Elayna Cristina da Silva Maciel. Animal Science, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil

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1 JOURNAL OF THE WORLD AQUACULTURE SOCIETY Vol. 45, No. 1 February, 2014 doi: /jwas Evaluation of the Effects of Different Stunning Methods on the Stress Responses and Meat Quality of the Amazon hybrid surubim, Pseudoplatystoma fasciatum female Leiarius marmoratus male Elayna Cristina da Silva Maciel Animal Science, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil Edivaldo Sampaio de Almeida Filho, William Bertoloni, and Janessa Sampaio de Abreu 1 Faculdade de Agronomia, Medicina Veterinária e Zootecnia (FAMEV), Universidade Federal de Mato Grosso (UFMT), Avenida Fernando Corrêa da Costa, 2367, Cuiabá, Mato Grosso , Brazil Abstract Different stunning methods (carbon dioxide [CO 2 ] and hypothermia) used in industrial fish processing were compared with asphyxia in air, which is used in traditional fishing, to evaluate the effects of these methods on the stress responses and the meat quality of the Amazon hybrid surubim, Pseudoplatystoma fasciatum female Leiarius marmoratus male. After the application, blood and behavioral indicators were evaluated. Following death by gill cutting, the fish were gutted, and analyses of muscle ph, rigor mortis, the exudation of the meat, and coloration were performed. Significantly higher levels of cortisol and glucose were exhibited by the fish subjected to asphyxia than by the fish stunned by either CO 2 or hypothermia. The fish subjected to asphyxia displayed decreasing muscle ph during the first 2 h after death and showed higher rates of rigor mortis after 3 h than the fish that were stunned by the other two methods. The asphyxia is a practice that exposes fish to suffering by causing an increase in stress responses, which affects meat quality. The immersion of fish in water and ice (hypothermia) for 5 min was more effective stunning method than the use of CO 2, resulting in higher loss of sensibility and greater welfare. The concept of welfare, as it applies to animal production, refers to the notion that animals must be properly treated during all stages of production, including at the time of slaughter. In fish, one of the critical steps that affect animal welfare and meat quality is the management of the fish when they are killed. If the procedures of this step are not applied properly, it can result in stress to the animals, compromising the quality of the meat that is obtained (Poli et al. 2005). Although fish constitute an important food for a large portion of the world, fish consumption is infrequent in Brazil, partially because of the nation s inadequate preservation and 1 Corresponding author. processing practices for fish. Studies have demonstrated that fish under stress experience a reduction in their muscle glycogen reserves and consequently have greater lactic acid buildup in their muscles than is observed under normal conditions. This phenomenon causes the ph of the meat from stressed fish to near neutral, which accelerates the action of muscle enzymes (auto-hydrolysis) and the growth of bacteria; this rapid degradation of the fish muscle produces early rigor mortis and changes in the texture and dripping of the meat, resulting in faster degradation and a shorter shelf life (Ashie et al. 1996; Roth et al. 2007). One of the techniques for minimizing the stress on fish is to use stunning methods that Copyright by the World Aquaculture Society

2 36 MACIEL ET AL. render the fish less sensitive to the stressful procedures that occur as part of the slaughtering process (Lambooij et al. 2002). Rapid stunning methods produce effective responses and contribute to the welfare of the fish (Marx et al. 1997). However, there is a growing market demand for less processed (in natura) fish, which are often derived from traditional fishing processes in which the fish are captured and exposed to open air prior to killing them; thus, these traditional processes entail a slow and painful procedure that does not maximize animal welfare. Currently, animal welfare has become a subject of study and concern in several countries, as scientists have recognized that animals are sentient beings and that the degree of animal welfare achieved is dependent not only on the physical health of animals but also on the behavioral and psychological aspects of animal treatment (Broom and Molento 2004; Oliveira and Galhardo 2007). The existing studies on animal welfare are mainly concentrated on mammals and birds destined for food production, and despite the existence of welfare-related regulatory legislation that includes all vertebrates, current knowledge about animal welfare in fish is still extremely scant (Braithwaite and Huntinford 2004). The use of the Amazon hybrid surubim, a cross between Pseudoplatystoma fasciatum female and Leiarius marmoratus male, has expanded in recent years, as the hybrid surubim s greater carcass yield and lower production costs have contributed to its rapid acceptance. At present, this hybrid is in high demand by many fish farms in Mato Grosso state and is commercialized throughout the country (Lopera-Barreto et al. 2011). In this study, two different methods of stunning (carbon dioxide [CO 2 ] and hypothermia) that are used in industrial fish processing were compared with the method of asphyxia in air that is used in traditional fishing to evaluate the effects of these methods on the stress response and meat quality of the Amazon hybrid surubim. Materials and Methods The Fish and Stunning Procedures The study was conducted in July 2011 in the fish-farming sector of the Experimental Farm of the School of Agronomy and Veterinary Medicine of the Federal University of Mato Grosso (FAMEV/UFMT). The Ethics Committee of Animal Research (CEPA/UFMT, /11-9 process number) approved the experimental protocol. A total of 80 Amazon hybrid surubim in the commercial size (average weight of 1.28 ± 0.45 kg and average standard length of ± 6.20 cm) were acquired from a commercial fish farm and stored in four net cages (each of 7.2 m 3 in volume) at a density of 20 fish/cage. During the adaptation period of 15 d, the fish remained in the net cage and were fed an extruded commercial feed for tropical omnivorous fish (28% crude protein, 6% crude fat, and 3000 kcal crude energy/kg) twice a day until satiation. Before performing the experiment, the fish were deprived of food for 24 h, in accordance with the normal routine of fish farming and transferred to cement tanks (each of 1 m 3 in volume) to be prepared for the treatment application. During experimental period, two stunning methods, immersion in water supersaturated with carbon dioxide (CO 2 ) (Treatment 1) and hypothermia (Treatment 2) were applied. For comparison, one group of fish was exposed to air (asphyxia), simulating the procedure that is often used by fishermen during traditional fishing (Treatment 3). A group of 10 non-stressed fish were quickly captured from the net cage with minimum disturbance and blood was drawn in less than 3 min to avoid possible handling effects in physiological stress indicators. This group served as a control to determine the basal physiological profiles and meat quality parameters for Amazon hybrid surubim. To implement the process of stunning with CO 2 gas, fish (n = 20) were transferred from cement tanks to a polyethylene box (containing 100 L of water), where they remained for 15 min in water supersaturated with CO 2 ;this water was obtained by diffusing CO 2 gas

3 EFFECTS OF STUNNING METHODS IN AMAZON HYBRID SURUBIM 37 Table 1. The responses of the Amazon hybrid surubim verified after the application of different stunning methods. Response Swimming Description Swimming movement Within the aquarium Balance The return of the fish to its initial position after positional modification Opercular beat Opercular movement Reaction to escape The escape reaction observed after the stimulation of fish with a needle in the tail area Outside the aquarium Ocular reflex The eye movement observed after the fish has been moved along its longitudinal axis Movement reflex The movement reflex observed after stimulation with a needle in the tail portion of the fish through the water until the ph of the water reached a mean value of 4.66, in accordance with the methods described by Van de Vis et al. (2003). To implement the process of hypothermia, the same polyethylene box was prepared with water and ice in a 1:1 ratio until it reached an average temperature of 3.4 C. The fish (n = 20) were then subjected to thermal shock for 5 min. To implement the process of asphyxia, the fish (n = 20) were captured from cement tanks and exposed to air for approximately 60 min. The duration for each method was established in preliminary experiments. Immediately after applying the treatments, the fish were removed from the polyethylene box and transferred to a glass aquarium for visual observations of their behavior and brain reflexes, based on the tests described by Kestin et al. (2002) to examine sensibility and brains function in exotic farmed fish. The description of each response assessed is summarized in Table 1. Only one person (the observer) performed the behavior monitoring for each fish inside and outside the aquarium and the same observer was maintained from beginning to the end of the experiment. The observations were rapidly made and were encoded in the following manner: a value of zero (0) was recorded for fish that did not respond to stimuli and/or demonstrated an absence of movement and reflexes, a value of one (1) was recorded for fish that exhibited responses considered to be lethargic and/or displayed abnormal movements and reflexes, and a value of two (2) was recorded for fish that had expressive responses to stimuli and/or normal movements and reflexes. Sampling After the behavioral observations, the fish were identified with a chip (AnimalTag AT01; Korth RFID Ltd., São Carlos, Brazil) and subjected to blood sampling by caudal puncture. They were then weighed (using a Toledo Scale 9094; São Bernardo do Campo, Brazil), measured (with an ichthyometer), killed by gill cutting, gutted, and analyzed for meat quality (measured by muscle ph, rigor mortis, meat color, and exudation of the filet). The measurement of post-mortem ph was performed at 0 (immediately after killing), 2, 3, 12, and 24 h after death, using an insertion electrode (Quimis Q400B; Diadema, Brazil) in the epaxial muscle of the fish. With the fish maintained in an acclimatized room (18 C), the rate of rigor mortis was evaluated at 0 (immediately after death), 1, 2, and 3 h after death by measuring the angle of sag (using a CARCI goniometer, Säo Paulo, Brazil) between the vertical axis of the table where the anterior portion of the fish was supported and the end of the tail (Fig. 1). A portion of the filet (65.40 ± g per portion) was taken from each fish, stored for 48 h at 4 C, and subsequently weighed to assess the water loss by exudation, which was calculated as the difference between the initial and final weights and expressed as a percentage of the initial weight, in accordance with the methods of Olivo et al. (2001). The color parameters of L* (lightness), a* (redness),

4 38 MACIEL ET AL. A B Figure 1. Measurement of the rate of rigor mortis of Amazon hybrid surubim submitted to different stunning methods (A). (B) Goniometer utilized to measure the angle of the bending degree during rigor mortis development. and b* (yellowness) were measured in the epaxial region of the filet with a manual colorimeter (Konica Minolta CR400; Tokyo, Japan) according to CIE L*a*b* (CIE 1976). In the blood samples, both hemoglobin (Labtest commercial kit; Labtest Diagnóstica S.A., Lagoa Santa, Brazil) and hematocrit (centrifugation for 5 min at 14,500 g in a Spin 1000 Microcentrifuge, Taiwan, China) were evaluated. Then, blood samples collected both with and without the anticoagulant EDTA (ethylenediaminetetraacetic acid) were centrifuged (HT CM-610 centrifuge; Biosystems, São José dos Pinhais, Brazil) for 10 min at 3300 g. The centrifugation induced plasma separation, allowing for the analysis of glucose and chloride (Labtest commercial kit), as well as separation of the serum for total protein analysis (Labtest commercial kit) and the quantification of cortisol by radioimmunoassay (Coat-A-Count RIA kit; Diagnostic Products Corporation, Los Angeles, CA, USA). During the experimental period, the water quality parameters were monitored in particular, and analyses of temperature and dissolved oxygen (dissolved oxygen meter YSI 55, Yellow Springs Instruments, Yellow Springs, OH, USA), ph (digital ph meter Quimis Q400BC), alkalinity (using the indicator solution of methyl orange), and total ammonia (Policontrol commercial kit; Policontrol Analytic Instruments, Diadema, Brazil) were performed. Statistical Analysis The samples were tested for differences using either analysis of variance (ANOVA) or nonparametric tests. To analyze the physiological and meat quality parameters, parametric statistics were applied using the SAS (Statistical Analysis System; SAS Institute Inc., Cary, NC, USA) software package, version 9.0. The results were expressed as the means ± SD, and comparisons among the means were performed using the Tukey test, with a significance level of The behavioral data were analyzed by the chi-squared nonparametric test with a significance level of 0.05, using the SAEG software package, version 9.1. Results The water quality parameters remained within the range considered to be suitable for tropical fish (Boyd 1982) (dissolved oxygen: 6.88 ± 1.25 mg/l, temperature: 27.3 ± 0.75 C, ph: 7.44 ± 1.05, alkalinity: ± 8.01 mg CaCO 3 /L, and total ammonia: 0.00 mg/l). Responses such as swimming reflex, balance, and reflex movements were significantly lethargic in fish stunned with CO 2 and absent in those subjected to hypothermia and asphyxia. In all treatments evaluated, the fish showed lethargic opercular movements. Normal eye movement was observed in fish that were either stunned with CO 2 or subjected to asphyxia, but these

5 EFFECTS OF STUNNING METHODS IN AMAZON HYBRID SURUBIM 39 eye movements were absent in fish subjected to hypothermia (Table 2). The serum cortisol and blood glucose were significantly higher in the fish subjected to asphyxia than in fish stunned with other methods or in fish of the control group (non-stressed fish), and these levels did not differ statistically between the group undergoing stunning with CO 2 and the group that experienced hypothermia. The cortisol levels did not differ among the group that experienced hypothermia and the control group. No significant differences were found in hemoglobin concentration, plasma chloride, and total protein among all of the groups examined (Table 3). The hematocrit levels were significantly higher compared with the control fish only for the fish exposed to hypothermia. Exudation and L* values for the filets of Amazon hybrid surubim did not differ among the tested treatments. The values of a* differed significantly between the methods tested and the control group (Table 4). There was interaction between treatments and times of measurement for the values of post-mortem muscle ph in Amazon hybrid surubim (Table 5). Immediately after killing, (0 h time point), the fish stunned with CO 2 and the fish subjected to hypothermia demonstrated muscle ph values significantly higher than the ph values of the asphyxiated and control groups, despite the lack of any significant difference in the sampling times for these groups. The highest value of muscle ph for the group subjected to asphyxia was observed 3 h after death. For the parameter of rigor mortis (Table 6), the control group displayed a significantly lower rigor index immediately after death (the 0 h time point) than the groups subjected to the other treatments. In the fish stunned by either CO 2 or hypothermia, the highest rigor indices were found at the 0 h time point and had decreased significantly by 1 h after death, with significantly lowest levels observed after 2 h. At this sampling time point (2 h), the fish subjected to asphyxia exhibited significantly higher index than the fish subjected to the other treatments. Table 2. The responses to stimuli of Amazon hybrid surubim submitted to different stunning methods. A value of zero (0) indicates fish that did not respond to stimuli and/or demonstrated an absence of movement and reflexes, a value of one (1) was recorded for fish that exhibited responses considered to be lethargic and/or displayed abnormal movements and reflexes, and a value of two (2) was recorded for fish that had expressive responses to stimuli and/or normal movements and reflexes. a,b Swimming movement Stunning methods CO 2 0 Bc 16 Aa 4 Ab Hypothermia 11 Aa 8 ABb 0 Bc Asphyxia 17 Aa 2 Bb 1 ABb Balance CO 2 3 Bb 14 Aa 2 Ab Hypothermia 15 Aa 5 Bb 0 Ac Asphyxia 14 Aa 5 Bb 0 Ac Reaction to escape CO 2 3 Aa 7 Aa 0 Aa Hypothermia 13 Aa 7 Aa 0 Aa Asphyxia 20 Aa 0 Aa 0 Aa Opercular beat CO 2 0 Bc 15 Aa 5 Ab Hypothermia 5 Ab 15 Aa 0 Bc Asphyxia 1 ABb 19 Aa 0 Bb Ocular reflex CO 2 1 Bb 4 Ab 13 Aa Hypothermia 14 Aa 6 Aa 0 Cb Asphyxia 2 Bb 11 Aa 7 Bab Movement reflex CO 2 8 Ba 6 Aa 0 Ab Hypothermia 19 Aa 1 Ab 0 Ab Asphyxia 17 ABa 1 Ab 1 Ab a Different uppercase letters in the columns indicate significant differences between treatments within the particular score. b Different lowercase letters in the lines indicate significant differences between the scores within the particular treatment.

6 40 MACIEL ET AL. Table 3. The blood parameters of Amazon hybrid surubim submitted to different stunning methods. a Stunning methods Control CO 2 Hypothermia Asphyxia Cortisol (ng/ml) ± 8.27 C ± B ± BC ± A Glucose (mg/dl) ± C ± B ± B ± A Hemoglobin (g/dl) 7.39 ± 1.41 A 7.04 ± 2.89 A 8.49 ± 2.58 A 7.61 ± 1.81 A Hematocrit (%) ± 4.21 B ± B ± A ± 4.63 B Chloride (meq/l) ± A ± A ± A ± A Total Protein (g/dl) 3.80 ± 0.56 A 4.81 ± 0.82 A 4.78 ± 0.85 A 4.66 ± 0.56 A a Different letters indicate significant differences between the treatments, as assessed by the Tukey test (P 0.05) (n = 10 for the control group and n = 20 for the other groups). Table 4. Color, as measured by the parameters L*, a*, and b*, and exudation in filets of Amazon hybrid surubim submitted to different stunning methods. a Stunning methods Control CO 2 Hypothermia Asphyxia Color L* ± 2.55 A ± 2.35 A ± 1.36 A ± 1.14 A a* 5.57 ± 1.09 A 3.15 ± 1.04 B 2.81 ± 0.79 B 2.95 ± 0.80 B b* 3.38 ± 1.04 B 4.37 ± 1.23 AB 5.22 ± 0.72 A 4.70 ± 0.82 AB Meat exudation (%) 1.00 ± 0.35 A 1.20 ± 0.42 A 1.01 ± 0.22 A 1.14 ± 0.39 A a Different letters indicate significant differences between treatments, as assessed by the Tukey test (P 0.05) (n = 10 for the control group and n = 20 for the other groups). Discussion Over a short period of time, the physiological changes caused by stress can be beneficial to the fish because these changes are adaptive and compensatory, allowing the animal to restore homeostasis. However, if the intensity of the stressor is high or exposure to the stressor is prolonged, the stress ceases to be adaptive and becomes harmful (Barton and Iwana 1991). In addition to the evaluation of the physical and physiological states of the fish, the behavioral aspects of these fish should be investigated as well, given that physiological stress is not always synonymous with suffering. From the viewpoint of animal welfare, therefore, any method of killing must incorporate a stunning procedure and unconsciousness before death (Pedrazzani et al. 2008). An important indicator of animal welfare that evaluates the humane killing of fish is the rate of sensitivity loss (Kestin et al. 2002). From the welfare perspective, the use of asphyxia appears to be directly related to the misunderstanding of fish sentience, as a similar method would never be countenanced for terrestrial vertebrates (Pedrazzani et al. 2008). In this study, although the fish exposed to air for approximately 60 min exhibited no deficiencies in their swimming reflexes, escape reactions, and reflex movements, these fish did display alterations in their original color, as they became grayish; moreover, these fish also displayed an expressive ocular reflex (vestibular-ocular reflex). These changes appear to demonstrate the pain and suffering of fish subjected to asphyxia. On the basis of neuroanatomy, neuropharmacology, and behavioral responses, there is evidence that fish can feel pain, which would make them sentient beings (Volpato 2009). According to Kestin et al. (2002), the ocular reflex response is based on tests used to evaluate both sensitivity and brain function. These authors found that trout, Oncorhynchus mykiss; salmon, Salmo salar; and sea bream, Sparus aurata, showed full awareness after exposure to air, with expressive ocular reflexes. The use of CO 2 was not a favorable stunning method for Amazon hybrid surubim because the observed responses were characterized by lethargic movements and expressive clinical signs, such as the ocular reflex, that indicate

7 EFFECTS OF STUNNING METHODS IN AMAZON HYBRID SURUBIM 41 Table 5. The post-mortem muscle ph values of Amazon hybrid surubim submitted to different stunning methods. a Stunning methods ph (h) Control CO 2 Hypothermia Asphyxia ± Ab 6.47 ± Aa 6.45 ± Aa 6.23 ± Bb ± Ab 6.44 ± Aa 6.41 ± Aab 6.24 ± Bb ± Bb 6.41 ± Aa 6.38 ± Aa 6.44 ± Aa ± Bb 6.30 ± Aa 6.31 ± Aa 6.26 ± ABa ± ABb 6.30 ± Aa 6.35 ± Aa 6.32 ± ABa a Different uppercase letters in the columns indicate significant differences between different sampling times within each treatment and different lowercase letters in the line indicate significant differences between treatments within each sampling time, as assessed by the Tukey test (P 0.05) (n = 10 for the control group and n = 20 for the other groups). Table 6. The rigor mortis development expressed as angle of the bending degree of Amazon hybrid surubim submitted to different stunning methods. a Stunning methods Rigor mortis (h) Control CO 2 Hypothermia Asphyxia ± 0.00 Bb ± 4.44 Aa ± 8.46 Aa ± 6.34 Aa ± 2.64 Aa ± 5.71 Bb ± 5.59 Bb ± 3.97 Bb ± 3.38 Abc ± 2.22 Bc ± 3.04 Bc ± 5.68 Aab ± 2.64 Bc ± 1.12 Bc ± 3.04 Bc ± 4.83 Ab a Different uppercase letters in the columns indicate significant differences between different sampling times within each treatment and different lowercase letters in the line indicate significant differences between treatments within each sampling time, as assessed by the Tukey test (P 0.05) (n = 10 for the control group and n = 20 for the other groups). potential suffering. The use of CO 2 is already prohibited as a method of commercial stunning in certain European countries; the trend in these countries is to utilize mechanical percussion and electric shock in the slaughtering process (Robb et al. 2000a; Lambooij et al. 2010). In Brazil, neither CO 2 nor electric shock is used in fish processing plants; instead, the most common stunning method utilizes water and ice with an average temperature of 0 C. In this study, hypothermia for 5 min caused a reduction in mobility, as well as lethargy in reactions of either escape or pain and the lack of an ocular reflex. However, Van de Vis et al. (2003) reported that the European eel, Anguilla anguilla, regained consciousness after immersion in water and ice despite its immobilization; thus, behavioral observations alone may not be sufficient for the evaluation of consciousness. The values obtained after leaving the fish without any stunning showed higher stress response. The results of this study indicate that approximately 60 min of asphyxia increased fish blood levels of cortisol and glucose by factors of 4.5 and 3.4, respectively, relative to the control, whereas for the stunning methods by either CO 2 or hypothermia, these quantities increased by approximately times relative to the control group. These results confirm that capture and exposure to air is a severe stressor. According to Robb and Kestin (2002), the time dependence of the effects produced by exposure to air vary based on the temperature of air and the species of fish examined, but asphyxia in any form appears to be a harmful practice for fish. In this study, stunning by either CO 2 or hypothermia resulted in cortisol and glucose levels for fish that were significantly lower than the same levels for fish subjected to asphyxia; thus, both CO 2 and hypothermia appear to be effective at reducing stress responses. This finding is consistent with the results of Ribas et al. (2007), who found significantly higher concentrations of cortisol and glucose in Solea senegalensis if the fish were killed by asphyxia than if the fish were subjected to hypothermia

8 42 MACIEL ET AL. (4 C). Poli et al. (2005) did not consider exposure to air to be a humane method of killing and reported that specimens of sea bass, Dicentrarchus labrax, subjected to asphyxia demonstrated high concentrations of plasma cortisol, glucose, and hematocrit. The high hematocrit levels found in this study in fish exposed to hypothermia may be the result a physiological response to stress of cold shock, that occurred when the fish had been acclimated to a specific water temperature on cement tanks (27.3 ± 0.75 C) and were subsequently exposed to a rapid decrease in temperature (3.4 C), during hypothermia stunning. The rapid decreases in water temperature may result in a cascade of physiological responses in fish and the elevation of the hematological indicators might be reflecting the higher need for oxygen to support this stressful condition (Donaldson et al. 2008). For fish, meat color can be an indicator of quality and is often the only variable considered by the consumer at the moment of purchase. The measurements of L*, a*, and b* provide information on the lightness, redness, and yellowness of the flesh and can be used to determine differences between treatment groups or to follow changes in these parameters during storage. The coloration in filets of Atlantic salmon was slightly more reddish in fish subjected to CO 2 stunning (a* values = 27.1 ± 0.1) compared with fish stunned in isoeugenol (a* values 26.4 ± 0.1) (Kiessling et al. 2004). Animals subjected to high levels of activity prior to death had changes in muscle color resulting in increase of L*, a*, and b* in meat (Robb et al. 2000b). According the authors, L*, a*, and b* system was lowered in flesh from rainbow trout, O. mykiss, that was anesthetized and had shown very low levels of activity before death. Similar results were obtained in our study, suggesting the possibility that all forms of management applied in this study led a decreased level of soluble muscle proteins in the flesh. Myoglobin is a protein complex of the muscle, formed by a globin polypeptide and a prosthetic group called heme (Fe), responsible for its color. The lactic acid formation and consequent ph drop post-mortem in situations of low animal welfare cause denaturation of globin and subsequent dissociation of oxygen from the heme group by changing the meat color and resulting in significant difference for the values of a* between the methods tested and the control group (unstressed animals). Stunning procedures, if applied correctly, can prevent stress induction by reducing the exhaustion of muscle energy and the lactic acid production of the fish, maintaining the equilibrium of muscle ph and decreasing the speed with which rigor mortis is established, resulting in improved physical properties of the meat (Conte 2004). The results of this study corroborate these statements because the control group was captured with minimal stress and killed immediately, indicating more energy reserves (glycogen) in the muscle, which results in a slower decrease in ph (minimal ph was reached 3 h after death). The fish subjected to asphyxia displayed decreasing muscle ph during the first 2 h after initiation due to the production of lactic acid by the intense muscular effort used by the animals during this procedure. Both stunning methods (CO 2 and hypothermia) ensured a management procedure that was less stressful to the fish, which led to little change in muscle ph for these treatments relative to the initial ph measured. The findings of this are similar to the results of Albuquerque et al. (2004), who found no differences in the ph values (which ranged between 6.18 and 6.77) for Nile tilapia, Oreochromis niloticus, stunned with CO 2 and stored on ice and concluded that muscle composition characteristics were not altered as a result of the system used for stunning. However, in European sea bass, stunning either with CO 2 or via asphyxiation resulted in a reduction of the muscle ph in the fish, as the ph values were significantly lower than the initial ph values by 9 h after death. (Acerete et al. 2009). The management of fish during slaughter or the way that the capture is performed may interfere with rigor mortis determinations. Fish subjected to greater stress during capture deplete their glycogen reserves, leading to

9 EFFECTS OF STUNNING METHODS IN AMAZON HYBRID SURUBIM 43 large decreases in ATP and shortening the time required before muscular rigor mortis occurs (Poli et al. 2005). In turbot, Scophthalmus maximus, stunning through bleeding and prolonged electrical stimulation led to a decline in muscle ph; in addition, these stunning processes produced the onset of rigor mortis in only 24 h, which was early relative to fish that did not undergo a stunning process, as the control fish exhibited rigor mortis 48 h after death (Roth et al. 2007). Asphyxia and hypothermia were highly stressful for sea bass and sea bream, S. aurata, and accelerated the establishment of rigor mortis (Bagni et al. 2007). In this study, fish subjected to asphyxia presented lower rigor mortis index at 1 h postslaughter; the rapid establishment of rigor may have been caused by stress, which can greatly influence fish post-mortem biochemical processes, mostly the anaerobic glycolysis and ATP degradation rate. This can markedly influence the onset and release of rigor mortis, which in turn largely determines the involution rate of fish freshness (Poli et al. 2005). The duration of the pre-rigor phase depends on ATP and glycogen reserves at slaughter. In this study, this finding provides a rationale for the observation that rigor mortis was established later than 1 h post-kill in fish of the control group and fish subjected to stunning with either CO 2 or hypothermia, for which the lowest indices were measured at 3 h post-kill. Acerete et al. (2009) tested the same treatments examined in the present study using European sea bass and found similar results, with higher values of rigor mortis occurring between 2 and 24 h for fish treated with hypothermia and at 72 h for fish desensitized with CO 2 ; these values can be compared with the rigor mortis results for the group subjected to asphyxia, which exhibited significantly higher rigor after only 2 h with a significant decrease in rigor mortis between 72 and 96 h post-kill. The results of this study support the conclusion that asphyxia may be considered to be a practice that exposes the fish to suffering, provoking an increase in stress response of these fish and thereby anticipating the establishment of rigor mortis. Immersion of fish in water and ice (hypothermia) for 5 min was demonstrated to be a more effective stunning method than the use of CO 2, as the hypothermia-based stunning resulted in higher loss of sensibility, providing an increase in the level of welfare. Acknowledgments The authors would like to thank the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq) for financial aid (process no /2010-9) and a scholarship provided to Elayna C. S. Maciel (process no /2009-4), the Delicious Fish Farm (Primavera do Norte, Mato Grosso, Brazil) for providing the fish used in the experiment, and the professors Gerusa Corrêa, Cláudio Vieira de Araújo, and Joadil Gonçalves de Abreu for their assistance with the statistical analyses. Literature Cited Acerete, L., L. Reig, D. Alvarez, R. Flos, and L. Tort Comparison of two stunning/slaughtering methods on stress response and quality indicators of European sea bass (Dicentrarchus labrax). Aquaculture 287: Albuquerque, W. F., J. F. F. Zapata, and R. Santos Almeida Estado de frescor, textura e composição muscular da tilápia do Nilo (Oreochromis niloticus) abatidos com dióxido de carbono e armazenados em gelo. Revista Ciência Agronômica 35: Ashie, I. N. A., J. P. Smith, and B. K. Simpson Spoilage and shelf-life extension of fresh fish and shellfish. Critical Reviews in Food Science and Nutrition 36: Bagni, M., C. Civitareale, A. Priori, A. Ballerini, M. Finoia, and G. Brambilla Procedures preslaughter crowding stress and killing affecting quality and welfare in sea bass (Dicentrarchus labrax)andsea bream (Sparus aurata). Aquaculture 267: Barton, B. A. and G. K. Iwana Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annual Review of Fish Diseases 1:3 26. Boyd, C. E Water quality management for pond fish culture. Elsevier Scientific Publishing Company, Amsterdam, the Netherlands. Braithwaite, V. A. and F. A. Huntinford Fish and welfare: do fish have capacity for pain perception and suffering? Animal Welfare 13:87 92.

10 44 MACIEL ET AL. Broom, D. M. and C. F. M. Molento Bem-estar animal: conceito e questões relacionadas. Archives of Veterinary Science 9:1 11. CIE Official recommendations on uniform color space, color difference equations and metric color terms. Supplement no. 2 to CIE Publication no. 15, Colorimetry. Commission International de l Éclairage, Paris, France. Conte, F. S Stress and welfare of culture fish. Applied Animal Behaviour Science 86: Donaldson, M. R., S. J. Cooke, D. A. Patterson, and J. S. MacDonald Cold shock and fish. Journal of Fish Biology 73: Kestin, S. C., J. W. Van de Vis, and D. H. F. Roob Protocol for assessing brain function in fish and the effectiveness of methods used to stun and kill them. Veterinary Record 150: Kiessling, A., M. Espe, K. Ruohnen, andt. Mørkøre Texture, gaping and color of fresh and frozen Atlantic salmon flesh as affected by preslaughter iso-eugenol or CO 2 anaesthesia. Aquaculture 236: Lambooij, E., J. W. Van de Vis, R. J. Kloosterboer, and C. Pieterse Evaluation of captive needle stunning of farmed eel (Anguilla anguilla L.) suitability for humane slaughter. Aquaculture 212: Lambooij, E., E. Grimsb, J. W. Van de Vis, H. G. M. Reimert, R. Nortvedt, andb.roth Percussion and electrical stunning of salmon (Salmo salar) after dewatering and subsequent effect on brain and heart activities. Aquaculture 300: Lopera-Barreto, M. N., R. P. Ribeiro, J. A. Povh, L. D. V. Mendez, and A. R. Poveda-Parra Visão da aquicultura no Brasil. Pages in M. N. Lopera-Barreto, R. P. Ribeiro, J. A. Povh, L. D. V. Mendez, and A. R. Poveda-Parra, editors. Produção de organismos aquáticos: Uma visão geral do Brasil e no Mundo. Agrolivros, Guaíba, Brazil. Marx, H., B. Brunner, W. Weinzierl, R. Hoffmann, and A. Stolle Methods of stunning freshwater fish: impact on meat quality and aspects of animal welfare. European Food Research and Technology 204: Oliveira, R. F. and L. Galhardo Sobre a aplicação do conceito de bem-estar a peixes teleósteos e implicações para piscicultura. Revista Brasileira de Zootecnia 36: Olivo, R., A. Soares, E. I. Ida, and M. Shimokomaki Dietary vitamin E inhibits poultry PSE and improves meat functional proprieties. Journal of Food Biochemistry 25: Pedrazzani, A. S., A. N. Ostrenky, P. C. F. Carneiro, M. V. Gayer, andc.f.m.molento Opinião pública e educação sobre abate humanitário de peixes no município de Araucária, Paraná. Ciência Animal Brasileira 9: Poli, M. B., G. Parisi, F. Scappini, and G. Zampacavallo Fish welfare and quality as affected by preslaughter and slaughter management. Aquaculture International 13: Ribas, L., R. Flos, L. Reig, S. MacKenzie, B. A. Barton, and L. Tort Comparison of methods for anaesthetizing Senegal sole (Solea senegalensis) before slaughter: stress response and final product quality. Aquaculture 269: Robb, F. H. D. and S. C. Kestin Methods used to kill fish: observations and literature reviewed. Animal Welfare 11: Robb, D. H. F., S. B. Wotton, J. L. Mckinstry, N. K. Sorensen, and S. C. Kestin. 2000a. Commercial slaughter methods used on Atlantic salmon: determination of the onset of brain failure by eletroencephalography. Veterinary Record 9: Robb, D. H. F., S. C. Kestin, and P. D. Warriss. 2000b. Muscle activity at slaughter: I. Changes in flesh color and gaping in rainbow trout. Aquaculture 182: Roth, B., A. Imsland, S. Gunnarsson, A. Foss, and R. Schelvis-Smit Slaughter quality and rigor contraction in farmed tubot (Scophthalmus maximus); a comparison between different stunning methods. Aquaculture 272: Van de Vis, H., S. C. Kestin, D. Robb, J. Oehlenschläger, B. Lambooij, W. Münkner, H. Kuhlmann, K. Kloosterboer, M. Tejada, A. Huidobro, H. Ottera, B. Roth, N. K. Sørensen, L. Akse, H. Byrne, and P. Nesvadba Is humane slaughter of fish possible for industry? Aquaculture Research 34: Volpato, G. L Challenges in assessing fish welfare. Institute for Laboratory Animal Research Journal 50: