PHB-10871; No of Pages 6 Physiology & Behavior xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

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Keywords: Hypothermia Turbot (Un)consciousness EEG and ECG Welfare

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1. Introduction

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Behavioural, neural and physiological aspects related to pre-slaughter cooling of turbot habituated to two environmental temperatures (18.7 and 12.0 °C) were investigated. Six fish in both treatments were immersed in ice water for 75 min. For control, four fish were immersed in water under their habituated environmental temperature. Turbot did not show a quick reduction of overall power in the EEG (electroencephalogram) to less than 10%, nor did the turbot show a shift in brain wave predominance from high to low frequency waves. At 15 min after immersion in ice water at least 7 out of 12 fish still showed total power values over 10% of pre-immersion values. Significant reductions in responsiveness to needle scratches and reduced breathing after immersion in ice water were observed, but none of these parameters had dropped to 0 even after 75 min in ice water. A significant reduction in gill score was found at 2 and 5 min after immersion in ice water compared to the control fish (p b 0.05). Heart rates significantly increased immediately after immersion in ice water and then decreased to a low basal value 30 min after immersion. The heart beat did not show major changes in regularity over time. Finally, at 15 and 75 min the turbot in ice water were significantly more responsive to vibration than to needle scratches. From these results we conclude that immersion in ice water may not induce unconsciousness, however, the brain activity does decrease to a lower level. The implication of this low brain activity with respect to welfare is not clear. Increased heart rates and maintained low brain activity and response to needle scratches during early immersion in ice water are indicative of a stress response appearing to affect welfare negatively. © 2015 Published by Elsevier Inc.

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(Salmo salar) are chilled in cold water the cortisol level increases which may indicate that this procedure is stressful [2,3]. For slaughter, fish are put in ice water as part of the killing procedure. Chilling fish to 4 °C decreases metabolism and facilitates handling but does not reduce the ability to feel pain [4,5]. Chilling of eels to a body temperature of b5 °C resulted in at least 5% of the fish remaining conscious [6]. An increase in heart rate occurred when catfish was placed in a cold restrainer and subsequently placed in ice water [7]. The heart rate is often measured as a response to stressors. It may either increase (tachycardia) or decrease (bradycardia). Changes in heart rate can be caused

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Article history: Received 26 March 2015 Received in revised form 16 May 2015 Accepted 19 May 2015 Available online xxxx

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Turbot habituated to a temperature of 12 or 19 °C was immersed in ice water. FFT analysis did not support the conclusion that they were unconscious. Heart rates first increased, and then decreased after immersion in ice water. After 75 min in ice water they were responsive to pain stimulation and vibration. Immersion in ice water is consistent with inactivity, but may be not humane.

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Wageningen UR Livestock Research, Wageningen UR, P.O. Box 338, 6700 H Wageningen, The Netherlands Akvaplan-niva, Bergen Office, Thormoehlensgate 53 D, N-5006 Bergen, Norway Akvaplan-niva, Iceland Office, Akralind 4, 201 Kópavogur, Iceland d Wageningen UR IMARES, P.O. Box 68, 1970 AB IJmuiden, The Netherlands

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Bert Lambooij a,d,⁎, Marc Bracke a,d, Henny Reimert a,d, Atle Foss b,d, Albert Imsland c,d, Hans van de Vis c,d

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Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water

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In the aquaculture industry and commercial fisheries cooling of fish is practised both in the slaughter process and in transportation [1]. For transportation purposes cooling is done to reduce the oxygen consumption, stress and metabolism of the fish, hence allowing longer transport durations at a lower cost. When carp (Cyprinus carpio) and Salmon ⁎ Corresponding author. E-mail address: [email protected] (B. Lambooij).

http://dx.doi.org/10.1016/j.physbeh.2015.05.019 0031-9384/© 2015 Published by Elsevier Inc.

Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Table 1 Average water values of RAS and tubs containing ice flakes in water before and after EEG recording. Temp.: temperature; SD: standard deviation; Sat.: saturation. Water temp. (C)

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0.06 0.00 0.29 0.05

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0.57 0.36 0.41 0.25

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2.1. Animals, treatments and read-out parameters

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For this study 34 (12 + 8 + 14) animals were used. The fish had been purchased from a commercial fish farm and transferred to the research lab of IMARES at Yerseke, The Netherlands where the experiment was conducted. The fish were acclimatised for 3 weeks in two separate recirculation aquaculture systems (RAS) filled with aerated seawater (tank dimensions: 0.6 × 2.5 × 2.5 m). The two RAS systems (S and W) were maintained at a high environmental temperature of 18.7 °C (S = ‘summer’) and at a low temperature of 12.0 °C (W = ‘winter’). For the experiment all fish were immersed in tubs with a water depth of 10 cm and a total volume of 30 l (salinity: 26.9 ± 2.2 ppt). More water values are given in Table 1. Twelve animals (6 S and 6 W turbot) were immersed individually in a tub containing water with ice flakes to record EEG and ECG values. The average body weights were 427 ± 85 g and 427 ± 40 g for S and W turbot, respectively. In each fish 3 needle scratches to the dorsolateral skin were applied at 2, 5, 15, 30, 45, 60 and 75 min after immersion in ice water. In addition at these time points gill movements (on a scale from 1 (minimal) to 4 (extensive gill movements)) as well as responses to tapping to the side of the tub, which resulted in a vibration in the water, were recorded. In addition, 8 fish (4 S and 4 W turbot) were immersed for a duration of 5 min in tubs filled with own RAS water. These groups may be considered as control treatments. In each of these groups 1 fish was fitted with EEG and ECG electrodes to record brain and heart activity in addition to behaviour. Unfortunately, the EEG recordings of these control fish could

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2.2. EEG and ECG records

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Prior to immersion in ice water, each fish was equipped with EEG and ECG electrodes, as described by Lambooij et al. [13]. The EEG electrodes (20 mm long and 1.5 mm diameter; 55% silver, 21% copper and 24% zinc) were placed percutaneous in the skull taking into account the position of the brain in this species, into the surface of the cortex. Two ECG electrodes (the same composition as the EEG electrodes) were placed subcutaneously, ventrally and dorsally of the upper pectoral fin. The earth electrode for both the EEG and ECG was placed subcutaneously near the tail. The EEG and ECG data were recorded using mobile data loggers [14]. Two fish (one in each treatment, S and W in ice water) were connected to a DI-720 data recording module with a WinDaq Waveform browser (Dataq Instruments, Akron, Ohio, USA). Two channels on the DI-720 data-recording module were used, with a 250 Hz sample frequency for each channel. The EEG and ECG recordings were screened using Labchart 7 Pro (version 7.3.7, AD Instruments, Cologne, Germany), and periods with clear artefacts lasting for more than a few seconds were identified and discarded in further analysis. In Labchart the EEG recordings were filtered (band pass: 0.5 to 45 Hz) and power analysis was done using the following settings: Hamming with a window overlap of 93.75%. The percentage of total power was determined for the following wave bands: 0.5–4 (Delta), 4–8 (Theta), 8–14 (Alpha), 14–30 (Beta), and 30-45 Hz (Gamma) (after [15]). Values for total power and percentage

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not be used for FFT analysis because of a pronounced saw tooth pattern in the EEG (probably related to relatively more pronounced breathing, see also Fig. 8). Finally, 14 fish were used to record the body temperature development (7 S and 7 W turbot, with average body weights of 400 ± 101 g and 361 ± 38 g respectively). These fish were immersed in ice water and sacrificed at fixed time points (5, 10, 15, 30, 45 and 60 min) to measure the deep body temperature. These fish were not fitted with electrodes. Upon completion of the experiments, the fish were killed by manual percussion with a club.

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by physical as well as psychological factors, and these factors may be difficult to distinguish [8]. The tachycardia observed in catfish can be classified as a sino atrial tachycardia, because this rhythm originated from the sino atrial node and may be caused by excitement [9]. Power spectral analysis is a method for the analysis of EEG signals. Spectral parameters can be used to quantify pharmacological effects of anaesthetics on the brain and the level of sedation. This method, in numerous variations, has been applied to monitoring the depth of anaesthesia and it has been incorporated into several commercially available EEG monitors [10]. EEG registrations are widely used to record brain activity under different circumstances to determine the state of consciousness and brain disorders in humans and animals. It has been stated that consciousness could be defined as awareness of the world around and of one's own body, whereas unconsciousness means unarguable unresponsiveness. In a state of unconsciousness, the EEG is always abnormal [11]. The electrical activity recorded on the EEG can be classified into δ (b4 Hz), τ (4 to 7 Hz), α (8 to 13 Hz), β (13 to 32 Hz) and γ (32 to 45 Hz) frequency bands. In α, β and γ rhythms, the animal is thought to be conscious [12]. Hence, while effects of cooling on general fish physiology are relatively well-known, less is known about the effects of rapid cooling on electrophysiology of the brain and heart. An important underlying issue concerns the question whether, and if so after which period of time, all fish will have lost consciousness when subjected to rapid cooling. In this experiment turbot kept at relatively low and high water temperatures were submerged in ice water to study effects on behavioural, neural and physiological parameters over a period of 75 min.

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RAS water of summer turbot RAS water of winter turbot Tub water before recording Tub water after recording

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Fig. 1. Fish (deep body) and water temperatures over time. The S (summer) and W (winter) turbot were collected from their own RAS water and subsequently immersed in ice water (n = 1 fish per time moment).

Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Fig. 2. Percentage of total power of individual summer (S) and winter (W) turbot in ice water. The pre-immersion (pre) values for individual fish have been set at 100%. Bars indicate transformed SD values over sampling intervals of 1 min. Significance levels are indicated as *: p b =0.05; #: p b 0.1. These symbols are placed as much as possible in between the two horizontal lines indicating which groups were compared. Dotted lines to the right indicate that the same p value applies in comparison to the most left-hand horizontal line.

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The heart beat was recorded by counting the number of QRS com- 162 plexes in the ECG. The heart beat regularity over that period was scored 163 using a subjective scale from 0 (very irregular) to 5 (regular). 164

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of total power were averaged for each fish over a period of up to 1 min before immersion in ice water, immediately after immersion and at 2, 5, 15, 30, 45 and 60 min after immersion in ice water.

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Fig. 3. Histogram of the percentage of total power of different brain wave frequency bands at fixed time points, before immersion (pre) and at 2, 5, 15, 30, 45 and 60 min after immersion in ice water, for summer and winter turbot. First average values were calculated over a 1 minute period around the sampling moment for each fish and these average values per fish were subsequently averaged over treatments (n = 6 fish per treatment). Standard deviation values are also shown.

Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Where possible numerical parameters were analysed using Student's t-tests in Microsoft Excel. When differences between treatments were not significant, treatments were lumped to examine effects over time. For effects over time, we focused on differences between adjacent time points and main contrasts (e.g. maximum versus minimum average values). Chi-square tests were done to examine proportions of positive responses, e.g. to compare treatment effects for responses to needle scratches.

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The reduction in body temperature over time for the summer (S) and winter (W) turbot immersed in ice water is presented in Fig. 1. By the end of the experiment at 75 min the deep body temperature of the fish in both groups was fairly close to the temperature of the ice water. No major differences could be observed between S and W turbot in the drop in total power after immersion in ice water compared to the values obtained before immersion (Fig. 2). At 2 min after immersion 3 fish (1 S and 2 W turbot) had values above 50% of pre-immersion values. By 5 min after immersion all fish had fallen below 50% of the preimmersion values. As of 15 min post-immersion three fish (S6, W2 and W3) had average values below 10% of pre-immersion values. By that time point S3 and W1 had values close to the 10% level. Since no differences were found between treatments, combined S and W values of total power were tested between time points. Pre-immersion power was significantly higher than all other time points (p b 0.05), the values at 2 min were significantly higher than all later time points (p b 0.05),

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Fig. 5. Regularity of the heart beat at fixed time points (before immersion (pre) and at 2, 5, 15, 30, 45 and 60 min after immersion in ice water) for summer and winter turbot.

except for 30 min, for which there was a trend (p b 0.1). The values at 5 min were higher than at 15 min (p b 0.05) and tended to be higher than at 60 min (p = 0.08). Major changes in the percentage of total power for the different brain wave bands compared to pre-immersion values were not observed (Fig. 3). In Fig. 4 the heart rate initially increased after immersion in ice water and then decreased over the next 30 min in ice water, where the regularity of the heart beat did not show a clear increasing or decreasing trend over time (Fig. 5). No differences were observed between S and W turbot in ice water, nor between S and W in RAS water. However, when data were pooled, we found that S and W turbot in ice water were significantly less responsive to the three needle scratches than the S and W fish in the warmer RAS water at 2 and 5 min after immersion. Tap responses were not observed at 2 and at 5 min after immersion. However, a substantial proportion of the fish responded to the tap vibration as of 15 min after immersion in ice water, and esp. at 30 and 75 min. At these latter time points (30 and 75 min) combined values for S + W were significantly higher than at 2 and 5 min (chi-square test). For W in ice water only, values were also significantly higher at 75 min compared to 2 or 5 min. For S in ice water only values at 75 and 15 min were significantly higher than at 5 min and there was a trend for the comparison with 2 min. Finally, at 45 and 60 min there was a trend effect of treatment in that at both points in time S turbot in ice water tended to show less behavioural responses to tapping than W turbot. When we compared the responsiveness of the fish to needle scratches (Fig. 6) and vibration/tapping (Figs. 7 and 8) we found that at 2 or 5 min after immersion in ice water for S and W combined responsiveness to needle scratches was not significantly higher than tap responsiveness. However, the S and W turbot immersed in RAS water did

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Gill movements and responses to tapping were recorded to show levels of behavioural quiescence and responsiveness. Movements of the gills were registered at 2, 5, 15, 30, 45, 60 and 75 min after immersion in ice water using a scale of scores from 0 to 4 (i.e. 0 — no movements; 1 — minor signs of breathing/single sporadic movements; 2 — slow movements; 3 — normal breathing; 4 — excessive gill movements). Tap responses (movements of the head, tail, fins, etc.; registered as responding yes/no) were tested at the same points in time by soft to moderate tapping/knocking on the outside wall of the polystyrene box, which caused vibration of the ice water. For turbot immersed in RAS water (of habituated temperature) needle scratches and gill scores were only tested at 2 and 5 min after immersion.

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Fig. 4. Average heart beat values at fixed time points (before immersion (pre) and at 2, 5, 15, 30, 45, 60 and 75 min after immersion in ice water) for summer and winter turbot. Standard deviation bars are for summer and winter turbot separately. Significant differences and trends for pairwise comparisons over time (where values for summer and winter turbot per time point were combined) are indicated as *: p b =0.05 and #: p b 0.1. These symbols are placed in between the two horizontal lines indicating which groups were compared. Dotted lines to the right indicate that the same p value applies in comparison to the most left-hand horizontal line.

Fig. 6. Percentage of positive nociceptive responses over time (between 2 and 75 min after immersion in ice water, and (only) at 2 and 5 min in RAS water (i.e. water of own recirculation system with initial temperatures of 18.7 and 12.0 °C in S and W respectively). Significant differences are indicated as *: p b =0.05. These symbols are placed in between the two horizontal lines indicating which groups were compared.

Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Fig. 8. Average gill scores (range 0–4) over time, i.e. between 2 and 75 min after immersion in ice water, and (only) at 2 and 5 min in RAS water (i.e. water of own recirculation system with initial temperatures of 18.7 and 12.0 °C in S and W respectively). Significant differences and trends are indicated as *: p b =0.05, **: p b 0.01, ***: p b 0.001; #: p b 0.1. These symbols are placed as much as possible in between the two horizontal lines indicating which groups were compared. Dotted lines to the right indicate that the same p value applies in comparison to the most left-hand horizontal line.

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Despite the higher and lower environmental temperatures the turbot expressed an early increase and an irregular decrease in heart rate during cooling down in ice water, which indicate stress. When cooled down the power frequency analyses did not shift to lower frequencies. However, the percentage of the total power did decrease in magnitude and also the responses of needle scratches and gill movements decreased. The tap responses increased during chilling. It is not clear in which way these responses affect the welfare of the fish. From an animal welfare standpoint it is recommended not to cool down the turbot as a pre-slaughter or transport method.

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Keeping fish alive for consumption is a common fish-handling practice both in developed and developing countries and at both artisanal and industrial levels. At present large numbers of species, including turbot, are kept alive and transported, very often from one country to another. There are wide differences in the behaviour and resistance of the various species. Therefore the method of keeping and transporting live fish should be tailored according to the particular species and the length of time it needs to be kept outside its natural habitat before slaughtering [16]. Therefore, summer environment and winter environment for turbot were simulated in a lab. It was observed that the

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show significantly more responses to needle scratches at these time points than tap responses. In addition, at 15 and 75 min after immersion in ice water S and W turbot combined were more responsive to tapping than to needle scratches (p b 0.01). Fig. 8 shows that the gill movements decreased over time after immersion in ice water, and remained detectable up to 75 min after immersion. No significant differences between treatments were found. However, when S + W in ice and in RAS were combined the turbot showed more respiration in RAS compared to ice water (at 2 and 5 min post-immersion).

internal cooling rate was faster when started at a high (summer) environmental temperature and was comparable for both summer and winter temperatures after 5 min in the ice water (Fig. 1). A higher or lower environmental temperature at start seemed to have little effect on the physiological processes seen in the brain (Figs. 2 and 3) as well as on heart activity (Figs. 4 and 5). On the other hand we found a trend effect (p b 0.1) at 45 and 60 min after immersion in ice water where W turbot tended to be more responsive to vibration (tapping) than S turbot. Fish may perceive vibrations in the water using the mechano-sensory lateral line, which consists of superficial and sub-dermal neuromasts — a system of hair-cell receptors [17]. We previously found that both turbot and sole immersed in ice water after electrical stunning were more responsive to vibration (tapping) than to needle scratches [18]. It is not clear to which extent the tap response may indicate consciousness, since spontaneous or reflex movements can occur even in “braindead” human patients [19,20,21]. Heart rate is often measured as a response to stressors and may show tachycardia (increased heart rate) or bradycardia (decreased heart rate). Physical as well psychological factors may affect changes in heart rate and these may be difficult to separate [8], moreover the occurrence of abnormal behaviours may be symptoms of stress [22]. When eels and catfish were placed in ice water, they showed abnormal behaviour and irregular heart rates, which may indicate stress [6,7]. In the present study an irregular heart rate during cooling was also observed, which again may indicate stress. However, the welfare implications of maintained responsiveness to tapping are not known. Hypothermia is not considered acceptable for euthanasia of fish, the period of consciousness is long and the ability to feel pain is not reduced [4]. The Farmed Animal Welfare Council [23] stated that live chilling of trout on ice should be prohibited. In African catfish, low brain activity and loss of responses to pain stimuli were observed after approximately 15 min. In both eel and catfish this physiological point was reached after a decrease in body temperature of 9 to 10 °C [6,7]. Low brain activity and no response to pain stimuli both on the EEG and in behaviour, support the assumption that the catfish were unconscious and insensible [11]. In turbot, low brain activity and response to needle scratches remained for at least 75 min with a body temperature equal to the temperature of ice water in our study. Welfare of fish should also be given moral consideration, as there is evidence that fish can perceive pain and fear [24,25, 26]. Contrary to the conclusions of these authors, Rose et al. [27] concluded that fish are not capable of pain and fear perception. For perception of pain and fear a living organism should possess cognitive capabilities. In this respect it is suggested [28,29] that fish have an ability to detect, conceptualise and respond to noxious stimuli. Especially, the application of pre-slaughter and slaughter methods can result in administering noxious stimuli to a fish.

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Fig. 7. Percentage of positive tap response, i.e. behavioural response to vibration over time, i.e. between 2 and 75 min after immersion in ice water, and (only) at 2 and 5 min in RAS water (i.e. water of own recirculation system with initial temperatures of 18.7 and 12.0 °C in S and W respectively). Note that all values were 0 at 2 and at 5 min. Significant differences and trends are indicated as *: p b =0.05 and #: p b 0.1 respectively. These symbols are placed as much as possible in between the two horizontal lines indicating which groups were compared. Dotted lines to the right indicate that the same p value applies in comparison to the most left-hand horizontal line.

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This research is supported by the European Research for SMEs pro- 314 ject MAXIMUS (FP7-SME-2011-286200). Thanks are due Mr. Youri 315 van Es and Mr. Angelo Hofman of IMARES Wageningen UR for 316

Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Please cite this article as: B. Lambooij, et al., Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.05.019

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Electrophysiological and behavioural responses of turbot (Scophthalmus maximus) cooled in ice water.

Behavioural, neural and physiological aspects related to pre-slaughter cooling of turbot habituated to two environmental temperatures (18.7 and 12.0°C...
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