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Concerns about the welfare of production animals have extended from farm animals to fish, but an overview of the impact of especially capture fisheries on fish welfare is lacking. This review provides a synthesis of 85 articles, which demonstrates that research interest in fish welfare in capture fisheries has increased over time and that research has focused more on trawls and hooks than on purse seines, gillnets, traps and seines. We found that various gear characteristics, fish characteristics and context variables affect external injuries and mortality. Although the influence of gear characteristics on injuries and mortality can by nature not be compared across gear types, synthesis of the articles reviewed shows that fish characteristics and context variables influence injuries and mortality across gear types. In terms of fish characteristics, decreasing fish length and certain fish species were associated with higher mortality. In terms of context variables, greater capture depth and a longer fishing duration were associated with more injuries and higher mortality, whereas a large change in water temperature, a longer duration of air exposure and a high density in the net were associated with higher mortality. These relations provide options to reduce injuries and mortality from commercial capture fisheries. Implementation of such options, however, would require analysis of potential trade-offs between welfare benefits, and ecological and economic consequences.

When fish are reared for food production in aquaculture, they can be held in different types of rearing systems and subjected to various husbandry routines and operations. Each of these systems or operations can present different welfare risks to the fish, which in turn are dependent upon both the species and its life stage. In this chapter, we address and outline potential welfare hazards the fish may encounter in a wide range of existing and emerging rearing systems used for on-growing. These systems include: (1) pond-based aquaculture, (2) flow-through systems, (3) semi-closed containment systems, (4) RAS, (5) net cages and (6) farming offshore using sea cages in exposed conditions. We also outline potential welfare hazards for two key farming operations: transport and slaughter. We present the tools the farmer can use to assess fish welfare during on-growing and also outline relevant welfare actions that can be taken to militate against welfare hazards.

Successive exposure to aquaculture-related stressors may compromise the allostatic capacity of African catfish and lead to allostatic overload and poor welfare. Therefore, we tested the effect of (i) feeding during the light or dark phase, (ii) density (51 fish per 140 L versus 51 fish per 43 L) and (iii) altered available resting space/shelter on stress handling were studied in African catfish (Clarias gariepinus). Available resting space/shelter was manipulated by providing PVC-tubes in the tanks. Growth, baseline stress load (cortisol and osmolality), metabolism (glucose, lactate and non-esterified fatty acids) and aggression (measured by skin scar incidence) were assessed. Upon completion of the experimental period, we determined the adaptive capacity of the fish by the response to air exposure. We show that night-feeding enhances growth and lowers feed conversion ratio compared to day-feeding. No effects of density were found for baseline stress hormones, basal metabolism or aggression. Low density seems to stimulate aggressive behaviour after air exposure. Unexpectedly, providing PVC-tubes under high-density conditions increased aggression, raised baseline cortisol levels and decreased basal metabolism as well as growth; also the endocrine response to air exposure was stronger. We argue that these effects relate to a reduction in available space, i.e. a further increase in density, or to an increased competition for shelter. Overall, our data suggest that night-feeding optimizes growth and that care should be taken in using objects as shelter/enrichment for African catfish.

The nitrite threshold concentration in rearing water of African catfish (Clarias gariepinus) was assessed. African catfish with an initial mean (SD) weight of 219.7 (57.8) g were exposed to an increasing range of water nitrite from 6 (Control) to 928 µM nitrite for 28 days. Mean (SD) plasma nitrite concentrations increased from 5.0 (3.6) to 32.5 (12.6) µM at 928 µM ambient nitrite. The increase in nitrite was accompanied by gradual increase in plasma nitrate from 41.6 (28.4) µM to 420.2 (106.4) µM. Haematocrit, haemoglobin, methemoglobin, plasma concentrations of cortisol, glucose, lactate, osmolality, gill morphology and branchial Na+/K+-ATPase activity were not affected. Feed intake, final weight, SGR, FCR and mortality were not affected. We advise not to exceed a water nitrite concentration of 43 µM (0.6 mg L-1 NO2--N) to prevent the risk of reduced growth and feed intake in African catfish aquaculture.

The aim of this study was to assess electrical stunning of Atlantic cod and turbot in seawater to develop a protocol for the process of stunning and killing. An induced general epileptiform insult (unconscious) had a duration of 40 ± 27 s (n =14) in cod (2.6 ± 0.5 kg) and 34 ± 18 s (n = 19) in turbot (520 ± 65 g). Seven cod and 3 turbot displayed a physical reaction, and 11 turbot registered an electroencephalogram (EEG) response to pain stimuli administered 30 s post-stun. The heart rate was 32 ± 6 beats/min in cod and 25 ± 7 beats/min in turbot prior to stunning. Post-stunning, the electrocardiogram (ECG) revealed fibrillation and reduced activity post-stun. EEG, ECG recordings, and behavioral observations indicate that when a bipolar square wave current was applied with a frequency of 133 Hz and 43% duty cycle side to side (turbot) and at 170 Hz and 33% duty cycle (cod) head to tail, both species were stunned in seawater at current densities of 3.2 A/dm2 and 2.5 A/dm2, respectively. For turbot, a 5 s exposure to electricity followed by chilling in ice water for 15 min is sufficient to prevent recovery. For cod, a killing method needs to be established.

Cod and haddock captured with commercial trawling gear were taken immediately after landing on deck to on-board storage in dry bins for measuring brain and heart activity, and behaviour. Other groups were first stored in holding tanks and then electrically stunned with a prototype "dry stunner". For stunning 52 V-rms was applied on individual fish for 1 s. As a result, the cod and haddock received an electrical current of 0.34 +/- 0.09 and 0.36 +/- 0.12 A(rms), respectively. Electrical activity in the brain and heart was measured before and after electrical stunning. The fish remained conscious for at least 2 h after landing and during on-board storage as indicated by the electrical activity measured in brain and heart. Behavioural responsiveness to administered stimuli was absent in both species. After electrical stunning, both species showed a general epileptiform insult which was characterised by a tonic phase followed by a clonic phase and terminating with an exhaustion phase. Since the fish remained conscious after landing and storage, electrical stunning and subsequent killing with a throat cut, may provide an option for improving fish welfare on-board commercial fishing vessels. In particular, we recommend to stun and kill wild cod and haddock as soon as possible after landing on deck using a dry stunner applying 52 V-rms (coupled AC/DC current) for more than 3 s. (C) 2012 Elsevier B.V. All rights reserved.

Animals should be rendered unconscious before slaughter in order to avoid suffering or pain. The objective of this study was to evaluate an electrical stunning after dewatering to induce instantaneous unconsciousness and insensibility in yellowtail kingfish (Seriola lalandi), common sole (Solea solea) and pike-perch (Stizostedion lucioperca). To kill without recovery, the current was applied for 5 s, followed by chilling in ice water for 10 min. Loss of consciousness and sensibility were assessed by neural (EEG), physiological (ECG) and behavioural parameters. An epileptiform insult was observed in all yellowtail kingfish (n = 27), common sole (n = 25) and pike-perch (n = 25) after administering a current of 0.54 +/- 0.12 A(rms) (124 V dc and 11 V-rms ac; 100 Hz), 0.65 +/- 0.23 A(rms) (98 V dc and 8.4 V-rms ac; 100 Hz) and 0.75 +/- 0.24 A(rms) (144 V dc and 13 V-rms ac; 100 Hz) during 1 s through the head of individual fish, respectively. When yellowtail kingfish (n = 11) was submitted to a 5 s electrical stun followed by chilling in ice water, this resulted in passing 0.72 +/- 0.13 A(rms) for 5 s and no recovery during chilling. In the case of common sole (n = 10) and pike-perch (n = 12), passing 1.4 +/- 0.64 and 0.75 +/- 0.24 A(rms) during 5 s followed by chilling in ice water for 10 min resulted in an irrecoverable stun in 9 out 10 fish and 11 out 12 fish, respectively. We conclude that for yellowtail kingfish the investigated combination of electrical stunning and chilling is suitable for humane slaughter, whereas for common sole and pike-perch this procedure needs to be optimised. (C) 2012 Elsevier B.V. All rights reserved.

Behavioural, neural and physiological parameters were scored after electro-stunning of Nile tilapia ( Oreochromis niloticus ) in three positions to an electric field in fresh water. An overall current density, 1.0 A rms /dm 2 , 50 Hz sinusoidal, applied for 1 s top-to-bottom in water (700 μS/cm), induced a general epileptiform insult on the EEG for 26 ± 10 s ( n = 24). The ECG revealed fibrillation for 9 ± 4 s in 15 out of 24 fish. To kill without recovery, the current was applied for 5 s, followed by chilling in ice water for 15 min. Killing of the stunned fish by gill-cutting instead of chilling is not recommended, as 2 out 4 tilapias responded 10 min post stun. When electricity was applied head-to-tail on tilapia ( n = 8), the overall current density could be reduced to 0.4 A rms /dm 2 , 50 Hz sinusoidal, to induce a general epileptiform insult, which lasted 27 ± 10 s. A pulsed square wave alternating current (overall density of 0.6 A rms /dm 2 , 133 Hz and 43% duty cycle) was applied for 1 s side-to-side on tilapia in water (650 μS/cm). This induced a general epileptiform insult for 51 ± 37 s ( n = 14). Fibrillation occurred for 16 ± 7 s ( n = 10). Free-swimming fish righted between 68 and 95 s ( n = 5) and between 438 and 1139 s ( n = 5) after 1 and 5 s electro-stunning, respectively. Our results show that protection of welfare of tilapia at slaughter can be obtained with electro-stunning followed by killing the stunned fish in ice water.

To protect the welfare of fish at slaughter, these animals should be rendered unconscious and insensible prior to killing. Furthermore, the state of unconsciousnessmust be long enough to allowkillingwithout recovery. The objective of this study was to determine the stunner settings for effective tail-first dry electrical stunning of turbot (Scophthalmusmaximus) and common sole (Solea solea). The fish were separated in two batches (B1 and B2). The turbot and sole in B1were subjected to a short tail-first stun lasting for 1 s and after 1 min of recovery to a second, longer (20 s) stun. The fish in B2were exposed to a single long (20 s) stun, whichwas tail-first in sole, but headfirst in turbot. The short stun was applied to verify that the loss of consciousness was instant (i.e. within 1 s), whereas the long stun (followed by immersion in ice water) was performed with the aim of showing that it is feasible to kill the fish without recovery. Loss of consciousness and sensibility were assessed using electrophysiological (EEG and ECG) and behavioural parameters. After administering a current of 2.39 ± 0.91 A rms by applying 125.5 ± 0.6 V rms (100 Hz) in turbot and 1.22 ± 0.68 A rms by applying 152.4 ± 0.5 V rms in sole for 1 s, 25 out of 26 turbot and 9 out of 10 sole in B1 exhibited EEG patterns showing that the fish were rendered unconscious instantly. The long tail-first exposure of turbot in B1 to 3.88 ± 1.26 A rms for 1 s, followed by 1.44 ± 0.41 A rms for 19 s, followed by immersion in ice water, led to an irrecoverable stun in 21 out of 22 fish, whereas the long head-first stunning of turbot in B2 (n = 13) resulted in passing 1.27 ± 0.40 A rms for 1 s and 0.65 ± 0.21 A rms for 19 s through the fish and no recovery during chilling. After the long, tail-first exposure of sole in B1 (n = 9) and B2 (n = 22) to 1.18± 0.49 A rms for 1 s+0.35 ± 0.22 A rms for 19 s, and 1.20± 0.59 A rms for 1 s + 0.36 ± 0.15 A rms for 19 s, respectively, none of the fish regained consciousness during the chilling. We conclude that the tail-first electrical stunning, followed by immersion in ice water can be developed into an effective stunning and killing method for turbot and sole.

The objective of this study was to assess whether high-pressure injection of air into the brain of African catfish (Clarias gariepinus) could render the animal unconscious and insensible immediately and permanently. In the study, 48 African catfish with a live weight of 900-1900 g were restrained and equipped with EEG and ECG electrodes and then stunned. The catfish were stunned mechanically using a captive needle pistol. The pressure to shoot the needle was 8 bar and that to inject the air was 3 bar for 1.5 s. The catfish behaviour was observed during and after stunning. and waves and spikes, which precede a stoppage in brain activity as measured on the EEG, were used as indices for the measurement of immediate induction of unconsciousness and insensibility In 23 of 42 fish, an iso-electric line was observed after an average of 13.4 s, while in the remaining fish the and waves and spikes remained on the EEG during the recording period. In all cases, the ECG showed an irregular heart rate with fibrillation and extra systolae. Moreover, the configuration showed ischaemia. Before the captive needle stunning, free-swimming fish (n=7) explored the tank for an average of 21±12 s before lying down at the bottom. After stunning, they showed clonic uncoordinated swimming movements. The movements stopped after an average of 38±50 s. In another group (n=7) that was stunned and subsequently placed in ice water, clonic cramps were observed in two out of seven animals. When taking into account the number of animals with a reliable EEG (n=42) and using 95onfidence intervals, it was concluded that at least 93f the catfish were effectively stunned using a correctly positioned captive needle pistol. Furthermore, it is recommended to immobilize the stunned fish by chilling, as the post-stun clonic cramps may hinder gutting and filleting

The inhibitory avoidance paradigm allows the study of mechanisms underlying learning and memory formation in zebrafish (Danio rerio Hamilton). For zebrafish, the physiology and behavior associated with this paradigm are as yet poorly understood. We therefore assessed the effects of environmental enrichment and fish age on inhibitory avoidance learning. Fish raised in an environmentally enriched tank showed decreased anxiety-like behavior and increased exploration. Enrichment greatly reduced inhibitory avoidance in 6-month (6M)- and 12-month (12 M)-old fish. Following inhibitory avoidance, telencephalic mRNA levels of proliferating cell nuclear antigen (pcna), neurogenic differentiation (neurod), cocaine- and amphetamine-regulated transcript 4 (cart4), and cannabinoid receptor 1 (cnr1) were lower in enriched-housed fish, while the ratios of mineralocorticoid receptor (nr3c2)/glucocorticoid receptor α [nr3c1(α)] and glucocorticoid receptor β [nr3c1(β)]/glucocorticoid receptor α [nr3c1(α)] were higher. This was observed for 6M-old fish only, not for 24-month (24 M) old fish. Instead, 24 M-old fish showed delayed inhibitory avoidance, no effects of enrichment, and reduced expression of neuroplasticity genes. Overall, our data show strong differences in inhibitory avoidance behavior between zebrafish of different ages and a clear reduction in avoidance behavior following housing under environmental enrichment.

It was observed that farmed eels could be rendered unconscious and insensible instantaneously by passing an electrical current through fresh water. The general epileptiform insult on the EEG was characterized by a tonic/clonic and an exhaustion phase. After stunning, the ECG (electro-cardiogram) revealed fibrillation. The electrical stunning parameters were on average 194 ± 4 V and 0.636 ± 0.040 A/dm2 for 1.6 ± 0.4 s. Within a confidence level of 95L taking into account the number of animals with a reliable EEG (n = 29), at least 93 f all eels are effectively stunned in fresh water by an average current of 0.636 ± 0.040 A/dm2. The behaviour of groups of five eels, which were able to move freely in the water was observed before and after stunning with 50 V and 0.17 A/dm2. After 3-s stunning, two eels were turned upside down. They changed to a normal position after 10 and 13 s respectively. Subsequently, all eels were very active in swimming behaviour and stopped swimming after 75 s. When stunned for a longer duration, all eels were turned upside down and stopped breathing for a limited period of time. In the last experiment the eels were stunned in fresh water (500 ?S) with a voltage of 200 V for approximately 1 s, which was followed by 50 V for 5 min. As soon as the stunning started the water was de-oxygenated by flushing nitrogen to kill the eels by suffocation during the period of unconsciousness and insensitivity. The oxygen saturation decreased from 74 ± 10 to 23 ± 11 t 22 °C. After stunning no brain activity and no responses to pain stimuli were observed on the EEG. The heart rate increased (P < 0.05) after stunning, which was followed by a significant decrease. Only 1 out of 18 eels returned partially from upside down to a normal position 2 h after stunning; however, the eel did not respond to pain stimuli in behaviour. The developed stunning procedure can be recommended for humane slaughter of 50-kg batches of eels.

The overall objective was to evaluate the suitability of electronarcosis as a stunning method for farmed eels. In the first experiment the minimum electrical current needed to induce a general epileptiform insult by head-only stunning was assessed. The individual eels (n = 40) with a live weight of 700-800 g were fixed in a specially designed re-strainer. The EEG (electroencephalogram) and ECG (electrocardiogram) recordings, observation of behaviour and responses to pain stimuli were used to assess unconsciousness, insensibility and cardiac function. The applied current of 150, 200 or 250 V, 50 Hz, AC was delivered via scissor-model stunning tongs for approximately 1 s. A general epileptiform insult was observed in 31 eels for which a successful EEG recording was obtained, using 255 ± 4 V, 545 ± 32 mA, for 1.2 ± 0.2 s. The general epileptiform insult as measured on the EEG was characterized by a tonic/clonic phase and an exhaustion phase. The behaviour showed one phase: tonic cramps alternated by clonic ones. The heart rate was 22 ± 8 beats min1 (n = 23) prior to stunning. After stunning the ECG revealed fibrillation. In the second experiment the behaviour of seven individual eels able to move freely in water was observed after head-only stunning (250 V). Two phases were distinguished. Limited tonic and clonic cramps combined with backward swimming were followed by heavy clonic cramps combined with unco-ordinated movements such as jumping out of the water. A distinct exhaustion phase was not observed in all animals. In the third experiment a head-to-tail electrical method was examined in 15 eels for rendering the eels unconscious and insensitive prior to slaughter. They were stunned by applying 253 V for 3 s followed by 50 V for 5 min. In the fourth experiment nine eels were head-only stunned with 260 V for 1 s immediately followed by 50 V for 5 min applied from head to tail. Results obtained in these two experiments were similar. After stunning no brain activity and no responses to pain stimuli on the EEG were observed and the ECG showed ventricular extra systolae. It was observed that it might take 60 ± 25 min or longer for a complete recovery. It can be concluded that for effective electrical stunning of eels with a weight of 700-800 g an average current of 545 ± 32 mA (at approximate 250 V, 50 Hz AC) is needed. In this case, within a confidence level of 95t least 91␘f the eels are effectively stunned (n = 31). Therefore, it is recommended to increase the minimum current for an effective stun in practice to 600 mA. Further research is needed to determine the conditions to induce permanent unconsciousness and insensibility of the eels to protect the animals at slaughter.

The current procedures for slaughtering European eels (Anguilla anguilla) for food are very slow and cause suffering. Although there is little legislation for protecting the welfare of fish at slaughter, the legislation covering farmed mammals and birds at slaughter is well defined, requiring that these animals be rendered insensible immediately or without fear or pain prior to being killed. For many mammals and birds this can be achieved using an electrical stun, which is then followed by a procedure that actually kills them, such as exsanguination. This paper reports the investigation of the possibility of using electricity to stun eels, rendering them insensible to pain. Using 1 s duration alternating currents at 50 Hz applied directly across the head of the fish, it was shown that it was possible to stun the fish with currents of 0.1 A and above. Increasing the applied current increased the length of the period of the stun. When the duration of the application of the current was increased to 30 s it was found that the fish could be killed using currents between 0.50 A and 0.95 A. These results show that it is possible to use electricity to instantly stun eels and also to kill them by using longer duration currents. The use of preslaughter electrical stunning at slaughter could allow the welfare of these fish at slaughter to be improved greatly.

The ammonia (NH 3 ) and nitrate (NO 3 − ) threshold concentrations in rearing water of juvenile pikeperch ( Sander lucioperca ) were assessed. Pikeperch with an initial mean (SD) weight of 17.7 (4.2) g were exposed to 0.9 (control), 3.6, 5.2, 7.1, 11.2 and 18.9 μM NH 3 in the water for 42 days. Plasma NH 4 + concentrations stayed at control levels (~ 650 μM) up to 11.2 μM NH 3 in the water. At the highest water NH 3 concentration tested, plasma NH 4 + had more than doubled to 1400 μM. Based on the specific growth rate, the EC 10 value for NH 3 was 5.7 μM. When pikeperch (initial mean (SD) weight of 27.0 (4.9) g) were exposed to 0.1 (control), 1.5, 2.3, 3.7, 6.1, 10.2, 15.8 and 25.6 mM NO 3 − for 42 days, mean (SD) plasma NO 3 − concentrations increased linearly from 88 (47) to 5993 (899) μM at the highest ambient NO 3 − level. Feed intake, specific growth rate and feed conversion ratio were not affected. Neither NH 3 nor NO 3 − exposure significantly affected haematocrit, plasma concentrations of cortisol, glucose, lactate, osmolality, gill morphology or branchial Na + /K + -ATPase activity in pikeperch. For juvenile pikeperch we advise not to exceed a water NH 3 concentration of 3.4 μM (0.05 mg NH 3 –N/L), the lower limit of the 95% confidence interval of the EC 10 value for SGR, to ensure proper physiology and growth. For NO 3 − we advise not to exceed 25 mM (350 mg NO 3 − –N/L). This criterion is based on the highest NO 3 − concentration tested (25.6 mM). As no negative effects were detected at the highest concentration tested, the actual NO 3 − threshold probably exceeds 25.6 mM.

In this article an approach is presented to implement stunning and killing of farmed fish in practice. First, in a laboratory setting, the conditions need to be established to achieve an effective stun without causing avoidable distress and discomfort. Product quality is evaluated to assess the impact of stunning method on carcass quality. During subsequent implementation in a commercial setting, it is likely that stress-physiological and neurophysiological measurements are not feasible and, therefore, only behavioral and physical measurements can be performed. Finally, stunning and killing of farmed fish in practice should be controlled using a process-oriented quality assurance system.

The objective was to take a first step in the development of a process-oriented quality assurance (QA) system for monitoring and safeguarding of fish welfare at a company level. A process-oriented approach is focused on preventing hazards and involves establishment of critical steps in a process that requires careful control. The seven principles of the Hazard Analysis Critical Control Points (HACCP) concept were used as a framework to establish the QA system. HACCP is an internationally agreed approach for management of food safety, which was adapted for the purpose of safeguarding and monitoring the welfare of farmed fish. As the main focus of this QA system is farmed fish welfare assurance at a company level, it was named Fish Welfare Assurance System (FWAS). In this paper we present the initial steps of setting up FWAS for on growing of sea bass (Dicentrarchus labrax), carp (Cyprinus carpio) and European eel (Anguilla anguilla). Four major hazards were selected, which were fish species dependent. Critical Control Points (CCPs) that need to be controlled to minimize or avoid the four hazards are presented. For FWAS, monitoring of CCPs at a farm level is essential. For monitoring purposes, Operational Welfare Indicators (OWIs) are needed to establish whether critical biotic, abiotic, managerial and environmental factors are controlled. For the OWIs we present critical limits/target values. A critical limit is the maximum or minimum value to which a factor must be controlled at a critical control point to prevent, eliminate or reduce a hazard to an acceptable level. For managerial factors target levels are more appropriate than critical limits. Regarding the international trade of farmed fish products, we propose that FWAS needs to be standardized in aquaculture chains. For this standardization a consensus on the concept of fish welfare, methods to assess welfare objectively and knowledge on the needs of farmed fish are required.

This study evaluated the industrial application of dry electro-stunning for Claresse® and African catfish via monitoring of brain and heart activities, behavioral responses to noxious stimuli and meat quality. Dry electro-stunning (150 V, AC + DC, 100 Hz) of Claresse® for 0.7 ± 0.2 s (mean ± SD) resulted in unconsciousness for 48 ± 15 s, fulfilling the welfare requirements for obtaining pre-slaughter unconsciousness in less than 1 s. Current input for 5.2 ± 0.5 s with a measured current of 0.57 ± 0.16 A followed by chilling, resulted in prolonged unconsciousness for 124 ± 20 s in Claresse®. Loss of behavioral responses to noxious stimuli and then death within 5 min in 9 out of 10 African catfish resulted from stunning for 9.1 ± 0.4 s (0.91 ± 0.18 A) followed by decapitation. The meat quality did not reveal any difference in color between electro-stunned (0.76 A, 150 V, AC + DC for 15 s) and chilled-only Claresse®. Although flesh acidification was significantly higher in the stunned group on the day of the experiment, after 24 h the groups showed similar pH changes for the next six days. We conclude that dry electro-stunning (150 V, AC + DC) followed by chilling and decapitation is a reliable method for industrial pre-slaughter unconsciousness of Claresse® and African catfish and stunning (150 V, AC + DC) leaves no significant effect on flesh quality of Claresse® compared to chilling method.

The fish welfare debate is intensifying. Consequently, more research is carried out to further our knowledge on fish welfare in aquaculture. We define here a series of key parameters to substantiate an acute response to a supposedly painful stimulus: a standardized tailfin clip. Ultrastructural analysis of common carp (Cyprinus carpio) tailfin indicates the presence of A-δ and C-type axons, which are typical for transmitting nociceptive signals in (higher) vertebrates. In Nile tilapia (Oreochromis niloticus), responses to a tailfin clip were studied and the unavoidable acute stress associated with the handling required for this procedure. A series of key parameters for further studies was defined. The responses seen in 'classical' stress parameters (e.g., changes in plasma cortisol, glucose and lactate levels) did not allow discrimination between the clipping procedure and the handling stress. However, three parameters indicated a differential, stronger response to the clip stimulus itself: first, swimming activity increased more and clipped fish spent more time in the light (in a tank where half the volume is covered by dark material); second, the gill's mucus cells released their content as observed 1h after the clip, and this response is transient (no longer observed at 6h post clipping). Third, branchial Na(+)/K(+)-ATPase activity assayed in vitro was not affected by the procedures, but a remarkable migration of Na(+)/K(+)-ATPase immunoreactive (chloride) cells into the lamellar epithelium was observed as of 6h post clipping. We conclude that the differential response to clipping supports that this is a painful procedure that evokes a transient specific physiological status.

The overall objective of the study was to evaluate a percussive and an electrical stunning method under laboratory conditions in Atlantic salmon. Evidence of unconsciousness and insensibility of the salmon was provided on the electroencephalogram (EEG) by the appearance of slow waves and spikes, followed by a strong depression in electrical activity. This phenomenon was observed in 17 salmon after percussive stunning using an air pressure of 8.1 to 10 bars, whilst 8 fish were considered conscious at pressures below 8.1 bars although some were seemingly unconscious on behaviour. Consequences were a haemorrhage in the brain cavity in 15 out of 17 fish, broken upper or lower jaws in 9 fish and eye burst in 8 fish. A general epileptiform insult (unconscious and insensible) was obtained by delivering a voltage, consisting of a direct current (DC) coupled with 100 Hz alternating current (AC) with a peak value of ≈112 volt (V), head to body, for ≈ 0.5 s. The total duration of the insult was 62 ± 44 s (mean ± SD; n = 25) which was followed by minimal brain activity in 19 fish. The heart rate was 20 ± 7 beats/min prior to stunning. After stunning the electrocardiogram (ECG) revealed fibrillation for 22 ± 15 s and became irregular and showed extra systolae (ventrical contraction) afterwards. Exposing the salmon for 5 s with electricity followed by a gill cut resulted that 1 out of 3 fish recovered temporary after 3 min. Haemorrhages were not observed in the fillets. Average current for head to body electrical dry stunning was 668 milliampere (mA) root mean square (RMS) with an average stunning voltage of 107.9 V rms . Electrical head to body stunning can be recommended when using coupled AC and DC current of 668 mA rms and ≈ 107 V rms . The salmon can be stunned in ≈ 0.5 s. However, a correct bleeding procedure should be developed. For percussive stunning we conclude that if sufficient force is used the fish will be rendered unconscious insensible which result in damage of the carcass, whereas a combined AC and DC can be recommended source for dry electrical head to body stunning.

The objective of the study was to evaluate the anaesthetic effects of Propiscin (2% etomidate) and 2-phenoxyethanol on common carp (Cyprinus carpio L.) by observing neural and behavioural measures. When exposed to the anaesthetic agent, the carp lost equilibrium after approximately 90 s of exposure. They did not breathe and the controls did not respond to tail pinch or prick or 6 V at the skin of the upper jaw. A shift to lower frequencies on the electroencephalogram (EEG) and computing fast Fourier transformation was observed when exposed to water containing Propiscin or 2-phenoxyethanol. Heart rate was reduced after placement in the water containing an anaesthetic agent. It may be concluded from our results that common carp were immobilized and sedated, when exposed to water containing 2 mL L¿1 Propiscin and immobilized and anaesthetized in water containing 0.5 mL L¿1 2-phenoxyethanol. In addition, the use of EEG provides for a sound assessment of exposure of carp to these anaesthetics.

The response of neutral sugar oligomers in different reducing sugar assays was determined. Monomers, dimers and trimers—as well as tetramers—of xylose and galactose were tested on a molar basis for their response in the Nelson-Somogyi assay, the neo-cuproine assay and the ferricyanide assay. The results show increasing response with increasing chain length of the oligomers in the ferricyanide assay and the neo-cuproine assay. The response of the dimers, trimers and tetramers are, however, the same in the Nelson-Somogyi assay. These differences are important when assaying, for instance, endo(1, 4)-β-xylanase and endo(1, 4)-β-galactanase activities or determining their kinetic parameters.

This study was performed to identify the electrical current and exposure duration that would instantaneously render broiler chickens unconscious at slaughter when using a head-to-cloaca water bath stunner. The water in which the head was immersed was one electrode, and a steel-coned or cutaneous U-shaped electrode penetrating the cloaca was the other electrode. When an electrode penetrating the cloaca was used, a 640-Hz sinusoidal current induced a tonic-clonic phase on the electroencephalogram that lasted for 10 +/- 3 s and an exhaustion phase that lasted for 34 +/- 12 s. The heart rate was 375 +/- 39 beats/min before stunning. After stunning, the electrocardiogram revealed fibrillating for 429 +/- 58 s, after which the heart activity stopped. When a U-shaped electrode was placed on the skin at the cloaca, the same phenomenon was induced. A general epileptiform insult was induced when using a pulsed alternating square wave current of 33 mA (peak 60 V, 600 Hz, and a duty cycle of 50%), which lasted, on average, for 25 s (n = 25). When the broilers were bled within 14 s after stunning, they remained unconscious and the heart activity stopped after 237 +/- 103 s. We concluded from this experiment that broilers were effectively stunned with an average current of 111 mA (50 V, 640 Hz, sinusoidal alternating current) for 1 s when using a water bath in which the head of the broiler was immersed in water, with the water being one electrode and a steel electrode penetrating the cloaca or placed around it being the other electrode. Energy use could be reduced when an alternating pulsed square wave is used when the broilers are stunned, by using a current of approximately 33 mA (peak of 60 V, frequency of 600 Hz, and a 50% duty cycle).

The overall objective of the study was to evaluate electrical and percussive stunning methods under laboratory conditions in common carps. The electrical current needed to induce a general epileptiform insult was assessed in 13 carps. The insult was obtained by delivering a current of 0.24 ± 0.03 A (163 ± 1 V; 50 Hz, a.c.) to the head, via scissor-model stunning tongs for approximately 1 s. The duration of the tonic, the clonic and the exhaustion phases were 11 ± 4 s, 5 ± 3 s and 20 ± 14 s on the EEG, respectively. An actual clonic phase was observed in two fish. The total duration of the insult was 31 ± 14 s. The heart rate was 22 ± 12 beats/min prior to stunning while after stunning, the ECG revealed fibrillation for 18 ± 7 s and was irregular. The behaviour of 10 individual carp, which were allowed to move freely in water, was recorded following head-only stunning (164 V, 0.23 ± 0.6 A). The duration of the tonic phase was approximately 10 s and the fish started fin movements after 48 ± 8 s and resumed swimming after 121 ± 83 s. A general epileptiform insult could be induced in carps (n = 25) by passing an electrical current through fresh water using 0.14 ± 0.03 A/dm2 (113 ± 17 V; electrode distance 16 cm) for 1 s at a water conductivity of 200 ¿S/cm. Twenty three carps were rendered unconscious and insensible during electrical stunning in fresh water, using 0.73 ± 0.05 A/dm2 (411 ± 2 V; electrode distance 16 cm) for 5 s at a water conductivity of 330 ¿S/cm, followed by chilling in ice water. As a second stunning procedure, carps were stunned mechanically using a percussion pistol at a maximal velocity of 10.99 ± 0.88 J. Evidence of unconsciousness and insensibility of the carps was provided by the appearance of theta, delta waves and spikes on the EEG, which were proceeded by no brain activity. However, two carp responded after 0.5 min to administered pain stimuli and one carp after 3 min. It may be concluded from this study that common carp are effectively stunned in fresh water. The application of an electrical current of 0.73 A/dm2 (26 V/cm, 50 Hz, a.c., 330 ¿S/cm) to individual carps in a tank in combination with chilling in ice water is an effective procedure for slaughter. The mechanical method of percussion stunning did not provide assurance of effective stunning as not all carp were unconscious and insensible after its administration.

The overall objective of the study was to evaluate loss of consciousness and sensibility after electrical stunning in fresh water and live chilling in ice water for slaughter of African catfish using measurement of electrical brain and heart activity. To provoke immediate loss of consciousness and insensibility, the minimum electrical current needed to induce a general epileptiform insult was assessed by placing a fish one by one in a tank between 2 plate electrodes in fresh water. The general epileptiform insult on the EEG (electroencephalogram) was characterised by a tonic, clonic and an exhaustion phase. After stunning, the ECG (electrocardiogram) revealed fibrillation. Within a confidence level of 95%, taking into account the number of animals with a reliable EEG ( n = 24), at least 88% of all catfish are effectively stunned in fresh water by an average current of 1.60 ± 0.11 A/dm 2 (50 Hz, sinusoidal, a.c.) at a conductivity of 876 μS of the water. After electrical stunning in combination with decapitation the fishes showed minimal brain activity until death by bleeding. For assessment of live chilling, 28 individual catfish kept in aerated tap water of 24 °C were placed one by one in ice water of 0.1 ± 0.5 °C for 30 min. The fishes showed swimming followed by clonic muscle cramps and became motionless at the end. No response on pain stimuli on the EEG appeared after a median of 12.5 min (5 to 20 min) at a body temperature of 13.7 ± 2.6 °C ( n = 22). When taking into account the number of animals with a reliable EEG ( n = 22) and using 95% confidence intervals, it was concluded that at least 87% of the catfish were unconscious and insensible at a decrease in body temperature of approximately 8.7 °C. Live chilling of African catfish resulted in an extremely high heart rate (tachycardia). Values between 294 ± 47 and 311 ± 38 beats/min ( n = 13) were measured, where a normal value is between 70 and 80 beats/min. It was observed that unconscious and insensibility can be induced instantaneously by electrical stunning in fresh water. To kill the fish for slaughter decapitation is an option. Live chilling appeared to be a slow method. Whilst the African catfish was still conscious and sensible muscle cramps and tachycardia both occurred which may indicate stress.

A method for the evaluation of brain function in fish has been developed which is based on assessments of self-initiated behaviours, responses to stimulation, and reflexes. These assessments were validated in several freshwater and marine species and applied to evaluate the brain function of fish while they were anaesthetised and while they were being killed by a variety of methods. The results of these investigations were compared with published neurophysiological observations and it is concluded that some of the tests can be used to identify, with reasonable confidence, the state of awareness, and thus the capacity to experience suffering of several species of fish.

The overall objective of the study was to evaluate a slaughter method of eels, which consisted of chilling until their body temperature was

In the study, 53 eels with a live weight of 700 to 800 g were restrained and equipped with EEG and ECG electrodes before stunning. The eels were mechanically stunned by a captive needle pistol using a shooting pressure of 8 bar and an air injection of 3 bar during 1.5 s. The behaviour was observed during and after stunning. Indices for the immediate induction of unconsciousness and insensitivity were the appearance of theta and delta waves tending to an iso-electric line, i.e. no brain activity. The duration of these waves averaged 11±8 s ( n =32) on the EEG. Another nine animals showed directly after stunning no brain activity. Due to severe clonic cramps five animals were stunned twice. The ECG showed fluttering of the heart configuration immediately after stunning in all eels. It was concluded that within a confidence level of 95%, taking into account the number of animals with a reliable EEG ( n =42), at least 93% of the eels were effectively stunned by a correctly positioned captive needle pistol. It was easy to stun eels with a captive needle pistol using air pressure; however, more research is needed for the development of a restraining and stunning device suitable for use in practice.

Two galactanases, purified from experimental enzyme preparations derived from Aspergillus niger and Aspergillus aculeatus were found to be similar in a number of properties. They had similar molecular weights (Mr = 42–43 kD) and both showed highest activity on 1,4-β- D -galactan. Optimal activity was measured at 50–55°C and pH 4·00–4·25; optimal stability was observed in the pH range of 5–7 at 30°C. A sodium acetate buffer was found to be the best incubation buffer. Activity and stability were affected by Pb2+ (endo-galactanase from A. aculeatus was inhibited completely) and to a lesser extent by Ag+ and Zn2+ ions. Digestion of (arabino)-1,4-β- D -galactan resulted initially in a big shift in the Mw value of the bulk of (arabino)-1,4-β- D -galactan and formation of low galacto-oligomers, mainly tetra- and trimers of galactose. In the final stage of the reaction mono- and dimer accumulated as end products. Therefore a multiple attack mechanism was suggested for the endo-1,4-β- D -galactanases. The galactanases did not hydrolyse arabino-1,3/6-β- D -galactan. The effect of the temperature on their stability, their specific activities and their affinity for potato arabinogalactan differed; in the absence of substrate the A. niger and A. aculeatus endo-galactanase were stable up to 60°C and 35°C, respectively. The specific activities on potato arabinogalactan in a sodium acetate buffer pH 5·0, 30°C were found to be 158 and 244 u/mg, respectively. The Km values estimated for potato arabinogalactan were 0·77 and 0·31 g/litre, respectively. For an optimal breakdown of potato arabinogalactan combined action of arabinanases from A. niger and endo-galactanase was required. Arabinofuranosidase B did not stimulate the degradation of potato arabinogalactan by the endo-galactanases under the conditions used, whereas endo-1,5-α- L -arabinanase had an immediate synergistic effect with both endo-galactanases, indicating the presence of linear 1,5-α- L -arabinan side chains.