Review: Assessing fish welfare in research and aquaculture,with a focus on European directives

The number of farmed fish in the world has increased considerably. Aquaculture is a growing industry that will in the future provide a large portion of fishery products. Moreover, in recent years, the number of teleost fish used as animal models for scientific research in both biomedical and ecological fields has increased. Therefore, it is increasingly important to implement measures designed to enhance the welfare of these animals. Currently, a number of European rules exist as requirements for the establishment, care and accommodation of fish maintained for human purposes. As far as (teleost) fish are concerned, the fact that the number of extant species is much greater than that of all other vertebrates must be considered. Of further importance is that each species has its own specific physical and chemical requirements. These factors make it difficult to provide generalized recommendations or requirements for all fish species. An adequate knowledge is required of the physiology and ecology of each species bred. This paper integrates and discusses, in a single synthesis, the current issues related to fish welfare, considering that teleosts are target species for both aquaculture and experimental models in biological and biomedical research. We first focus on the practical aspects, which must be considered when assessing fish welfare in both research and aquaculture contexts. Next, we address husbandry and the care of fish housed in research laboratories and aquaculture facilities in relation to their physiological and behavioural requirements, as well as in reference to the suggestions provided by European regulations. Finally, to evaluate precisely which parameters described by Directive 2010/63/EU are reported in scientific papers, we analysed 82 articles published by European researchers in 2014 and 2015. This review found that there is a general lack of information related to the optimal environmental conditions that should be provided for the range of species covered by this directive.     

Tail docking in pigs: acute physiological and behavioural responses

Tail docking of piglets is a routine procedure on farms to control tail-biting behaviour; however, docking can cause an acute stress response. The objectives of this research were to determine the stress responses to tail docking in piglets and to compare two methods of tail docking; cautery iron (CAUT) and the more commonly used blunt trauma cutters (BT). At approximately 6 days of age, piglets were tail docked using CAUT (n = 20), BT (n = 20) or sham tail docked with their tails remaining intact (CON; n = 40). Blood samples were taken prior to tail docking and at 30, 60 and 90 min after tail docking to evaluate the effect of tail docking on white blood cell (WBC) measures and cortisol concentrations. The above experiment was repeated to observe behaviour without the periodic blood sampling, so as not to confound the effects of blood sampling on piglet behaviour. Piglet behaviour was recorded in the farrowing crate using 1 min scan-samples via live observations for 60 min prior to and 90 min after tail docking. Total WBC counts were reduced (P > 0.05) among BT and CAUT compared with CON piglets 30 min after tail docking. Cortisol concentrations were higher (P < 0.01) among BT compared with CON and CAUT piglets 60 min after tail docking. Cautery and BT-docked piglets spent more (P < 0.05) time posterior scooting compared with CON piglets between 0 and 15 min, and 31 and 45 min after tail docking. Piglets tail docked using CAUT and BT tended to spend more (P < 0.07) time sitting than CON piglets between 0 and 15 min post tail docking. Elevated blood cortisol can be reduced by the use of the CAUT rather than the BT method of tail docking. Although the tail docking-induced rise in cortisol was prevented by using CAUT, the behavioural response to BT and CAUT docking methods was similar.     

Hot-water spraying is a sensitive test for signs of life before dressing and scalding in pig abattoirs with carbon dioxide (CO2) stunning

This study investigated the benefits of hot-water spraying (HWS) as a diagnostic test to verify the absence of signs of life (SOL) before scalding in pigs slaughtered with carbon dioxide (CO₂) stunning. A total of 37 108 finishing pigs from five German abattoirs (A to E) operating at 55 to 571 pigs per hour were assessed. Suspended pigs were sprayed onto the muzzle, head and front legs (143 to 258 s post sticking for 4 to 10 s, 57°C to 72°C). Any active movements during HWS were rated as positive test outcomes. In comparison, SOL were considered to be absent if a subsequent manual examination was negative and no active movements were observed following HWS. The incidence of pigs with activity during hot-water spraying (PWA) was restricted to two abattoirs (B: 0.25%; D: 0.02%; A, C, E: 0.00%). PWA showed movements of facial muscles (88%), mouth opening (78%), righting reflex (63%), isolated leg movements (35%) and vocalization (4%). The manual examination was positive in 71% of PWA (corneal/dazzle reflex: 67%/53%, nasal septum pinch: 33%), whereas all inactive pigs tested negative (P<0.001). The sensitivity for HWS as a test for SOL was calculated as 100%, dropping to 75% when only obvious and strong movements were taken into account. The specificity was >99.9% in either case. Any positive manual findings as well as any respiratory activity were instantly terminated using a penetrating captive bolt. Active movements triggered by the shot were shown to be an indicator for SOL (P<0.001). Video analyses revealed that spontaneous movements (SM) following sticking were present in 100% of PWA as opposed to 3.1% in pigs without such activity (controls). Results for different categories of SM in PWA v. controls were as follows: 100% v. 2.6% for mouth opening, 16.0% v. 0.1% for righting reflex and 22.0% v. 0.9% for isolated leg movements (all P<0.001). First mouth opening after sticking was observed later in PWA (28±24 v. 10±7 s), but mouth openings were observed for a longer period of time (141±44 v. 27±25 s) (both P<0.001). PWA with shorter mouth-opening intervals showed higher movement intensities during HWS and more positive manual findings (P<0.05). We conclude that HWS is a promising test for SOL. SM and sustained mouth opening in particular are indicators for compromised animal welfare and affected pigs should be shot by captive bolt.     

Behavioural and physiological stress responses to handling in wild guanacos

Both ethology and physiology can provide insights about stress conditions in wild animals during handling. Lama guanicoe (guanaco) is the major large herbivore inhabiting arid zones in Argentina. Since 2005, the Payún Matrú Cooperative, a business established and run by native people, has captured and handled guanacos for shearing at Payunia Reserve, Mendoza Province, Argentina, to market the species’ fibre for clothing and other products. The aim of our study was to evaluate the behavioural and physiological stress responses to handling of wild guanacos during these shearing activities. We evaluated stress conditions in guanacos by monitoring the frequency of agonistic behaviour in the holding corrals, following capture, the rate of vocalisation and agonistic behaviour during handling, and by estimating cortisol levels in handled animals. Crowding increased agonistic behaviour; this appeared to be related to dominance conflicts between males in the corrals. Time of corralling increased rate of agonistic behaviour and vocalisations during handling. The rate of behavioural stress responses was negatively related to cortisol levels in males; which may be due to animal fatigue or because behavioural stress responses may help mitigate physiological stress responses. Incorporating our results into animal welfare practices could help reduce stress conditions in wild guanacos. Our results suggest that both behavioural and physiological responses are important measures of stress in handled animals, and that behavioural stress responses cannot be used as a simple surrogate for glucocorticoid levels.     

A review of some physiological and toxicological responses of freshwater fish to acid stress

Synopsis Data relating to the specific effect of low pH on growth of freshwater fishes are ambiguous. Reproductive failure resulting from acid stress appears to be related to an upset in calcium metabolism and to faulty deposition of protein in developing oocytes. It appears that the ’no effect‘ level of pH depression for successful reproduction is around 6.5. Data on behaviorial responses of freshwater fish to acid stress and CO₂ are described. Most fish appear to be indifferent to pH within the range of approximately 10.5 to 5.5 and between 7.4 and 4.5 CO₂ appears to be the main directive factor. In cases of severe acid stress alteration of gill membranes and/or coagulation of gill mucus occurs and death due to hypoxia may result from a lengthening of the water-blood diffusion distance. Several reports agree that acid stress causes an upset of electrolyte homeostasis in fish but effects of low pH on osmotic permeability are largely lacking. Most hatcheryreared salmonids can tolerate pH 5.0 indefinitely but below this level the homeostatic electrolyte and osmotic regulatory mechanisms become inadequate. When fish are subjected to debilitating acid stress blood pH decreases possibly as the result of flux of H⁺ ions across gill membranes into the blood. This could change transepithelial potential and allow a blood, to-water diffusion of Na⁺ ions down an electrochemical gradient. Lowered ambient pH may interfere with gill calcium levels increasing permeability to both H⁺ and Na⁺ ions or an acidemia may occur as the result of a decrease in the excretion of metabolically produced H⁺ ions and CO₂. When the capacity of the buffer mechanisms is exceeded the blood pH drops and the capacity of hemoglobin to transport oxygen is decreased.     

Temperature increase and its effects on fish stress physiology in the context of global warming

The capacity of fishes to cope with environmental variation is considered to be a main determinant of their fitness and is partly determined by their stress physiology. By 2100, global ocean temperature is expected to rise by 1–4°C, with potential consequences for stress physiology. Global warming is affecting animal populations worldwide through chronic temperature increases and an increase in the frequency of extreme heatwave events. As ectotherms, fishes are expected to be particularly vulnerable to global warming. Although little information is available about the effects of global warming on stress physiology in nature, multiple studies describe the consequences of temperature increases on stress physiology in controlled laboratory conditions, providing insight into what can be expected in the wild. Chronic temperature increase constitutes a physiological load that can alter the ability of fishes to cope with additional stressors, which might compromise their fitness. In addition, rapid temperature increases are known to induce acute stress responses in fishes and might be of ecological relevance in particular situations. This review summarizes knowledge about effects of temperature increases on the stress physiology of fishes and discusses these in the context of global warming.     

The onset of piscivory in a freshwater fish species: analysis of behavioural and physiological traits

The onset of piscivory in fish, resulting in a shift from zooplankton or invertebrate to fish prey, was studied on pikeperch (Sander lucioperca) larvae displaying and not displaying piscivorous behaviour at different ages (23, 30, 37, 44 and 52 days post-hatching) using behavioural (attack, capture and swimming activity), morphological (allometry) and digestive enzymatic (trypsin, α-amylase and pepsin) analyses. The shift from zooplanktonic food items (Artemia nauplii) to a piscivorous diet did not occur at the same time for all individuals within the same cohort. Predation tests, conducted under controlled conditions (20°C; ad libitum feeding), showed that some larvae attacked fish prey as early as the age of 3 weeks [11.0 ± 1.3 mm total length (TL)], whereas others did not start until the age of 6 weeks (16.6 ± 1.9 mm TL). Piscivorous individuals were bigger, with larger heads, longer tails, higher acid protease and lower alkaline protease activities, than non-piscivorous conspecifics. In conclusion, high interindividual variability in morphological and digestive system developments linked to the development of predatory abilities could induce cannibalism in fish.     

Effects of repeated stress during pregnancy in ewes on the behavioural and physiological responses to stressful events and birth weight of their offspring

Stressors are frequently encountered during the pregnancies of farm animals. However, very few studies have investigated the effects of repeated stress in pregnant females on their offspring. Forty-eight ewes at 2.5 months of pregnancy were selected for their high or low cortisol response to isolation. Half of the ewes were subjected, twice a week during the last 5 weeks of gestation, to isolation. The first isolation bout of each week consisted only of isolation from conspecifics, while the second bout of each week consisted of isolation in the presence of a dog. The cortisol responses of ewes were measured during the treatment. The other half of the ewes was drafted and released without further disturbance. The emotional reactivity of the lambs to a light contrast test, a startling stimulus test and a social isolation test were studied at 25 days of age. The lambs were retested at 8 months of age in the same tests and also in a novel arena and a novel object tests. Cardiac responses to the startling stimulus were studied at 8 months of age. Basal cortisol concentrations and cortisol response after the isolation test were studied at both ages and cortisol response to ACTH was studied at 25 days of age. Ewes repeatedly exposed to the treatment of isolation plus presence of a dog habituated to the treatment. Moreover, the initial differences between the high and low reactivity ewes were not apparent when the stress treatment was subsequently imposed. Lambs prenatally stressed were heavier at birth than control lambs. The prenatally stressed lambs showed more exploration and locomotion in the behavioural tests at 8 months of age but not at 25 days of age. At 25 days of age, the basal cortisol concentrations were higher in the prenatally stressed lambs compared to the control lambs. Whereas these results suggest that mild prenatal stress of lambs has no adverse effect on the behavioural and cortisol responses to a range of stressors, there is evidence that this treatment increases birth weight and exploratory behaviour of prenatally stressed lambs. Clearly these results require confirmation.     

CO2麻醉对中华蜜蜂处女蜂王卵巢发育的影响

【目的】明确中华蜜蜂Apis cerana cerana蜂王羽化后生殖系统的动态变化,探索CO₂麻醉对中华蜜蜂处女蜂王卵巢发育的影响。【方法】通过人工育王技术和蜂王的储存获取不同日龄的中华蜜蜂处女蜂王,观察蜂王羽化后生殖系统的形态,测定生殖系统各项形态学指标(体重、卵巢重、受精囊直径和卵巢管数目)。中华蜜蜂处女蜂王连续2 d(分别于5和6日龄时)接受不同浓度(50%, 75%与90%)CO₂麻醉8 min, 以及不同日龄处女蜂王接受75% CO₂麻醉1~3次(每次8 min),待蜂王发育至10日龄时,利用石蜡切片技术与HE染色法观察蜂王卵巢形态、测定蜂王卵巢重和蜂王卵巢最大卵母细胞长度,通过实时荧光定量PCR(RT-qPCR)测定10日龄蜂王头部卵黄原蛋白基因(Vg)的相对表达量。【结果】中华蜜蜂蜂王的卵巢管数目与受精囊直径不会随着日龄发生明显的波动,但蜂王卵巢重在6-12日龄明显增加,之后将趋于稳定。与对照组(未接受CO₂麻醉处理)相比,50%, 75%与90% CO₂麻醉后中华蜜蜂蜂王卵巢重与最大卵母细胞长度均增加,并且头部Vg基因的相对表达量上调。其中CO₂浓度为75%,麻醉次数2次显著促进了中华蜜蜂蜂王的卵巢发育。【结论】中华蜜蜂蜂王羽化后卵巢会发生明显的发育变化,而CO2麻醉能加速卵巢的发育。     

不同耐性水稻幼苗根系对镉胁迫的形态及生理响应

采用水培试验,以两个耐镉性不同的水稻品种为材料,研究了不同浓度镉胁迫对水稻幼苗根系形态、根系活力、游离脯氨酸含量及抗氧化酶活性的影响。结果表明:低于5 μmol/L Cd胁迫对2个水稻品种总根长、根表面积、根体积、根干重、根系活力无明显影响,在1 μmol/L Cd时,甚至起促进作用。随Cd浓度增加表现出一定的抑制效应,秀水63在10 μmol/L Cd胁迫下根系形态、根系活力明显受到抑制,而秀水09在25 μmol/L Cd胁迫下明显受抑。随Cd胁迫浓度的增加,游离脯氨酸含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性均呈上升趋势,两品种相比,秀水09的游离脯氨酸含量、SOD和POD增幅大于秀水63,而MDA含量增幅小于秀水63;过氧化氢酶(CAT)活性变化表现为先上升后下降,10-100 μmol/L Cd胁迫下秀水63根系中CAT活性明显低于秀水09。总之,水稻对Cd毒害响应存在明显的品种差异,且Cd胁迫下根系生理响应的差异是品种间耐性差异的重要原因之一。     

Cultured fish: integrative biology and management of domestication and interactions with wild fish

Fish aquaculture for commodity production, fisheries enhancement and conservation is expanding rapidly, with many cultured species undergoing inadvertent or controlled domestication. Cultured fish are frequently released, accidentally and deliberately, into natural environments where they may survive well and impact on wild fish populations through ecological, genetic, and technical interactions. Impacts of fish released accidentally or for fisheries enhancement tend to be negative for the wild populations involved, particularly where wild populations are small, and/or highly adapted to local conditions, and/or declining. Captive breeding and supplementation can play a positive role in restoring threatened populations, but the biology of threatened populations and the potential of culture approaches for conserving them remain poorly understood. Approaches to the management of domestication and cultured‐wild fish interactions are often ad hoc, fragmented and poorly informed by current science. We develop an integrative biological framework for understanding and managing domestication and cultured‐wild fish interactions. The framework sets out how management practices in culture and for cultured fish in natural environments affect domestication processes, interactions between cultured and wild fish, and outcomes in terms of commodity production, fisheries yield, and conservation. We also develop a typology of management systems (specific combinations of management practices in culture and in natural environments) that are likely to provide positive outcomes for particular management objectives and situations. We close by setting out avenues for further research that will simultaneously improve fish domestication and management of cultured‐wild fish interactions and provide key insights into fundamental biology.     

High physiological demands in intensively raised pigs: impact on health and welfare

Genetic selection and better control of the environment of the pigs have resulted in increased production levels concerning both reproduction and growth. Such high performances imply high physiological demands that may deteriorate health and welfare. The aims of this paper are to review the physiological challenges that pigs are facing, to identify possible consequences on health and welfare, to propose ways of detecting and correcting problems whenever possible. At weaning, piglets are submitted to abrupt changes in food supply, housing and social environment. Behavioural changes and efficient adaptations of the digestive tract are critical for their health and welfare. Physiological demands to support these adaptations and risks of failure are inversely related to the age of the pigs. During fattening, modern pigs have high daily weight gain especially of lean tissue as well as elevated feed conversion rate. These high growth performances are suspected to favour stress and disease susceptibility, undesirable behaviours as well as leg weakness, but further experimental data are necessary to validate these effects and find their origin. In reproductive females, high prolificacy generates elevated foetal demands for nutrients and space that are not fully met as shown by an increased number of light piglets having difficulties to adapt successfully to the neonatal life. During lactation, sows with high milk production have high nutrient requirements leading to intense catabolism that may affect their health, welfare and future reproductive abilities.     

To change or not to change experimenters: caveats for repeated behavioural and physiological measures in Montagu's harrier

Animals facing anthropogenic disturbances may exhibit different anti‐predator responses depending on their perception of the risk. Experimental measures of behavioural and physiological traits may be impacted by the disturbance due to experimenter handlings and activities. In this study, we assessed the behavioural and physiological responses of Montagu's harrier chicks Circus pygargus from 14 nests, visited four times from hatching to fledgling by either the same (group I) or different (group NI) experimenters. Escape, aggressiveness and stress‐induced corticosterone concentration increased for both groups over time but chicks from group I showed a higher increase in their behavioural and physiological responses than chicks from group NI. This increase occurred after the second visit when experimenters took the first blood sampling on nestlings. Handling by the same experimenters throughout the study did not result in habituation but in sensitization. Our results suggest that Montagu's harrier nestlings may discriminate between the people who visit their nest and respond to experimenters’ identity. Therefore, we urge researchers working on behaviour and/or the physiology of stress including repeated measurements (such as animal personality studies for instance) to consider whether the experiments should or should not be conducted by the same experimenters (as usually done to minimize bias) as it can influence the outcomes of the experiments.     

Sphagnum physiological responses to elevated temperature,nitrogen, CO2 and low moisture in laboratory and in situ microhabitats: a review

Aquatic Ecology - Sphagnum mosses are considered peatland engineers because of their ability to create conditions inducing carbon accumulation. Here, we report on a review of the effects of four...     

A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function

Resorption and remodelling of skeletal tissues is required for development and growth, mechanical adaptation, repair, and mineral homeostasis of the vertebrate skeleton. Here we review for the first time the current knowledge about resorption and remodelling of the skeleton in teleost fish, the largest and most diverse group of extant vertebrates. Teleost species are increasingly used in aquaculture and as models in biomedical skeletal research. Thus, detailed knowledge is required to establish the differences and similarities between mammalian and teleost skeletal remodelling, and between distantly related species such as zebrafish (Danio rerio) and medaka (Oryzias latipes). The cellular mechanisms of differentiation and activation of osteoclasts and the functions of teleost skeletal remodelling are described. Several characteristics, related to skeletal remodelling, distinguish teleosts from mammals. These characteristics include (a) the absence of osteocytes in most species; (b) the absence of haematopoietic bone marrow tissue; (c) the abundance of small mononucleated osteoclasts performing non‐lacunar (smooth) bone resorption, in addition to or instead of multinucleated osteoclasts; and (d) a phosphorus‐ rather than calcium‐driven mineral homeostasis (mainly affecting the postcranial dermal skeleton). Furthermore, (e) skeletal resorption is often absent from particular sites, due to sparse or lacking endochondral ossification. Based on the mode of skeletal remodelling in early ontogeny of all teleosts and in later stages of development of teleosts with acellular bone we suggest a link between acellular bone and the predominance of mononucleated osteoclasts, on the one hand, and cellular bone and multinucleated osteoclasts on the other. The evolutionary origin of skeletal remodelling is discussed and whether mononucleated osteoclasts represent an ancestral type of resorbing cells. Revealing the differentiation and activation of teleost skeletal resorbing cells, in the absence of several factors that trigger mammalian osteoclast differentiation, is a current challenge. Understanding which characters of teleost bone remodelling are derived and which characters are conserved should enhance our understanding of the process in fish and may provide insights into alternative pathways of bone remodelling in mammals.     

Finding the optimal vertical distribution: behavioural responses of Daphnia pulicaria to gradients of environmental factors and the presence of fish

1. The vertical distribution of zooplankton results from active habitat choice aiming to optimise fitness gain in a system of trade‐offs. 2. Using large, controlled indoor mesocosms (Plön Plankton Towers), we monitored the behavioural response of Daphnia pulicaria to vertical gradients of temperature, food, oxygen and light, in the presence and absence of fish predation. 3. In the absence of fish, Daphnia distributed as predicted by an ideal ‘free distribution with costs’. If the food was distributed homogeneously, they stayed in the warm epilimnion, while they balanced their time dwelling in epi‐ and hypolimnion if the food was concentrated in a deep‐water maximum. 4. However, oxygen depletion in the hypolimnion, representing an additional cost, prevented Daphnia from completely exploiting the hypolimnetic food maximum. Consequently, the proportion dwelling in the hypolimnion was larger if oxygen was not limiting. 5. Fish predation had an overwhelming effect, driving Daphnia into the hypolimnion under all experimental conditions. If permitted by oxygen availability, Daphnia used the whole hypolimnion, but oxygen depletion reduced their possible habitat to the upper hypolimnion with oxygen concentrations above c. 0.7 mg L^?1. As fish were less tolerant of low oxygen, the layer below the thermocline formed a predation refuge for Daphnia.