Embracing prospects for reducing the numbers of animals used in aquaculture research.

The principles of three Rs-REPLACEMENT, REDUCTION, and REFINEMENT-govern the protection and use of animals, including fish, for research purposes in the European Union and Norway. In this paper, we discuss some straightforward steps to simplify the delivery of these principles at the idea stage and adapt some of these examples for conducting fish trials related to health and welfare. Although some of the approaches are well established in other animal science arenas, we believe there can be a timely recap of their key facets. We discuss a number of simple strategies to emphasize how a reduction in fish numbers can be achieved from initial project conception to implementation, highlighting not only their advantages but also their limitations. We also highlight the role that funding agencies can play in the implementation of the 3R principles in aquaculture research. These simple points can be used in frameworks to initiate a broader and dynamic intersectoral dialogue among stakeholders of aquaculture research on how to promote ethics and embrace opportunities for this within the tenets of the 3Rs.

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production-related stressors.

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). Nonetheless, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. The authors collected skin from lumpfish from both research facilities (tank-reared fish) and commercial production (cage-reared fish) and used operational welfare indicators, in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2 O2 ) and peracetic acid. Lumpfish sampled from the farm were classified as clinically healthy or weak based on their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced the migration capacity of keratocytes, a weakened epidermal barrier, and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank-reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.

A Framework for ExacTrac Dynamic Commissioning for Stereotactic Radiosurgery and Stereotactic Ablative Radiotherapy.

This paper aims to provide guidance and a framework for commissioning tests and tolerances for the ExacTrac Dynamic image-guided and surface-guided radiotherapy (SGRT) system. ExacTrac Dynamic includes a stereoscopic X-ray system, a structured light projector, stereoscopic cameras, thermal camera for SGRT, and has the capability to track breath holds and internal markers. The system provides fast and accurate image guidance and intrafraction guidance for stereotactic radiosurgery and stereotactic ablative radiotherapy. ExacTrac Dynamic was commissioned on a recently installed Elekta Versa HD. Commissioning tests are described including safety, isocenter calibration, dosimetry, image quality, data transfer, SGRT stability, SGRT localization, gating, fusion, implanted markers, breath hold, and end-to-end testing. Custom phantom designs have been implemented for assessment of the deep inspiration breath-hold workflow, the implanted markers workflow, and for gating tests where remote-controlled movement of a phantom is required. Commissioning tests were all found to be in tolerance, with maximum translational and rotational deviations in SGRT of 0.3 mm and 0.4°, respectively, and X-ray image fusion reproducibility standard deviation of 0.08 mm. Tolerances were based on published documents and upon the performance characteristics of the system as specified by the vendor. The unique configuration of ExacTrac Dynamic requires the end user to design commissioning tests that validate the system for use in the clinical implementation adopted in the department. As there are multiple customizable workflows available, tests should be designed around these workflows, and can be ongoing as workflows are progressively introduced into departmental procedures.

Synthesis, characterization and biological activities of semicarbazones and their copper complexes.

Substituted semicarbazones/thiosemicarbazones and their copper complexes have been prepared and several single crystal structures examined. The copper complexes of these semicarbazone/thiosemicarbazones were prepared and several crystal structures examined. The single crystal X-ray structure of the pyridyl-substituted semicarbazone showed two types of copper complexes, a monomer and a dimer. We also found that the p-nitrophenyl semicarbazone formed a conventional 'magic lantern' acetate-bridged dimer. Electron Paramagnetic Resonance (EPR) of several of the copper complexes was consistent with the results of single crystal X-ray crystallography. The EPR spectra of the p-nitrophenyl semicarbazone copper complex in dimethylsulfoxide (DMSO) showed the presence of two species, confirming the structural information. Since thiosemicarbazones and semicarbazones have been reported to exhibit anticancer activity, we examined the anticancer activity of several of the derivatives reported in the present study and interestingly only the thiosemicarbazone showed activity while the semicarbazones were not active indicating that introduction of sulphur atom alters the biological profile of these thiosemicarbazones.

Investigation of biogeochemical functional proxies in headwater streams across a range of channel and catchment alterations.

Historically, headwater streams received limited protection and were subjected to extensive alteration from logging, farming, mining, and development activities. Despite these alterations, headwater streams provide essential ecological functions. This study examines proxy measures of biogeochemical function across a range of catchment alterations by tracking nutrient cycling (i.e., inputs, processing, and stream loading) with leaf litter fall, leaf litter decomposition, and water quality parameters. Nutrient input and processing remained highest in second growth forests (the least altered areas within the region), while recently altered locations transported higher loads of nutrients, sediments, and conductivity. Biogeochemical functional proxies of C and N input and processing significantly, positively correlated with rapid assessment results (Pearson coefficient = 0.67-0.81; P = 0.002-0.016). Additionally, stream loading equations demonstrate that N and P transport, sediment, and specific conductivity negatively correlated with rapid assessment scores (Pearson coefficient = 0.56-0.81; P = 0.002-0.048). The observed increase in stream loading with lower rapid assessment scores indicates that catchment alterations impact stream chemistry and that rapid assessments provide useful proxy measures of function in headwater ecosystems. Significant differences in nutrient processing, stream loading, water quality, and rapid assessment results were also observed between recently altered (e.g., mined) headwater streams and older forested catchments (Mann-Whitney U = 24; P = 0.01-0.024). Findings demonstrate that biogeochemical function is reduced in altered catchments, and rapid assessment scores respond to a combination of alteration type and recovery time. An analysis examining time and economic requirements of proxy measurements highlights the benefits of rapid assessment methods in evaluating biogeochemical functions.

Injuries and deformities in fish: their potential impacts upon aquacultural production and welfare.

Fish can be the recipients of numerous injuries that are potentially deleterious to aquacultural production performance and welfare. This review will employ a systematic approach that classifies injuries in relation to specific anatomical areas of the fish and will evaluate the effects of injury upon production and welfare. The selected areas include the (1) mouth, (2) eye, (3) epidermis and (4) fins. These areas cover a large number of external anatomical features that can be injured during aquacultural procedures and husbandry practices. In particular, these injuries can be diagnosed on live fish, in a farm environment. For each anatomical feature, this review addresses (a) its structure and function and (b) defines key injuries that can affect the fish from a production and a welfare perspective. Particular attention is then given to (c) defining known and potential aquacultural risk factors before (d) identifying and outlining potential short- and long-term farming practices and mitigation strategies to reduce the incidence and prevalence of these injuries. The review then concludes with an analysis of potential synergies between risk factors the type of injury, in addition to identifying potential synergies in mitigation strategies. The paper covers both aquaculture and capture-based aquaculture.

Demand feeding and welfare in farmed fish.

Following the development of demand-feeding systems, many experiments have been conducted to explore feeding motivation and feed intake in farmed fish. This work aims to review a selection of studies in the field, focusing on three key factors, related to demand feeding and fish welfare. Firstly, we outline how demand feeders should be considered when developing feed management strategies for improving welfare in production conditions. Secondly, via laboratory demand-feeding experiments, we show self-feeding activities depend not only on feeding motivation and social organisation, but also on individual learning capacity and risk-taking behaviour. Thirdly, we report encouraging results demonstrating that when presented with two or more self-feeders containing complementary foods, fish select a diet according to their specific nutritional requirements, suggesting that demand feeders could be used to improve welfare by allowing fish to meet their nutritional needs.

Behavioural indicators of welfare in farmed fish.

Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare. This review summarises the main findings on how behavioural changes have been used to assess welfare in farmed fish, using both functional and feeling-based approaches. Changes in foraging behaviour, ventilatory activity, aggression, individual and group swimming behaviour, stereotypic and abnormal behaviour have been linked with acute and chronic stressors in aquaculture and can therefore be regarded as likely indicators of poor welfare. On the contrary, measurements of exploratory behaviour, feed anticipatory activity and reward-related operant behaviour are beginning to be considered as indicators of positive emotions and welfare in fish. Despite the lack of scientific agreement about the existence of sentience in fish, the possibility that they are capable of both positive and negative emotions may contribute to the development of new strategies (e.g. environmental enrichment) to promote good welfare. Numerous studies that use behavioural indicators of welfare show that behavioural changes can be interpreted as either good or poor welfare depending on the fish species. It is therefore essential to understand the species-specific biology before drawing any conclusions in relation to welfare. In addition, different individuals within the same species may exhibit divergent coping strategies towards stressors, and what is tolerated by some individuals may be detrimental to others. Therefore, the assessment of welfare in a few individuals may not represent the average welfare of a group and vice versa. This underlines the need to develop on-farm, operational behavioural welfare indicators that can be easily used to assess not only the individual welfare but also the welfare of the whole group (e.g. spatial distribution). With the ongoing development of video technology and image processing, the on-farm surveillance of behaviour may in the near future represent a low-cost, noninvasive tool to assess the welfare of farmed fish.