The behavior and welfare of neglected species: some examples from fish and mammals.

Animal welfare is the state of an animal's body and mind and the level to which its requirements are satisfied. Animal welfare is affected by human decisions and actions. Numerous decisions concerning animals are driven by human desires to enhance their own lives, and some of these decisions may be influenced by self-interest or a strong emphasis on economic factors. How to assess the welfare state of animals is a central issue in animal welfare science. Two critical questions can be used to address animal welfare: first, is the animal healthy, and second, does the animal have what it needs? Both of these questions can potentially be answered using the study of animal behavior. The development of behavioral methodologies is crucial for evaluating welfare in contexts where concern for animal welfare is often highest, such as on intensive modern farms and sites where working animals are used. Herein, we discuss animal welfare by focusing on some of its major concepts and explanations. Later, to illustrate key aspects of animal welfare, we chose to examine the information that is available for some 'neglected' livestock species, which are commercially important on a global basis and found in large numbers: buffaloes (Bubalus bubalis), camels (Camelus dromedarius), donkeys (Equus asinus), mules (Equus asinus × Equus caballus), and lumpfish (Cyclopterus lumpus). We chose these species because there are major ongoing concerns about their welfare, and more research is required to help solve the various problems. Overall, there are strong imbalances in terms of the species that are usually studied in terms of animal welfare research, and we call for greater attention to those that have traditionally been neglected.

Differential metabolic responses in bold and shy sea anemones during a simulated heatwave.

As climate change-induced heatwaves become more common, phenotypic plasticity at multiple levels is a key mitigation strategy by which organisms can optimise selective outcomes. In ectotherms, changes to both metabolism and behaviour can help alleviate thermal stress. Nonetheless, no study in any ectotherm has yet empirically investigated how changing temperatures affect among-individual differences in the associations between these traits. Using the beadlet anemone (Actinia equina), an intertidal species from a thermally heterogeneous environment, we investigated how individual metabolic rates, linked to morphotypic differences in A. equina, and boldness were related across changing temperatures. A crossed-over design and a temporal control were used to test the same individuals at a non-stressful temperature, 13°C, and under a simulated heatwave at 21°C. At each temperature, short-term repeated measurements of routine metabolic rate (RMR) and a single measurement of a repeatable boldness-related behaviour, immersion response time (IRT), were made. Individual differences, but not morphotypic differences, were highly predictive of metabolic plasticity, and the plasticity of RMR was associated with IRT. At 13°C, shy animals had the highest metabolic rates, while at 21°C, this relationship was reversed. Individuals that were bold at 13°C also exhibited the highest metabolic rates at 21°C. Additional metabolic challenges during heatwaves could be detrimental to fitness in bold individuals. Equally, lower metabolic rates at non-stressful temperatures could be necessary for optimal survival as heatwaves become more common. These results provide novel insight into the relationship between metabolic and behavioural plasticity, and its adaptive implications in a changing climate.

Pain management in zebrafish : Report from a FELASA Working Group.

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe.

Acetic acid-induced pain elicits stress-, and camouflage-related responses in zebrafish: Modulatory effects of opioidergic drugs on neurobehavioral phenotypes.

While pain results from the activation of nociceptors following noxious stimuli, mounting evidence links pain- and stress-related responses in mammals. In zebrafish, the activation of hypothalamic-pituitary-interrenal (HPI) axis may also regulate body pigmentation (the camouflage response). Here, we aimed to investigate a putative relationship between pain-, stress-, and camouflage-related parameters in adult zebrafish. To answer this question, we assessed whether intraperitoneal acetic acid injection can activate the HPI axis, measuring whole-body cortisol and the camouflage response as physiological endpoints in the presence or absence of morphine or naloxone, an opioid antagonist. Acetic acid induced a stereotypic circling behavior in the top of the tank, accompanied by abdominal writhing-like response, a specific phenotype that reflects local nociceptive effect. Both whole-body cortisol levels and camouflage response increased in the acetic acid group, while morphine prevented these responses, and naloxone antagonized morphine-induced effects. Moreover, we observed positive correlations between representative behavioral, physiological and skin coloration endpoints, and a "pain index" was proposed to summarize phenotypic profile of zebrafish under different pharmacological manipulations. Collectively, these findings suggest a coordinated activation of pain, camouflage- and stress-related pathways following acetic acid injection in zebrafish. Our data also support that camouflage response represents a novel and relevant biomarker for future probing pain and stress neurobiology, with a robust sensitivity to opioidergic drugs.

Methods to Induce Analgesia and Anesthesia in Crustaceans: A Supportive Decision Tool.

Methods to induce analgesia and anesthesia for research purposes, handling, transport, or stunning have been used in 71 species of crustaceans. A non-systematic literature search was conducted on crustacean anesthetic methods. This review presents a comprehensive evaluation of drugs and non-chemical methods used to provide analgesia and anesthesia in many crustacean species rather than just decapod crustaceans. This information allows users to select an appropriate method or agent for their species of interest. We prepared an on-line tool based on datasette, a no-code open-source solution for simple web-based database frontends that allows exploration and downloading data by method, analgesic/anesthetic, species, life stage, or sex, as well as other data including environmental conditions (temperature, salinity, light), route of administration, dosage, and induction and recovery times. These values can be selected to filter the dataset and export it to CSV or JSON formats. Currently, several techniques and chemicals are, in our opinion, unsuitable for use as anesthetics in crustaceans, and the basis for these opinions are presented. Given the evidence of a pain-like experience in crustaceans, we propose that researchers should treat crustaceans humanely, applying the principles of good handling, care, and the management of stress and pain to safeguard their welfare.

Pain Recognition in Fish.

Empirical evidence has demonstrated that fish experience pain, and so to ensure their good welfare, it is vital that we can recognize and assess pain. A range of general, behavioral, and physiologic indicators can be used when assessing pain in fish. Many of these can be used at the tank side and are termed operational welfare indicators, whereas some require further computer or laboratory analysis. Behavioral indicators are valid and have been shown to profoundly differ between nonpainful and painful treatments in fish. However, these are not universal, and species-specific differences exist in behavioral responses to pain.

Impact of Lidocaine on Pain-Related Grooming in Cuttlefish.

Nociception is the neural process of encoding noxious stimuli and is typically accompanied by a reflex withdrawal response away from the potentially injurious stimulus. Studies on nociception in cephalopods have so far focused on octopus and squid, with no investigations to our knowledge on cuttlefish. Yet, these are an important species both in scientific and commercial use. Therefore, the present study demonstrated that a standard pain stimulus, acetic acid, induced grooming behaviour directed towards the injection site in cuttlefish and that the injection of lidocaine reduces grooming behaviours in acetic-acid-injected cuttlefish. Wound-directed behaviour demonstrates that the animal is aware of the damage; thus, when subjecting these animals to any painful treatments in the laboratory, researchers should consider alleviating pain by the administration of pain-relieving drugs.

Understanding early-life pain and its effects on adult human and animal emotionality: Translational lessons from rodent and zebrafish models.

Critical for organismal survival, pain evokes strong physiological and behavioral responses in various sentient species. Clinical and preclinical (animal) studies markedly increase our understanding of biological consequences of developmental (early-life) adversity, as well as acute and chronic pain. However, the long-term effects of early-life pain exposure on human and animal emotional responses remain poorly understood. Here, we discuss experimental models of nociception in rodents and zebrafish, and summarize mounting evidence of the role of early-life pain in shaping emotional traits later in life. We also call for further development of animal models to probe the impact of early-life pain exposure on behavioral traits, brain disorders and novel therapeutic treatments.

The Use of Zebrafish as a Non-traditional Model Organism in Translational Pain Research: The Knowns and the Unknowns.

The ability of the nervous system to detect a wide range of noxious stimuli is crucial to avoid life-threatening injury and to trigger protective behavioral and physiological responses. Pain represents a complex phenomenon, including nociception associated with cognitive and emotional processing. Animal experimental models have been developed to understand the mechanisms involved in pain response, as well as to discover novel pharmacological and non-pharmacological anti-pain therapies. Due to the genetic tractability, similar physiology, low cost, and rich behavioral repertoire, the zebrafish (Danio rerio) is a powerful aquatic model for modeling pain responses. Here, we summarize the molecular machinery of zebrafish responses to painful stimuli, as well as emphasize how zebrafish-based pain models have been successfully used to understand specific molecular, physiological, and behavioral changes following different algogens and/or noxious stimuli (e.g., acetic acid, formalin, histamine, Complete Freund's Adjuvant, cinnamaldehyde, allyl isothiocyanate, and fin clipping). We also discuss recent advances in zebrafish-based studies and outline the potential advantages and limitations of the existing models to examine the mechanisms underlying pain responses from evolutionary and translational perspectives. Finally, we outline how zebrafish models can represent emergent tools to explore pain behaviors and pain-related mood disorders, as well as to facilitate analgesic therapy screening in translational pain research.

Personality-specific carry-over effects on breeding.

Carry-over effects describe the phenomenon whereby an animal's previous conditions influence its subsequent performance. Carry-over effects are unlikely to affect individuals uniformly, but the factors modulating their strength are poorly known. Variation in the strength of carry-over effects may reflect individual differences in pace-of-life: slow-paced, shyly behaved individuals are thought to favour an allocation to self-maintenance over current reproduction, compared to their fast-paced, boldly behaved conspecifics (the pace-of-life syndrome hypothesis). Therefore, detectable carry-over effects on breeding should be weaker in bolder individuals, as they should maintain an allocation to reproduction irrespective of previous conditions, while shy individuals should experience stronger carry-over effects. We tested this prediction in black-legged kittiwakes breeding in Svalbard. Using miniature biologging devices, we measured non-breeding activity of kittiwakes and monitored their subsequent breeding performance. We report a number of negative carry-over effects of non-breeding activity on breeding, which were generally stronger in shyer individuals: more active winters were followed by later breeding phenology and poorer breeding performance in shy birds, but these effects were weaker or undetected in bolder individuals. Our study quantifies individual variability in the strength of carry-over effects on breeding and provides a mechanism explaining widespread differences in individual reproductive success.

The impact of personality, morphotype and shore height on temperature-mediated behavioural responses in the beadlet anemone Actinia equina.

Between-individual variation in behavioural phenotype, termed personality, is an important determinant of how populations cope with acute environmental fluctuation related to climate change. Personality in the beadlet sea anemone Actinia equina is linked to genetically distinct morphotypes, which are associated with different heights on the shore. In the intertidal zone, high-shore environments experience more environmental fluctuation due to longer periods of exposure, and animals adapted to live in these environments are predicted to deal more effectively with environmental perturbation than their low-shore counterparts. We collected beadlet anemones of two different morphotypes from three different shore heights. We investigated variation in two behaviours at three different temperatures and in a temporal control treatment where the temperature was not changed: startle response time, the time it took an anemone to re-extend its tentacles after a threatening stimulus, and immersion response time, the time to re-extend tentacles after simulated tidal immersion. These behaviours reflect risk-taking and allow individuals to be categorized as bold, shy or intermediate based upon response times. Both behaviours showed significant changes as the temperature increased. For immersion response, the morphotype associated with the low-shore-lengthened response times at high temperatures. For startle response, all animals lengthened their response times at high temperatures but animals collected from the low-shore lengthened theirs to the greatest degree. At the individual level, although control individuals exhibited temporal changes in their response times, a clear effect of temperature was present in both behaviours. Shy and bold individuals became more intermediate at higher temperatures in immersion response (this effect was present to a lesser degree in control individuals), while intermediate individuals raised their response times at higher temperatures for startle response. Given that prolonged tentacle retraction reduces foraging opportunities and can negatively impact respiratory efficiency, our data suggest that some individuals within a single population of A. equina, particularly those associated with the lower shore, may exhibit less effective behavioural responses to temperature shifts than others. These findings demonstrate that acute temperature changes influence risk-taking, and could have profound short and long-term implications for survival in the face of climate change.

Acute and chronic stress prevents responses to pain in zebrafish: evidence for stress-induced analgesia.

The state of an animal prior to the application of a noxious stimulus can have a profound effect on their nociceptive threshold and subsequent behaviour. In mammals, the presence of acute stress preceding a painful event can have an analgesic effect whereas the presence of chronic stress can result in hyperalgesia. While considerable research has been conducted on the ability of stress to modulate mammalian responses to pain, relatively little is known about fish. This is of particular concern given that zebrafish (Danio rerio) are an extensively used model organism subject to a wide array of invasive procedures where the level of stress prior to experimentation could pose a major confounding factor. This study, therefore, investigated the impact of both acute and chronic stress on the behaviour of zebrafish subjected to a potentially painful laboratory procedure, the fin clip. In stress-free individuals, those subjected to the fin clip spent more time in the bottom of the tank, had reduced swimming speeds and less complex swimming trajectories; however, these behavioural changes were absent in fin-clipped fish that were first subject to either chronic or acute stress, suggesting the possibility of stress-induced analgesia (SIA). To test this, the opioid antagonist naloxone was administered to fish prior to the application of both the stress and fin-clip procedure. After naloxone, acutely stressed fin-clipped zebrafish exhibited the same behaviours as stress-free fin-clipped fish. This indicates the presence of SIA and the importance of opioid signalling in this mechanism. As stress reduced nociceptive responses in zebrafish, this demonstrates the potential for an endogenous analgesic system akin to the mammalian system. Future studies should delineate the neurobiological basis of stress-induced analgesia in fish.

Personality predicts foraging site fidelity and trip repeatability in a marine predator.

Animal populations are often comprised of both foraging specialists and generalists. For instance, some individuals show higher foraging site fidelity (spatial specialization) than others. Such individual differences in degree of specialization can persist over time-scales of months or even years in long-lived animals, but the mechanisms leading to these different individual strategies are not fully understood. There is accumulating evidence that individual variation in foraging behaviour is shaped by animal personality traits, such as boldness. Despite this, the potential for boldness to drive differences in the degree of specialization is unknown. In this study, we used novel object tests to measure boldness in black-legged kittiwakes (Rissa tridactyla) breeding at four colonies in Svalbard and deployed GPS loggers to examine their at-sea foraging behaviour. We estimated the repeatability of foraging trips and used a hidden Markov model to identify locations of foraging sites in order to quantify individual foraging site fidelity. Across the breeding season, bolder birds were more repeatable than shy individuals in the distance and range of their foraging trips, and during the incubation period (but not chick rearing), bolder individuals were more site-faithful. Birds exhibited these differences while showing high spatial similarity in foraging areas, indicating that site selection was not driven by personality-dependent spatial partitioning. We instead suggest that a relationship between boldness and site fidelity may be driven by differences in behavioural flexibility between bold and shy individuals. Together, these results provide a potential mechanism by which widely reported individual differences in foraging specialization may emerge.

Assessment of behaviour in groups of zebrafish (Danio rerio) using an intelligent software monitoring tool, the chromatic fish analyser.

BACKGROUND: Zebrafish (Danio rerio) are an increasingly popular model species within a variety of biomedical and neurobiological contexts. Researchers are required to prevent any negative states, such as pain, when using experimental animals to optimise fish welfare but analysis tools for zebrafish are lacking. NEW METHOD: The chromatic fish analyser (CFA) is a computer-based monitoring system that has the potential to identify changes in fish behaviour via spatial chromatic analysis of video images. The CFA was used to monitor the behaviour of groups of six fish, where none, one, three or six fish were given a fin clip. Additionally a drug with pain-relieving properties, lidocaine, was administered to determine if this ameliorated any alterations in behaviour. The CFA measured hue horizontally and vertically reflecting the position of the fish in their tank. Saturation (indicates clustering distribution) and lightness were measured to reflect overall zebrafish activity. RESULTS: Changes in vertical hue demonstrated that all fin clipped animals were closer to the bottom of the tank relative to pre-treatment; this was not observed in control groups, and was alleviated in those treated with lidocaine. Saturation (clustering) and lightness alterations indicated fin clipped groups reduced activity after receiving the fin clip. Lidocaine was effective in preventing the behavioural changes when 1 or 3 fish were clipped. CONCLUSIONS: The CFA proved powerful enough to identify significant changes in behaviour taken directly from video images. With further development this monitoring tool represents a step forward in detecting behavioural changes in groups of zebrafish indicating welfare.

Evolution of nociception and pain: evidence from fish models.

In order to survive, animals must avoid injury and be able to detect potentially damaging stimuli via nociceptive mechanisms. If the injury is accompanied by a negative affective component, future behaviour should be altered and one can conclude the animal experienced the discomfort associated with pain. Fishes are the most successful vertebrate group when considering the number of species that have filled a variety of aquatic niches. The empirical evidence for nociception in fishes from the underlying molecular biology, neurobiology and anatomy of nociceptors through to whole animal behavioural responses is reviewed to demonstrate the evolutionary conservation of nociception and pain from invertebrates to vertebrates. Studies in fish have shown that the biology of the nociceptive system is strikingly similar to that found in mammals. Further, potentially painful events result in behavioural and physiological changes such as reduced activity, guarding behaviour, suspension of normal behaviour, increased ventilation rate and abnormal behaviours which are all prevented by the use of pain-relieving drugs. Fish also perform competing tasks less well when treated with a putative painful stimulus. Therefore, there is ample evidence to demonstrate that it is highly likely that fish experience pain and that pain-related behavioural changes are conserved across vertebrates. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Automated monitoring of behaviour in zebrafish after invasive procedures.

Fish are used in a variety of experimental contexts often in high numbers. To maintain their welfare and ensure valid results during invasive procedures it is vital that we can detect subtle changes in behaviour that may allow us to intervene to provide pain-relief. Therefore, an automated method, the Fish Behaviour Index (FBI), was devised and used for testing the impact of laboratory procedures and efficacy of analgesic drugs in the model species, the zebrafish. Cameras with tracking software were used to visually track and quantify female zebrafish behaviour in real time after a number of laboratory procedures including fin clipping, PIT tagging, and nociceptor excitation via injection of acetic acid subcutaneously. The FBI was derived from activity and distance swum measured before and after these procedures compared with control and sham groups. Further, the efficacy of a range of drugs with analgesic properties to identify efficacy of these agents was explored. Lidocaine (5 mg/L), flunixin (8 mg/L) and morphine (48 mg/L) prevented the associated reduction in activity and distance swum after fin clipping. From an ethical perspective, the FBI represents a significant refinement in the use of zebrafish and could be adopted across a wide range of biological disciplines.

Ethical considerations in fish research.

Fishes are used in a wide range of scientific studies, from conservation research with potential benefits to the species used to biomedical research with potential human benefits. Fish research can take place in both laboratories and field environments and methods used represent a continuum from non-invasive observations, handling, through to experimental manipulation. While some countries have legislation or guidance regarding the use of fish in research, many do not and there exists a diversity of scientific opinions on the sentience of fish and how we determine welfare. Nevertheless, there is a growing pressure on the scientific community to take more responsibility for the animals they work with through maximising the benefits of their research to humans or animals while minimising welfare or survival costs to their study animals. In this review, we focus primarily on the refinement of common methods used in fish research based on emerging knowledge with the aim of improving the welfare of fish used in scientific studies. We consider the use of anaesthetics and analgesics and how we mark individuals for identification purposes. We highlight the main ethical concerns facing researchers in both laboratory and field environments and identify areas that need urgent future research. We hope that this review will help inform those who wish to refine their ethical practices and stimulate thought among fish researchers for further avenues of refinement. Improved ethics and welfare of fishes will inevitably lead to increased scientific rigour and is in the best interests of both fishes and scientists.

An investigation on the impact of substrate type, temperature, and iodine on moon jellyfish production.

Jellyfish are a popular public aquarium species, however, their collection from natural populations is undesirable due to impact on species abundance and bycatch. Thus, a sustainable supply of jellyfish bred in-house would be highly desirable. Here we describe an investigation into developing a sustainable moon jellyfish, Aurelia aurita, breeding program by determining the impact of substrate type on reattachment of polyps and the influence of iodine and temperature on strobilation and ephyra production. To test whether reattachment and growth of moon jellyfish polyps are influenced by substrate type polyps were provided with anthropogenic and natural substrates after being dislodged in experimental aquaria. Polyps selectively re-attached to plastics rather than natural materials. However, polyp growth was similar on all tested substrates. We tested whether cooling and addition of iodine affected strobilation. A period of cooling of around 10 °C while also introducing soluble iodine to the polyps enhanced strobilation rate. This method produced ephyra at a reliable rate in captivity negating the need for collection of wild individuals providing a supply of individuals for exhibit and for conservation research within a public aquarium. These results demonstrate that plastics should be adopted as an easier to colonize substrate and the use of cooling with iodine addition can enhance sustainable breeding protocols of moon jellyfish and may be relevant to the production of comparable jellyfish species.

Comparative Physiology of Nociception and Pain.

The study of diverse animal groups allows us to discern the evolution of the neurobiology of nociception. Nociception functions as an important alarm system alerting the individual to potential and actual tissue damage. All animals possess nociceptors, and, in some animal groups, it has been demonstrated that there are consistent physiological mechanisms underpinning the nociceptive system. This review considers the comparative biology of nociception and pain from an evolutionary perspective.

Where to draw the line? Should the age of protection for zebrafish be lowered?

Zebrafish are not protected by legislation in many countries until they reach the first feed stage, typically at five days post-fertilisation. If they exhibit similar responses to adults when responding to pain and other stimuli should they be given more protection?