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Research involving animals that occurs outside the laboratory raises an array of unique challenges. With regard to UK legislation, however, it receives only limited attention in terms of official guidelines, support, and statistics, which are unsurprisingly orientated towards the laboratory environment in which the majority of animal research takes place. In September 2019, four social scientists from the Animal Research Nexus program gathered together a group of 13 experts to discuss nonlaboratory research under the Animals (Scientific Procedures) Act (A(SP)A) of 1986 (mirroring European Union (EU) Directive 2010/63/EU), which is the primary mechanism for regulating animal research in the UK. Such nonlaboratory research under the A(SP)A often occurs at Places Other than Licensed Establishments (POLEs). The primary objective of the workshop was to assemble a diverse group with experience across a variety of POLEs (e.g., wildlife field sites, farms, fisheries, veterinary clinics, zoos) to explore the practical, ethical, and regulatory challenges of conducting research at POLEs. While consensus was not sought, nor reached on every point of discussion, we collectively identified five key areas that we propose require further discussion and attention. These relate to: (1) support and training; (2) ethical review; (3) cultures of care, particularly in nonregulated research outside of the laboratory; (4) the setting of boundaries; and (5) statistics and transparency. The workshop generated robust discussion and thereby highlighted the value of focusing on the unique challenges posed by POLEs, and the need for further opportunities for exchanging experiences and sharing best practice relating to research projects outside of the laboratory in the UK and elsewhere.

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment 1 . In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes 1 . Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Cephalopods are the first invertebrate class regulated by the European Union (EU) under Directive 2010/63/EU on the protection of animals used for scientific purposes, which requires prospective assessment of severity of procedures. To assist the scientific community in establishing severity classification for cephalopods, we undertook a web-based survey of the EU cephalopod research community as represented by the participants in the European COoperation on Science and Technology (COST) Action FA1301, Cephs In Action'. The survey consisted of 50 scenarios covering a range of procedures involving several cephalopod species at different life stages. Respondents (59 people from 15 countries) either allocated a severity classification to each scenario or indicated that they were unable to decide (UTD). Analyses evaluated score distributions and clustering. Overall, the UTD scores were low (7.0 ± 0.6%) and did not affect the severity classification. Procedures involving paralarvae and killing methods (not specified in Annexe IV) had the highest UTD scores. Consensus on non-recovery procedures was reached consistently, although occasionally non-recovery appeared to be confused with killing methods. Scenarios describing procedures above the lower threshold for regulation, including those describing behavioural studies, were also identified and allocated throughout the full range of severity classifications. Severity classification for scenarios based on different species (e.g. cuttlefish vs. octopus) was consistent, comparable and dependent on potentially more harmful interventions. We found no marked or statistically significant differences in the overall scoring of scenarios between the demographic subgroups (age, sex, PhD and cephalopod experience). The COST Action FA1301 survey data provide a basis for a prospective severity classification for cephalopods to serve as guide for researchers, project assessors and regulators.

Background: Obese, type two diabetics are at an increased risk for fracturing their limb bones in comparison to the general population. Phytoestrogens like as the soy isoflavone genistein have been shown to protect against bone loss. In this study, we tested the effects of genistein treatment on femurs of ob/ob mice, a model for obesity and type two diabetes mellitus., Methods: Twenty six-week-old female mice were divided into obese (ob/ob) control, obese genistein-treated, lean (ob/+) control, and lean genistein-treated groups (n = 5 each). Treatment with genistein consisted of 600 mg genistein/kg diet. Control mice were given standard rodent chow. At the end of a four-week treatment period, bone histomorphometric and three-point bending properties were compared among groups., Results: Obese mice had larger bone areas (B.Ar.; P < 0.05) and total areas (Tt.Ar.; P < 0.05), but similar bone volume (B.Ar./Tt.Ar.; P > 0.05) of the proximal femoral epiphysis in comparison to lean mice. Treatment with genistein decreased Tt.Ar. and femur length, and increased ultimate force required to fracture the femur and the maximum deformation to failure (P < 0.05)., Conclusions: Genistein improves resistance to fracture from bending loads.

Acute inhalation studies are conducted in animals as part of chemical hazard identification and characterisation, including for classification and labelling purposes. Current accepted methods use death as an endpoint (OECD TG403 and TG436), whereas the fixed concentration procedure (FCP) (draft OECD TG433) uses fewer animals and replaces lethality as an endpoint with 'evident toxicity.' Evident toxicity is defined as clear signs of toxicity that predict exposure to the next highest concentration will cause severe toxicity or death in most animals. A global initiative including 20 organisations, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has shared data on the clinical signs recorded during acute inhalation studies for 172 substances (primarily dusts or mists) with the aim of making evident toxicity more objective and transferable between laboratories. Pairs of studies (5 male or 5 female rats) with at least a two-fold change in concentration were analysed to determine if there are any signs at the lower dose that could have predicted severe toxicity or death at the higher concentration. The results show that signs such as body weight loss (>10% pre-dosing weight), irregular respiration, tremors and hypoactivity, seen at least once in at least one animal after the day of dosing are highly predictive (positive predictive value > 90%) of severe toxicity or death at the next highest concentration. The working group has used these data to propose changes to TG433 that incorporate a clear indication of the clinical signs that define evident toxicity., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

The severity classification of procedures using animals is an important tool to help focus the implementation of refinement and to assist in reporting the application of the 3Rs (replacement, reduction and refinement). The recently revised Directive that regulates animal research and testing within the European Union requires Member States to ensure that all procedures are classified as 'non-recovery', 'mild', 'moderate' or 'severe', using assignment criteria set out by the European Commission (EC). However, these are focused upon terrestrial species, so are of limited relevance to fish users. A Working Group set up by the Norwegian Consensus-Platform for the 3Rs (Norecopa) has produced guidance on the classification of severity in scientific procedures involving fish, including examples of 'subthreshold', 'mild', 'moderate', 'severe' and 'upper threshold' procedures. The aims are to complement the EC guidelines and help to ensure that suffering in fish is effectively predicted and minimized. Norecopa has established a website (www.norecopa.no/categories) where more information on severity classification for procedures using fish, including field research, will be made available.

The refinement of husbandry and procedures to reduce animal suffering and improve welfare is an essential component of humane science. Successful refinement depends upon the ability to assess animal welfare effectively, and detect any signs of pain or distress as rapidly as possible, so that any suffering can be alleviated. This document provides practical guidance on setting up and operating effective protocols for the welfare assessment of animals used in research and testing. It sets out general principles for more objective observation of animals, recognizing and assessing indicators of pain or distress and tailoring these to individual projects. Systems for recording indicators, including score sheets, are reviewed and guidance is set out on determining practical monitoring regimes that are more likely to detect any signs of suffering. This guidance is intended for all staff required to assess or monitor animal welfare, including animal technologists and care staff, veterinarians and scientists. It will also be of use to members of ethics or animal care and use committees. A longer version of this document, with further background information and extra topics including training and information sharing, is available on the Laboratory Animals website.

The current method for Paralytic Shellfish Poisoning (PSP) testing in shellfish is based on the mouse bioassay (MBA), which involves injecting shellfish extract into a conscious mouse, and then converting its time to death into PSP toxicity using Sommer's table. To improve animal welfare, the present study investigated the use of anaesthesia. A saxitoxin (STX) calibration study was conducted where known amounts of STX were injected into both unanaesthetised and anaesthetised mice. Death time was approximately doubled when mice were anaesthetised. Both unanaesthetised and anaesthetised animals showed a linear relationship between the inverse death time and log(STX). Based on these data, new calibration curves were developed. This study revealed that the current method employing Sommer's table underestimates toxicity by up to 50% for higher toxin levels. Subsequently, shellfish samples were tested on both unanaesthetised and anaesthetised mice. Using the new calibration curves, the numbers of samples exceeding the field closure limit were similar for unanaesthetised and anaesthetised mice, and were nearly two-fold higher than those obtained with the current method. The studies showed that the bioassay gives variable results for both unanaesthetised and anaesthetised animals. Anaesthesia forms a viable and more ethical alternative to the current bioassay, at least in the short term. A practical summary on how to conduct this method is given.

It is not known if the ruminant animal gastrointestinal tract (GIT) can oxidise essential amino acids (AA) other than leucine. Therefore, the oxidation of four essential AA (leucine, lysine, methionine and phenylalanine), supplied systemically as labelled 1-13C forms, was monitored across the mesenteric-drained viscera (MDV; small intestine) and portal-drained viscera (PDV; total GIT), as part of a Latin square design, in four wether sheep (35-45 kg) fed at 1.4 x maintenance. Oxidation was assessed primarily by appearance of 13CO2, corrected for sequestration of [13C]bicarbonate. The GIT contributed 25 % (P<0.001) and 10 % (P<0.05) towards whole-body AA oxidation for leucine and methionine respectively. This reduced net appearance across the PDV by 23 and 11 % respectively. The contribution of MDV metabolism to total PDV oxidation was 40 % for leucine and 60 % for methionine. There was no catabolism of systemic lysine or phenylalanine across the GIT. Production and exchange of secondary metabolites (e.g. 4-methyl-2-oxo-pentanoate, homocysteine, 2-aminoadipate) across the GIT was also limited. Less AA appeared across the PDV than MDV (P<0.001), indicative of use by tissues such as the forestomach, large intestine, spleen and pancreas. The PDV: MDV net appearance ratios varied (P<0.001) between AA, e.g. phenylalanine (0.81), lysine (0.71), methionine (0.67), leucine (0.56), histidine (0.71), threonine (0.63) and tryptophan (0.48). These differences probably reflect incomplete re-absorption of endogenous secretions and, together with the varied oxidative losses measured, will alter the pattern of AA net supply to the rest of the animal.

Both plasma and red blood cells contain amino acids (AA), but the relative amount of AA transferred from each vascular compartment to the tissues remains unclear. For splanchnic tissues, the relative transfers between the plasma, the red blood cells and the tissues may vary with nutritional state, but whether the same situation pertains for other tissues is not known. The current study focused on the transfer of lysine from plasma and red blood cells across the hindquarters of sheep offered four levels of intakes (0.5, 1.0, 1.5 and 2.5 x maintenance energy). This design, coupled with use of [U-13C]lysine as tracer, also allowed the effect of intake on protein kinetics to be examined. At all intakes, the concentration of lysine in the sheep' red blood cells exceeded that in plasma by 50% (P<0.001), while the distribution of labelled lysine between the plasma and the red blood cells was 0.71:0.29. Net lysine uptake by the hindquarters increased in a linear manner (P<0.001) with intake, with more than 90% extracted from the plasma. Free lysine enrichments in plasma from the posterior vena cava were less than that from the artery (P<0.001), but those in red blood cells were not different between the artery and vein. The red blood cells thus play a minor role in the transfers to and from the hindquarter tissues, regardless of intake. Based on plasma transfers and the enrichment of lysine in arterial plasma, hindquarter protein synthesis increased linearly with intake (P<0.001). In contrast, protein breakdown was unaffected by intake. The contribution of hindquarter protein synthesis to whole-body lysine flux remained unchanged with intake (18-20%).

Recent studies have demonstrated the occurrence of elevated levels of higher chlorinated PCDDs in the coastal environment of Queensland, Australia. This study presents new data for OCDD contamination and full PCDD/F profile analysis in the environment of Queensland. Marine sediments, irrigation drain sediments and topsoil were collected from sites that were expected to be influenced by specific land-use types. High OCDD concentrations were associated mainly with sediments collected near the mouth of rivers which drain into large catchments in the tropical and subtropical regions. Further, analysis of sediments from irrigation drains could be clearly differentiated on the basis of OCDD contamination, with high concentrations in samples from sugarcane drains collected from coastal regions, and low concentrations in drain sediments from drier inland cotton growing areas. PCDD/F congener-specific analysis demonstrated almost identical congener profiles in all samples collected along the coastline. This indicates the source to be widespread. Profiles were dominated by higher chlorinated PCDDs, in particular OCDD whereas 2,3,7,8-substituted PCDFs were below the limit of quantification in the majority of samples. The full PCDD/F profile analysis of samples strongly resemble those reported for lake sediments from Mississippi and kaolinite samples from Germany. Strong similarities to these samples with respect to congener profiles and isomer patterns may indicate the presence of a similar source and/or formation process that is yet unidentified.

Objective: As Helicobacter pylori infection is associated with an elevation in plasma gastrin with normal antral gastrin cell counts, an abnormality in antral somatostatin cells may be associated with the infection. We evaluated the effect of eradication of H. pylori on antral somatostatin cell density in the light of antral gastrin cell density and plasma gastrin levels., Design: Prospective study., Methods: Of 25 dyspeptic patients with H. pylori infection, nine had H. pylori successfully eradicated and the rest remained infected. Antral biopsies were immunostained for somatostatin cells and plasma gastrin measured before and 4 weeks after H. pylori eradication therapy. Ten other dyspeptic patients without H. pylori infection had their somatostatin cell density evaluated as controls., Results: Somatostatin cell density in the patients without H. pylori infection at the outset was significantly higher than that in the patients with H. pylori infection at the outset (median 57 [18-83] vs. 37 [6-80] cells/mm) respectively (P <0.05). Somatostatin cell density increased after H. pylori eradication (before treatment, median 50 [15-72]; after treatment 71 [39-107] cells/mm) (P < 0.05) but was unchanged with persistent H. pylori infection. Plasma gastrin decreased after H. pylori eradication (before treatment, median 70 [45-100]; after treatment 30 [10-100] ng/l) (P < 0.05) but was unchanged with persistent H. pylori infection., Conclusions: Following eradication of H. pylori, there is an increase in somatostatin cell density with a fall in plasma gastrin. This supports the theory that H. pylori infection results in a decrease in somatostatin cell density and, as the latter is an inhibitor of gastrin cells, this results in an increased plasma gastrin.