EFFECT OF DIFFERENT STUNNING OR/AND KILLING TREATMENTS ON THE NEUROTRANSMITTERS OF THE CENTRAL NERVOUS SYSTEM IN SEA BASS, Dicentrarchus labrax L.

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1 106 ΔΠΙΓΡΑΗ ΓΙΑΦΟΡΔΣΙΚΩΝ ΥΔΙΡΙΜΩΝ ΑΝΑΙΘΗΣΟΠΟΙΗΗ Η/ΚΑΙ ΘΑΝΑΣΩΗ ΣΟΤ ΝΔΤΡΟΓΙΑΒΙΒΑΣΔ ΣΟΤ ΚΔΝΣΡΙΚΟΤ ΝΔΤΡΙΚΟΤ ΤΣΗΜΑΣΟ ΣΟΤ ΛΑΒΡΑΚΙΟΤ, Dicentrarchus labrax L. Τζοπελάκος A. 1, Μήλιου Ε. 1 *, Δάλλα Χ. 2, Παπαδοπούλου-Νηαϊθώηη Ζ. 2 1 Γεσπνληθό Παλεπηζηήκην Αζελώλ, Τκήκα Επηζηήκεο Ζσηθήο Παξαγσγήο θαη Υδαηνθαιιηεξγεηώλ, Εξγαζηήξην Εθεξκνζκέλεο Υδξνβηνινγίαο, Θεξά Οδόο 75, Βνηαληθόο, Αζήλα, Εζληθό θαη Καπνδηζηξηαθό Παλεπηζηήκην Αζελώλ, Θαηξηθή Σρνιή, Εξγαζηήξην Φαξκαθνινγίαο, Μηθξάο Αζίαο 75, Γνπδή, Αζήλα, Περίληψη Μειεηήζεθε ε επίδξαζε δηάθνξσλ ρεηξηζκώλ αλαηζζεηνπνίεζεο ή/θαη ζαλάησζεο ζηα επίπεδα ησλ λεπξνδηαβηβαζηώλ ληνπακίλε (DA), λνξαδξελαιίλε (NA) θαη ζεξνηνλίλε (5-HT) θαζώο θαη ησλ κεηαβνιηηώλ ηνπο δη-πδξνμπ-θαηλπιαθεηηθό όμπ (DOPAC) θαη νκνβαληιηθό νμύ (HVA) γηα ηελ ληνπακίλε θαη 5-πδξνμπτλδνινμηθό νμύ (5-HIAA) γηα ηε ζεξνηνλίλε. Οη ρεηξηζκνί πεξηειάκβαλαλ αλαηζζεηνπνίεζε κε γαξηθαιέιαην ή κε θαηλνμπαηζαλόιε, ρηύπεκα ζην θεθάιη, ηνπνζέηεζε ζε παγσκέλν λεξό ή ζε πάγν θαη ηειηθά ζπλδπαζκό αλαηζζεηνπνίεζεο κε γαξηθαιέιαην θαη ζαλάησζεο ζε πάγν. Σε θάζε ρεηξηζκό ρξεζηκνπνηήζεθαλ δύν πιεζπζκνί ησλ 8 αηόκσλ. Ο ιόγνο DOPAC:DA πνπ απνηειεί έλδεημε ληνπακηλεξγηθήο ιεηηνπξγίαο βξέζεθε ζρεδόλ δηπιάζηνο θαηά ηελ αλαηζζεηνπνίεζε κε θαηλνμπαηζαλόιε απ όηη κε γαξηθαιέιαην, ππνδείρλνληαο εληνλόηεξε ζηξεζζνεπαγώκελε αληίδξαζε ησλ ςαξηώλ ζην πξώην αλαηζζεηηθό. Η ηνπνζέηεζε ζε παγσκέλν λεξό παξνπζίαζε ηε κεγαιύηεξε ζεξνηνληλεξγηθή ιεηηνπξγίαο (5-HIAA:5-HT) ζε ζρέζε κε ηνπο ππόινηπνπο ρεηξηζκνύο, πνπ ηειηθά επέθεξαλ ην ζάλαην. Ωζηόζν, ε πξνζζήθε γαξηθαιέιαηνπ πξηλ ηελ ηνπνζέηεζε ζε παγσκέλν λεξό θάλεθε λα κεηώλεη ηε ζεξνηνληλεξγηθή ιεηηνπξγία ζηα επίπεδα ησλ ππόινηπσλ ρεηξηζκώλ ζαλάησζεο. Μεηαμύ ηνπ ρηππήκαηνο ζην θεθάιη θαη ηεο ηνπνζέηεζεο ζε πάγν, δελ παξνπζηάζηεθε ζηαηηζηηθά ζεκαληηθή δηαθνξά ηόζν ζηε ζεξνηνληλεξγηθή, όζν θαη ηελ ληνπακηλεξγηθή ιεηηνπξγία. Τα απνηειέζκαηα κπνξνύλ λα ζπκβάιινπλ ζηνλ εκπινπηηζκό ηεο γλώζεο θαη ηε ζέζπηζε θαλόλσλ επδσίαο ή επζαλαζίαο θαηά ηηο πδαηνθαιιηεξγεηηθέο πξαθηηθέο ή ηνλ πεηξακαηηθό ζρεδηαζκό. Λέξεις κλειδιά: λαβράκι, νεσροδιαβιβαζηές, θανάηωζη. *Σπγγξαθέαο επηθνηλσλίαο: Μήιηνπ Ειέλε (elenmi@aua.gr). EFFECT OF DIFFERENT STUNNING OR/AND KILLING TREATMENTS ON THE NEUROTRANSMITTERS OF THE CENTRAL NERVOUS SYSTEM IN SEA BASS, Dicentrarchus labrax L. Tsopelakos A. 1, Miliou H. 1 *, Dalla C. 2, Papadopoulou-Daifoti Z. 2 1 Agricultural University of Athens, Faculty of Animal Science and Aquaculture, Department of Applied Hydrobiology, Iera Odos 75, Votanikos, Athens, 11855, Greece 2 National and Kapodestrian University of Athens, Medical School, Laboratory of Pharmacology, Mikras Asias 75, Goudi, Athens, 11527, Greece Abstract The effect of different stunning or/and killing treatments on the levels of brain neurotransmitters dopamine (DA), norepinephrine (NE), serotonin (5-HT), as well as their metabolites di-hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) for dopamine, and 5-hydroxy-indoleacetic acid (5-HIAA) for serotonin, was investigated in sea bass. Treatments involved anaesthesia with clove oil or 2-phenoxyethanol, percussive stunning, immersion in ice/water mixture (1:2), chilling in ice and a combination of clove oil anaesthesia and immersion in ice/water slurry. In every treatment, two groups of 8 fish were used. The DOPAC:DA ratio, which represents the dopaminergic activity, was found almost

2 107 two-fold higher in 2-phenoxyethanol than in clove oil anaesthesia, indicating a more intense response of fish to the stress induced by this anaesthetic. Immersion in ice/water slurry showed higher serotonergic activity (5-HIAA:5-HT) than the other slaughter treatments and moreover than anaesthesia. Sedation with clove oil before the immersion in ice/water slurry reduced the serotonergic activity to the levels of the remaining slaughter treatments. There were no significant differences between percussive stunning and chilling in ice for both serotonergic and dopaminergic activities. These results may contribute to the enrichment of scientific knowledge and the adoption of rules for welfare or euthanasia of reared sea bass. Keywords: sea bass, neurotransmitters, slaughter. *Corresponding author: Miliou Helen (elenmi@aua.gr) 1. Introduction From an animal welfare point of view, slaughter methods must cause immediate loss of consciousness, rendering the fish quickly insensitive to pain and suffering until death, or causing the death of a fish preventively anaesthetised or effectively stunned (EFSA 2008). To date, most of the studies on slaughter methods are referred to the plasma cortisol levels, the induced secondary stress responses and the flesh quality of fish, while little research has been conducted on the brain neurotransmitters. Behavioural, psychological and physiological responses to stress have common control mechanisms in the brain, regulated by the monoamine neurotransmitters serotonin (5- HT) and its metabolite 5-HIAA, dopamine (DA) and its metabolites (DOPAC and HVA), and norepinephrine (NE) (Ashley 2007). Knocking or percussive stunning is a frequently used slaughter method, mainly for large fish, and is considered as humane (Poli 2009). For the Mediterranean small-sized finfish, including sea bass, immersion in water/ice slurry is the current commercial slaughter method (Poli et al. 2005, Bagni et al. 2007). Chilling in ice is the traditional slaughter method in selective fisheries. The objective of this study was to investigate commercial and research stunning/killing methods for sea bass with respect to the brain monoaminergic activities (serotonergic 5- HIAA:5-HT and dopaminergic DOPAC:DA), aiming to get more insight to the psychological well-being of fish. 2. Materials and Methods Fish were kept for 5 weeks in 12 rearing tanks of l (8 fish per tank, 2 tanks per treatment). Initial body weight (mean ± SE) was ± 8.89 g and final ± 9.65 g per tank, not deferring among tanks (P>0.05). Six treatments were performed. For those involving anaesthetics, fish were netted from the rearing tank and placed in a holding tank containing the dissolved anaesthetic: (1) 105 ppm clove oil or (2) 1100 ppm 2-phenoxyehtanol. These concentrations render the fish unconscious and insensible in less than 2min. For the percussive stunning treatment (3), fish were serially removed from the rearing tank and received quickly a blast to the head. For the live chilling treatments, fish were netted and transferred in a holding tank filled with ice/water mixture (1:2) ( 0 o C) (4), where they rapidly immobilised and remained for 10 min (until unconsciousness) or into a case filled with ice (5), where they remained for 20min (until unconsciousness). For the last treatment (6), fish were anaesthetized (as in treatment 1), were immediately immersed in ice/water slurry (as in treatment 4) and remained for 10 min. Upon determination (EFSA 2008) of the unconscious state (absence of breathing and opercular movement, eyes fixed, absence of response to painful stimuli and loss of balance), or at the end of the time set (treatment 6), fish were serially removed from the holding tanks (treatments 1, 2, 4, 6) or cases (treatment 5).

3 108 Body weighting and brain sampling was carried out (<2 min per fish) immediately after the removal of each specimen from the holding tank (treatments 1, 2, 4, 6), holding case (treatment 5) or rearing tank (treatment 3). Brain sampling duration from the first to the last fish of each group varied approximately from 2 up to 16 min. Brain monoamine levels were determined as described by (Papadopoulou-Daifotis et al. 1995) with minor modifications. Comparison of means was performed by ANCOVA (Bonferroni test). 3. Results Brain sampling duration or fish body weight did not affect (covariates) significantly (P>0.05) the levels of the neurotransmitters and their activities. Brain norepinephrine (NE) levels (Fig. 1a) showed no significant differences (P>0.05) among different treatments. Dopamine (DA) levels (Fig. 1b) were elevated in treatments (1) and (2) (P<0.0001), while those of its metabolite DOPAC (Fig. 1c) were highest in treatment (2) (P<0.0001). DOPAC:DA ratio (Fig. 1h) was significantly lower (P<0.0001) in treatment (1) compared to the other treatments. HVA levels (Fig. 1d) were lowest in treatment (6) (P<0.05). Serotonin (5-HT) showed (Fig. 1e) the lowest levels in treatment (4) (P<0.05), while its metabolite 5-HIAA (Fig. 1f) the highest (P<0.0001) in the same treatment. 5-HIAA:5-HT ratio (Fig. 1g) was significantly higher (P<0.0001) in treatment (4) than in (3), (5), (6) and moreover than (1) and (2). 4. Discussion The decreased brain dopaminergic activity (DOPAC:DA) in sea bass anaesthetized with clove oil compared to 2-phenoxyethanol confirms that this organic product has stress-reducing capabilities. Moreover, clove oil is an effective anaesthetic at relatively low dosage and is safe for both environment and user (Iversen et al. 2003, Mylonas et al. 2005). Dopaminergic activity in sea bass immersed in ice/water slurry was similar to that in fish stunned by a blast to the head, indicating that the former method does not seem particularly stressful for the Mediterranean species, as it has been previously suggested (Poli et al. 2005). However, the increased levels of the brain serotonergic activity in fish chilled in ice/water slurry, demonstrate a psychological stress, as has been shown in subordinate fish (Ashley 2007). The pre-mortem time interval observed in this treatment (10 min) was shorter than that previously reported for sea bass immersed in ice/water slurry: 20 min (Bagni et al. 2007), 23±5 min (EFSA 2008) or 34±0.36 min (Acerete et al. 2009). This was probably due to the relatively higher levels of temperature or/and salinity in the rearing tanks. However, this method seems not appropriate for the welfare of sea bass from an ethical point of view. Sedation of sea bass with clove oil prior to transfer in ice/water slurry seems to reduce brain serotonergic activity to the levels obtained by percussive stunning. This combined procedure can be considered humane, as it renders the fish unconscious and minimizes its distress during chilling in ice/water slurry (hypothermia and anoxia) until death. It has been shown that rested harvesting prior to slaughter may reduce the stress response and also improves flesh quality (Ashley 2007).

4 109 Figure 1: Mean values (± SE) of brain neurotransmitter levels and ratios: (a) NE, (b) DA, (c) DOPAC, (d) HVA, (e) 5-HT, (f) 5-HIAA, (g) 5-HIAA:5-HT and (h) DOPAC:DA in sea bass subjected to different stunning/killing treatments: 1) clove oil anaesthesia, 2) 2-phenoexyethanol anaesthesia, 3) percussive stunning, 4) immersion in ice/water slurry, 5) clove oil anaesthesia followed by immersion in ice/water slurry and 6) chilling in ice. Different letters on the top of the bars denote that the means are statistically different (P<0.05). References Acerete L., Reig L., Alvarez D., Flos R., Tort L. (2009) Comparison of two stunning/slaughtering methods on stress response and quality indicators of European sea bass (Dicentrarchus labrax). Aquaculture 287(1-2): Ashley P.J. (2007) Fish welfare: Current issues in aquaculture. Applied Animal Behaviour Science 104(3-4): Bagni M., Civitareale C., Priori A., Ballerini A., Finoia M., Brambilla G., Marino G. (2007) Pre-slaughter crowding stress and killing procedures affecting quality and welfare in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Aquaculture 263(1-4):

5 110 EFSA (2008) Scientific Opinion of the Panel on Animal Health and Welfare on a request from the European Commission on animal welfare aspects of husbandry systems for farmed European sea bass and gilthead sea bream. The EFSA Journal 844: Iversen M., Finstad B., McKinley R.S., Eliassen R.A. (2003) The efficacy of metomidate, clove oil, Aqui-S and Benzoak as anaesthetics in Atlantic salmon (Salmo salar L.) smolts, and their potential stress-reducing capacity. Aquaculture 221(1-4): Mylonas C.C., Cardinaletti G., Sigelaki I., Polzonetti-Magni A. (2005) Comparative efficacy of clove oil and 2-phenoxyethanol as anesthetics in the aquaculture of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) at different temperatures. Aquaculture 246(1-4): Papadopoulou-Daifotis Z., Antoniou K., Vamvakides A., Kalliteraki I., Varonos D. (1995) Neurochemical changes in dopamine and serotonin turnover rate in discrete regions of rat brain after the administration of glycinergic compounds. Acta therapeutica 21(1): Poli B.M. (2009) Farmed fish welfare-suffering assessment and impact on product quality. Italian Journal of Animal Science 8(1s): Poli B.M., Parisi G., Scappini F., Zampacavallo G. (2005) Fish welfare and quality as affected by pre-slaughter and slaughter management. Aquaculture International 13(1):