post-operative. There were also significant decreases in serum Macrophage Inflammatory Protein -2 (MIP-2) (p=0.001), Platelet Derived Growth Factor (PDGF) (p=0.012), Matrix Metalloproteinase-9 (MMP-9) (p=0.002) and Vascular Endothelial Growth Factor C (VEGF-C) (p=0.04) serum concentration. Trends of decreasing concentration in MMP-2 (p=0.79), MMP-3 (p=0.15), interleukin-6 (IL-6) (p=0.26), interleukin-8 (IL-8) (p=0.15) and tumour necrosis factor-α (TNF-α) (p=0.69) were observed in BC patients up to 6 months post-operative. There was significant immunoreactivity of CD31 (p< 0.001), CD34 (p< 0.001), Human epidermal growth factor receptor-2 (HER-2) (p=0.032), S100P (p< 0.001), Cyclooxygenase-2 (COX-2) (p< 0.001), VEGFR-3 (p< 0.001), SOX-2 (p< 0.001) and thrombomodulin (p=0.010) in bladder tumours. Although recurrence was significantly associated with cancer grade, there was no association with antibody immunoreactivity. Conclusion Findings from the present study may indicate an alternative approach in the monitoring and management of patients with BC. It is proposed that by allowing urological surgeons access to laboratory markers such as MIP-2, MMP-9, PDGF, SCF, HER-2, Thrombomodulin and CD31 (biomarker profile), potentially, in the future, these biomarkers may be used in addition to, or in combination with, currently used scoring systems to predict cancer recurrence and progression. However, verification and validation of these biomarkers are needed using larger cohorts.
  • A Geoconservation perspective on the trace fossil record associated with the end – Ordovician mass extinction and glaciation in the Welsh Basin

    Burek, Cynthia; Hosie, Lottie; Nicholls, Keith H. (University of Chester, 2019-03-24)
    In this thesis I have illustrated the value of our geological heritage and geodiversity by focussing on a particular detailed aspect of the geological and palaeontological record, i.e. the trace fossil record associated with the end Ordovician (Hirnantian) global glaciation and extinction episode. The major elements of this work that are new are: • a significantly improved understanding of the nature of the soft sediment deformation, and in particular the role of “debrites” as basal landslide decollements in the Lower Palaozic Llangrannog rock succession of West Wales, • a much more detailed description of the trace fossil ichnocoenose present in the Llangrannog succession than has previously been published • an improved understanding of the nature of the ecological perturbation associated with the Hirnantian (Late Ordovician) Glacial “ice-house”, and the apparent role of an opportunistic soft body fauna in filling ecological niches vacated as a consequence of the associated extinction. • Considerable thought has been given to the question of how to value abiotic nature, and it is argued that the methods of conservation valuation associated with “Geosystem services” and in particular “Natural Capital” hold considerable potential for the Geoconservation community to engage with the public and with policy makers. • As a direct result of this research, two formal proposals have been put forward for new RIGS sites, together with a new geological SSSI.
  • The Use of Qualitative Risk Analysis Methods to Facilitate Decision Making in the Management of Health and Welfare in Wildlife

    Hill, Sonya; Smith, Tessa; Hartley, Matt (University of Chester, 2018-10-08)
    This thesis is composed of a series of papers, all of which have been published in peer reviewed publications. The papers use the recognised process of qualitative risk assessment in a range of scenarios in the field of wildlife health and welfare in both in situ and ex situ environments. Chapter 1 discusses the challenges faced regarding availability of empirical data in field of wildlife and zoological health and welfare and justifies the exploration of techniques to assist with decision making. The development of risk analysis and its integration with risk management and risk communication to become risk assessment is described before being put into the specific context of wildlife and zoological disease. Chapters 2 and 3 consider two scenarios where disease risk assessment is well established as a tool, importation across national borders and in conservation interventions. Chapter 2 develops the standard import risk assessment approach to include multiple species and multiple diseases. Chapter 3 reviews developments made over the last 25 years and proposes best practice approaches to implement. Chapter 4 describes how the risk assessments formulated as described in Chapter 3 are used for licensing purposes emphasising the importance of risk management and communication. This theme is continued in Chapter 6 where the integration of risk assessment and evidence based decision making is considered in the broad context of a strategic approach to wildlife health bringing together the outcomes and processes described in Chapters 2, 3, 4 and 5. The papers in Chapters 2,5 and 8 are focused on how risk analysis aids in development of disease control approaches and policy. The evidence base is composed primarily of peer-reviewed literature supported by expert review of the finalised assessment. Chapter 7 uses risk assessment in an applied scenario, taking the recognised process and modifying it to structure an active disease investigation demonstrating the versatility of the technique. Chapter 9 takes this a step further by again adapting the methodology which, has historically been used primarily for infectious diseases, to consider reproduction and assess risks to welfare rather than purely health. The paper in Chapter 9 builds on the methodology by combining existing peer-reviewed literature with data collected specifically for the purpose of feeding into the assessment and utilising a stakeholder and expert opinion elicitation workshop to obtain data too. These process are proposed and described in Chapter 3. The final chapter critically reviews risk assessment, highlighting three key areas of potential weakness and proposing approaches to address these criticisms. The value of the approach in wildlife and zoological health and welfare as demonstrated by this series of papers is described
  • The genetics and evolution of the critically endangered Trinidad Piping Guan Pipile pipile, synonym Aburria pipile.

    McDowall, Ian; Hosie, Charlotte A.; Grass, Amelia (University of Chester, 2018-02)
    The Trinidad Piping Guan, Pipile pipile synonym Aburria pipile (Jaquin, 1784) is the only endemic Cracid on the island of Trinidad. The species is currently listed as Critically Endangered and is considered to be in ‘on-going decline’ by the IUCN, BirdLife International and Cracid Specialist Group. This study aims to examine aspects of genetic variation and the evolution of the mitochondrial genome in the Trinidad Piping Guan utilising, for the first time, samples collected from individuals in the wild and reference specimens of the genus Pipile sourced from museum collections. In this study the complete mitochondrial genome of the Trinidad Piping Guan was sequenced for the first time. Analysis of intra-specific variation of wild Trinidad Piping Guan individuals using single nucleotide polymorphisms demonstrates extremely limited variation within the genes of the mitochondrial genome and nuclear gene intron sequences. Limited variation within this population is consistent with both historical and contemporary contractions of populations within a restricted island system, which may have serious implications for the future of this species in terms of both genetic diversity and conservation management. Phylogenetic analysis of the complete mitochondrial genome of the Trinidad Piping Guan enabled placement of the genus Pipile within the Galliforme evolutionary tree for the first time, and subsequently places the genus within the broader context of the Aves class. Mito-genomic analysis confirms that the Cracids are one of the basal Galliforme clades, and sister taxa to the Megapodidae. Phylogenetic placement of the Pipile genus is basal to that of the Crax species within the Cracidae family, indicative of an earlier evolutionary origin of the Piping Guans. The inclusion of the Trinidad Piping Guan, in the avian evolutionary tree using the whole mitochondrial genomes expands the current genetic phylogeny of the Cracid family, yielding a better understanding of evolutionary relationships among the Galliforme order and the diversification of modern avian lineages. This study has established novel molecular techniques for the analysis of mitochondrial DNA in historical specimens of the genus Pipile from museum reference collections. The analysis of inter-specific relationships within the genus Pipile has clarified the evolutionary and biogeographic relationships between the Piping Guan species. Additionally, the Trinidad Piping Guan is genetically defined for the first time as an evolutionarily significant unit, which represents a unique evolutionary pathway within this important genus in a closed island system on the island of Trinidad.
  • Production of DNA aptamers with specificity for bacterial food pathogens

    Bonwick, Graham A.; Drasbek, Mette R.; Young, Niall; Smith, Christopher; McDowall, Ian; Kärkkäinen, Riikka M. (University of Chester, 2012-09)
    Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.
  • The Role of Anaerobic Digestion in Achieving Soil Conservation and Sustainable Agriculture for Sustainable Development in the UK

    Burek, Cynthia V.; Bonwick, Graham A.; Alexander, Roy; Duruiheoma, Franklin I. (University of Chester, 2015-12)
    The subjection of soils to degradation directly and indirectly from rising world food demand and resultant intensified agricultural production, population growth, and climate change, demand that soils are better protected. The role of AD in addressing this challenge is examined using a pragmatic research paradigm and the questions: How can we raise awareness of AD in the UK? What factors motivate and hinder farmers towards adopting improved technology and sustainable agricultural practises? What is the perception of farmers about soils? To what extent does sustainable agriculture incorporate soil conservation in theory and practice? What role can legislation and policies play in AD adoption in the UK? The research was in two phases; qualitative and quantitative. The qualitative phase involved interviews with 21 AD stakeholder in the UK using electronic mail. The stakeholders who were divided into groups according to their expertise, were interviewed to explore their views on the areas of focus in the UK strategy and action plan regarding raising awareness of the technology, soil conservation, sustainable agriculture and sustainable development. Thematic analysis of interview data was carried out using MAXQDA 11 statistical software. The quantitative phase involved an online survey of 283 UK farmers aided by Yellow Pages directory for UK, Natural England directory, Twitter and electronic mail. Using SPSS 22.0 statistical software, the Chi square test was used to check for relationships between the variables measured at 95% confidence level (p<.05). Relationship strength was measured by means of Cramer’s V and Phi values. Answers to the 1st research question showed that: aligning AD with sustainable development goals, community AD and localism, small AD plants, provision of an available market for AD products, building UK skills and diversifying biogas use from AD are positive options for raising awareness of AD. Response to 2nd research question revealed: significant relationships between interests in agricultural technology and gender, level of education, and farm size; between knowledge of what AD is and gender, level of education and farm size; between interest in AD and age; between willingness to invest in AD if it improved soil properties and farm ownership; and between organic farming practice and age, farm type and farm size. Responding to the third research question, farmers’ describe soils in abstract, scientific, physical attribute and functional terms; awareness of soil benefits other than crop production was significantly related to age, and farm ownership; educational level was significantly related to familiarity with soil conservation, and opinion on whether soil should be protected like other natural resources. Findings regarding the 4th and 5th research questions showed: limited understanding of soil matters as a key challenge that has restricted the priority given to soil conservation, while level of education, knowledge of soil conservation and sustainable development and understanding of sustainable agriculture were also identified as influencing factors; digestate from AD is the main benefit viewed to contribute to soil conservation; finance, policy and legislation, low awareness and understanding, lack of feedstock and market, land use conflict and inefficiency of AD plants were identified as barriers to AD in the UK; promoting AD, providing finance, minimizing bureaucracy and simplification of AD systems are options for promoting AD adoption. This thesis also documents the implications of these findings for knowledge, policy and practice, and based on these recommendations are made, some of which are: better engagement of farmers in policy development for AD and soil management; use of small AD plants, demonstration, networking and training for AD adoption; promote soil conservation in theory and practice; and provision of enhanced support for owners, potential investors and farmers through incentives, simplified planning approval process, and available market for AD product.
  • Investigating Non-invasive Measures of Stress in Ornamental Fish

    Wolfenden, David C. C. (University of Chester, 2014)
    The transport of ornamental marine fish may cause stress, which to date has been the subject of limited research. The present study aimed to characterise the behavioural and physiological responses to simulated transport stress in the common clownfish Amphiprion ocellaris (Cuvier, 1830) with the additional goal of validating non-invasive measures of water cortisol in a marine teleost for the first time. Behaviour and physiology of the animals was measured at different stages of transport (from initial capture and handling up to 72 hours transport time) and water quality measurements were taken from the transport water at key sampling points. In a second experiment biological filtration materials (“Bioballs” with denitrifying bacteria) were added to the transport bag to determine if stress was reduced when water quality was improved. The results of the study suggest that capture, handling and transport are stressful for clownfish, and the stress response appears to peak between 24 and 48 hours after the onset of the stressor. Water-borne cortisol was found to be a valid alternative to invasive methods of sampling, although only an average of 53% cortisol was recovered from sea water. Although handling and confinement appeared to be highly significant factors in eliciting the stress response water quality measurements revealed that fish are temporarily subjected to relatively high concentrations of ammonia as transport time increased, which may contribute to long-term effects on the health of the animals. This was reflected in an increased latency to feed and reduced social behaviours in fish transported for 24 hours or longer. Improving water quality did reduce the concentration of ammonia present; however, fish still exhibited elevated cortisol excretion suggesting that water quality is not the primary stressor associated with transport. Thus, the duration of transport should be restricted to a maximum of 24 hours to reduce the stress associated with this practice. A separate study investigated the potential for beauty treatment ‘fish spas’ to elicit stress in the freshwater cyprinid fish Garra rufa (Heckel, 1843). Water cortisol was measured non-invasively to determine if stress was reduced through the provision of environmental enrichment / furnishings, and whether stocking density influenced stress. Water quality was monitored to determine the effects of stocking density on environmental parameters (pH, ammonia, nitrite and nitrate). Finally, the effect of ii human hands being placed into the aquarium was investigated, to determine whether this influenced stress. Three hundred G. rufa were used in total, with groups being allocated randomly to one of four treatment groups: OP/B (optimum stocking density / barren tank – i.e. no enrichment); OP/E (optimum stocking density / enriched tank); OS/B (overstocked / barren tank); and OS/E (overstocked / enriched tank). Human hands were placed in each tank, and water samples were collected before and after for measurement of cortisol by ELISA, and to determine water quality parameters. The results revealed that overstocking tanks with G. rufa produced relatively higher baseline cortisol levels, suggesting that stocking density may have a significant effect on stress levels. The addition of furnishings into the aquarium did not reduce baseline cortisol levels in the fish prior to the introduction of human hands. However, fish maintained under enriched conditions exhibited a greater cortisol response when compared to individuals in barren tanks. It is hypothesised that the provision of enrichment reduces the available space for fish following the introduction of human hands, thereby increasing stress. Further studies are required to attempt to determine the effect of enrichment based upon the results of the present study. Feeding on human hands resulted in an elevated cortisol response from three out of the four treatment groups (with the exception of OP/B), with the results suggesting that either 1) the lower (i.e. optimal) stocking density and lack of enrichment in holding tanks is preferable for G. rufa fish welfare, or 2) the elevated cortisol reflected a response to a rewarding stimulus and is linked to increased foraging. Overall, the results of these studies have shown that water cortisol measurements are a valid means of assessing physiological stress in two species of fish in different contexts. This negates the need for invasive sampling and is an important refinement to existing protocols where fish are killed for plasma or whole body samples. The results also highlight the welfare impacts of transport and overstocking of ornamental fish providing valuable evidence that may be relevant to improving the husbandry and guidelines with respect to the ornamental fish industry.
  • The classification and management of limestone pavements - an endangered habitat

    Burek, Cynthia V.; Alexander, Roy; Thom, Tim; Willis, Sue D. M. (University of Liverpool (University of Chester), 2011-01)
    This thesis describes an in-depth study of limestone pavements across North West England and North Wales. The aim was to combine elements of geodiversity and biodiversity in order to create a holistic limestone pavement classification to inform future management. A field-based research protocol was used to assess a stratified random sample (46 pavements), accounting for approximately 10% of the limestone pavements in the geographical area. Detailed analyses of key elements are presented, along with important issues that continue to pose threats to this Annex One Priority Habitat. This research resulted in a comprehensive classification, using TWINSPAN analysis and Nonmetric Multidimensional Scaling, identifying six distinct holistic functional groups. The prime factors driving limestone pavement morphology, and hence the classification, were established to be lithology, proximity to structural fault, altitude and human intervention, particularly in terms of grazing intensity. Three upland, open limestone pavement classes were formed. Of these, the richest in terms of geodiversity and biodiversity was the group with the thickest bedding planes and hence the deepest grikes, typically greater than 1m. The class that was most species-poor was "at the highest altitude (above 450m), formed on the thin limestones of the Yoredales. These were characterised by shallow, wide grikes. The third upland limestone pavement group had mid-range grikes, generally 0.5-1m in depth, and small clints. Two wooded classes were identified. One was a lowland 'classic' wooded limestone pavement group with deep, narrow grikes and shallow soils. Indicator species included Juniperus communis and Taxus baccata. The second wooded group was situated proximal to a major structural fault. In this group the pavement dip ranged between 10°-40° with well-runnelled clints that were heavily moss-covered. The sixth group was low altitude, proximal to the coast, characterised by low moss growth, un-vegetated clints and the presence of Ulex europaeus. Conservation management was identified as key to the quality of the limestone pavement habitat and this thesis identifies best management practises and links these to the holistic limestone pavement classification. Finally, as a sample case study, this thesis presents mollusc species and diversity from eleven of the Yorkshire limestone pavements. Analysis establishes significant links between geodiversity and mollusc populations, with key drivers for mollusc communities echoing those of plant species on limestone pavement.