ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Transcription

1 ENVIRONMENT, WELL-BEING, AND BEHAVIOR Cone restraining and head-only electrical stunning in broilers: Effects on physiological responses and meat quality E. Lambooij, 1 H. G. M. Reimert, M. T. W. Verhoeven, and V. A. Hindle Livestock Research of Wageningen UR, PO Box 65, 8200 AB Lelystad, the Netherlands INTRODUCTION There are immense variations in the definition of acceptable animal welfare conditions due to cultural, philosophical, or religious differences between individuals. Besides ethical aspects, humane treatment of animals in the production chain is an important component of the quality and safety of meat. Animals should be protected from any anthropogenic excitement, pain or suffering during transport, lairaging, restraint, stunning, slaughter, or killing (EFSA AHAW/04 027; EFSA, 2004). Consumers concerns for animal welfare have important implications for the future consumption of meat and for producers and retailers of animal-based food products within the European Union. ABSTRACT Two experiments were conducted to evaluate a new electrical stunning system for broilers. The objective of the first experiment was to evaluate the behavioral, neural, and physiological responses of 27 broilers after head-only electrical stunning while their bodies were restrained in cone-shaped holders. In the second experiment, quality of meat from 30 broilers after headonly electrical stunning in a cone-shaped restrainer was compared with that from 30 broilers stunned in a conventional water bath. Broilers were restrained in the cone with their heads positioned to facilitate a correct stun, followed by a neck cut by hand. After stunning, each broiler displayed a tonic phase, followed by minimal brain activity during bleeding. On average, heart rate was 258 ± 51 beats/min before stunning. The heart was observed to malfunction after cutting. According to the correlation dimension analyses, the score remained low. Within a confidence limit of 95%, taking into account the number of birds with a reliable electroencephalogram (n = 27), the chance of an effective stun and exsanguination with all broilers lies between 0.90 and 1.00 using a sinusoidal AC current of 264 ± 29 ma (~130 V). After a brief learning period, operators were able to easily position the broilers in the cone in a commercial setting. The ph after chilling was 0.5 units lower (P < 0.05) in the head-only stunned group compared with the group stunned in a conventional water bath. After head-only stunning, 60% of breast fillets showed no blood splashes and 3% showed severe blood splashes compared with 20 and 27% after conventional water bath stunning. No differences in temperature and color were observed between the 2 groups. It is concluded that broilers could be restrained in a cone, followed by correct head-only stunning, neck-cutting, and unconscious shackling afterward under laboratory and commercial slaughterhouse conditions. When this procedure was used, meat quality was better compared with broilers stunned in the conventional water bath. Key words: broiler, cone-shaped restrainer, electrical stunning, meat quality, welfare 2014 Poultry Science 93 : Poultry Science Association Inc. Received May 15, Accepted November 24, Corresponding author: bert.lambooij@wur.nl Prior to slaughter, broilers are subjected to an array of stressful events on their last day of life (Nijdam et al., 2004, 2005). Feed withdrawal is the first stressful occurrence before catching and placing in crates or containers (Nijdam et al., 2004), followed by transportation (Kettlewell and Hallworth, 1990). Broilers are transported alive in crates from the farm of origin to the processing plant were they are held until required for slaughter. This process of (manual) catching, crating, and transportation leads to stress and physical damage (i.e., bruising, dislocations, and bone breakage; Nijdam et al., 2005). A large proportion of the stunning, killing, and slaughter process has been automated. However, transfer of the broilers from the transport crates to the shackles remains a manual exercise. This physically demanding exercise involves lifting live, struggling broilers of 2 to 3 kg from waist to shoulder height by the slaughterhouse personnel to hang them in a moving shackle line that conveys them through the water bath stunner (Kettlewell and Hallworth, 1990). Inversion, hanging upside down, is also a physiologically ab- 512

2 RESTRAINING AND STUNNING OF BROILERS normal posture for broilers. Handling, inversion, and shackling are all traumatic and stressful experiences for animals (Shields and Raj, 2010). The rate of acidification after death is controlled by the degree of hormonal and contractile status of muscles immediately before and during slaughter (Monin and Ouali, 1992). Color and water-binding capacity are determined by protein denaturation, caused by a rapid acidification. It is assumed that stress before slaughter leads to an increased breakdown of glycogen and a greater depletion of the energy store. In broilers, this results in pale, soft, and exudative (PSE) meat. This explanation must not be generalized, as the physiological response to stressors from the environment is partly influenced by the genotype of the animal (Nicol and Scott, 1990). In well-fed, rested animals, meat ph falls to about 5.5 to 5.8 after slaughter (Hillebrand, 1993). Comparative experiments involving a cone-shaped restrainer, standing, and shackling have also been performed (Hillebrand et al., 1996). Use of the coneshaped restrainer in combination with captive bolt stunning resulted in less (P < 0.01) hemorrhaging of breast and thigh muscles compared with whole-body electrical stunning in shackles and in similar levels of hemorrhaging compared with head-only electrical stunning using a cone-shaped restrainer. Head-only electrically stunned broilers placed in a cone displayed fewer convulsions than broilers stunned with a captive bolt. Hillebrand et al. (1996) considered that placing broilers in a cone during stunning and exsanguination limited convulsions, making this, in their view, more aesthetically acceptable. However, this design still involves inversion and the authors did not comment further on the welfare aspects of this restraining method, other than to speculate that the reduction in hemorrhaging is probably related to fewer convulsions. It is likely that broilers are easier to place in a cone than in shackles. This is a welcome development because it has been recognized that the inverted shackling of live broilers is detrimental to their welfare (Debut et al., 2005; Bedanova et al., 2007). A recent survey of poultry slaughterhouses in the Netherlands showed serious concerns about the welfare of broilers and the effectiveness of conventional stunning methods (Hindle et al., 2010). Present water bath stunning equipment in slaughterhouses does not allow accurate and rapid measurement and control of individual current supply and bird impedance. The actual current that a bird receives during electrical water bath stunning will vary according to the number of broilers in the water bath at any one time. Individual variability in impedance dictates whether or not some broilers are effectively stunned. It is stated that any new electrical stunning systems should be tested under abattoir conditions (EFSA, 2012). It would be unnecessary to perform electroencephalogram (EEG) measurements to verify adequate performance if a system could measure and guarantee adequate current supply to each bird. In a previous study (Lambooij et al., 2010), it was concluded that broilers may be insensible and unconscious after head-only electrical stunning with pin-electrodes using an average current of 190 ± 30 ma for 0.5 s. For practical implementation, a set current of 250 ma (average + 2*SD) was recommended to overcome individual differences in resistance, and to prevent recovery the stun should be followed by an immediate neck cut. Because carcass quality was only slightly compromised, such equipment should be further developed for practical application and commercial use. The present experiment was conducted in a laboratory setting to evaluate behavioral, neural, and physiological responses of broilers after placing in a cone-shaped restrainer, followed by head-only electrical stunning and neck cutting. A second experiment was conducted in a commercial setting to compare the quality of meat from broilers placed in a cone-shaped restrainer, followed by head-only electrical stunning and a neck cut with that from broilers stunned in a conventional water bath. Analyses of ph and meat color were performed on fillets during storage, together with scoring for blood splashes in fillets and legs. MATERIALS AND METHODS Experiment 1: Behavioral, Neural, and Physiological Responses 513 Birds. Twenty-seven Ross broilers (2.2 ± 0.2 kg) were randomly selected from flocks that were delivered to a slaughter plant in the Netherlands. Broilers were transported in standard crates from the slaughter plant to the experimental facilities, where the effectiveness of the new head-only stunning system was tested. Prior to delivery at the slaughterhouse, the broilers had been fasted for a period of 6 h. Experimental Design. Each broiler was placed in a specially developed cone-shaped restrainer with the legs free between the rods of the shackle (Figure 1). The broiler was stunned using a head-only electrical stunner via pin electrodes on both sides of the head. The stunning current ran through the head for 3 s. A neck cut was performed on average 14 ± 2 s after stunning to let the broiler bleed out. Before stunning, each individual broiler was equipped with EEG and electrocardiogram (ECG) electrodes. The EEG electrodes (10 mm long and 1.5 mm in diameter; 55% silver, 21% copper, 24% zinc) were placed by pressing them through the skin and skull: one electrode 0.3 cm to the right and one electrode 0.3 cm to the left of the sagittal suture and 0.5 cm left and right of an imaginary transverse line at the caudal margin of the eyes. The ECG electrodes (35 mm long and 1.5 mm in diameter, with the same metal composition) were placed subcutaneously at the left and right side of the breast directly under the wing. The ground electrode for the EEG and the ECG was placed subcutaneously lateral to the right leg. Both EEG and ECG were recorded from 30 s before to 2 min after stunning. The response of each bird to a

3 514 Lambooij et al. pain stimulus (comb pinching) was observed at 30 s and at 1 and 2 min after the stun to assess unconsciousness. The data recorder used was a DI 720 data-recording module with a WinDaq Waveform browser (Dataq Instruments, Akron, OH). The EEG and ECG recordings were analyzed for changes in the waveforms, frequency, and suppression afterward. Prior to stunning, the cone with the instrumented bird was transported to the stunning unit. The EEG and ECG wires were shortly connected to handheld switch box, which was connected with the measuring equipment by a covered wire of 5 m. The head of the broiler was fixated by electrodes on both sides of the head outside the cone (Tiel Engineering BV, Tiel, the Netherlands; Figure 1). The measured electrical current averaged 264 ± 29 ma (~130 V, 50 Hz, AC) and was delivered for 3 s. The current was stabilized in relation to the measured impedance. The impedance changed with each bird and varied during the stunning process. The behavior of the birds was monitored for the occurrence of tonic cramp (severe tension of all muscles), clonic cramps (uncontrolled severe muscle contractions), exhaustion (muscle flaccidity), and recovery (wake up with righting). Experiment 2: Meat Quality Birds. A prototype of the specially developed coneshaped restrainer and electrical head-only stunner with pin-electrodes on both sides of the head was placed in a slaughterhouse in the Netherlands. In the second experiment, a random selection of 60 Ross 308 broilers (average weight 1.9 kg) was taken from a single flock delivered to the slaughterhouse. Experimental Design. Half (n = 30) of the group was placed in the cones of the head-only stunner with electrical parameters set at 0.5 s at 130 V, 1 s at 110 V, followed by 1.5 s at 90 V and 50 Hz (~271 ± 81, 358 ± 72, 311 ± 70 ma). Because the area of electrodes was Ethics The experiments were approved beforehand by the Ethical Committee of the Animal Sciences Group of Wageningen UR (experimental code ). Statistical Analyses Each broiler represents an experimental unit with a probability P that the broiler is unconscious during a general epileptiform insult. For n broilers, which are treated independently, the number x, which are unconscious, is binomially distributed with total n and probability P. A CI can be calculated for probability P based on a relationship between the binomial and β distribution. Therefore, the number of effective stuns follows a Figure 1. The sequence of the cone and head-only stun process is as follows: 1) broilers placed in a cone, 2) with heads positioned to allow a correct head stun (left photo), 3) head-only stunning (right photo), 4) followed by neck cutting of blood vessels by hand, and 5) automatic shackling. Color version available in the online PDF. increased, the current settings were adapted to prevent too high currents. The remaining broilers were used as controls stunned in the conventional water bath of the slaughterhouse with electrical parameters set at 140 ma, 399 Hz for 12 s, according the European Council (2009) regulation. After stunning, the broilers were slaughtered according to the regular procedure of the slaughterhouse. The time between stunning and cutting was 26 to 28 s in the cone head-only stunner and 6 s in the water bath stunner. Temperature, ph, and color in breast muscle (pectoralis major) were measured after evisceration and chilling. The ph was also measured at 1 d postmortem and color was also measured 2, 3, and 6 d postmortem. The ph was measured with a knick ph meter connected to an Ingold electrode (Xerolyt, type LOT 406-M6-DXK-S7/25), and temperature was measured with a Pt 1000 electrode. Primary color coordinates L*, a*, and b* were assessed [on CIE (Commission Internationale de l Eclairage) LAB space] in the breast muscle (ventral side of pectoralis major; presented as a mean value of the measurements in 2 locations equally distributed over the muscle) using a Spectrophotometer Minolta CM-525i (Minolta, Osaka, Japan; light source D65). Chromatic values are given in the CIE L*a*b* system. The L* value is a measure for lightness. The a* and b* are the chromaticity coordinates. The a* and b* indicate color directions: +a* indicates a tendency toward red, a* toward green, +b* toward yellow, and b* toward blue. Hemorrhages in breast (dorsal side of pectoralis major and minor) and left and right thigh muscles (medial side) were quantified using a visual grading system (Veerkamp et al., 1987). The classification was performed independently by 2 observers. For classification, a threshold model consisting of a discontinuous 5-point scale with 4 cut-off points was used. Cut-off points were formed by photographs of breast and thigh muscles showing a particular severity of hemorrhaging: class 1 represents hemorrhage-free muscles and class 5 represents muscles with numerous and severe hemorrhages (Lambooij et al., 1999).

4 RESTRAINING AND STUNNING OF BROILERS 515 binomial distribution. A 95% confidence limit on the probability for an effective stun and exsanguination can be obtained by means of a well-known relationship with the β distribution (Johnson and Kotz, 1969). The EEG traces were subjected to correlation dimension (CD) analysis. This analysis provides a nonlinear (fractal) measure of signal complexity (for algorithm see van den Broek et al., 2005). Correlation dimension analysis is a relatively new technique that has been customized to measure depth of anesthesia in humans (van den Broek, 2003). The small amplitude, high frequency (awake) EEG signal is more complex than the large amplitude, low frequency (unconscious) EEG signal. Therefore, high CD values are taken to indicate awareness, whereas low values indicate a state of unconsciousness. It is suggested that chickens are awake, drowsy and sleep at a CD score of 7, 6.6, and 6, respectively (Coenen and van den Broek, 2005). The number is 18 out of 27 animals due to failure of the analyses caused by the artifacts of neck cutting. The effect of stunning procedure on ph and meat color parameters were analyzed according to 2-sided Student s t-test of sample populations with similar variance using the P-value. displayed wing flapping in the cone, which was reduced in number and limited during bleeding out. After stunning, all broilers displayed a tonic phase followed by minimal brain activity during bleeding out (Figure 2). No responses to pain stimuli were detected on the EEG, although there was a suspicion of a behavioral response in one bird. According to the CD analyses (Figure 3), the score remained low; however, there was a short increase after cutting. Within a confidence limit of 95%, taking into account the number of animals with a reliable EEG (n = 27), the chance of an effective stun and exsanguination with all broilers lies between 0.90 and 1.00 using a sinusoidal AC current of 264 ± 29 ma (~130 V). Average heart rate was 258 ± 51 beats/min before stunning. After stunning, the heart showed fibrillation for 9 ± 2 s, and 30 s after stunning the heart rate was RESULTS Experiment 1: Behavioral, Neural, and Physiological Responses Broilers could easily be placed in the cone and have their heads positioned to allow a correct head stun (Figure 1) followed by a neck cut by hand. After the cut, all broilers were shackled automatically out of the cone-shaped restrainer. During stunning, the broilers Figure 3. Correlation dimension analysis scores (±SD) before and after head-only stunning with a current of 264 ± 29 ma for 3 s with neck cutting within on average 14 ± 2 s. Figure 2. Electroencephalogram (EEG; upper trace) and electrocardiogram (ECG; lower trace) recordings before and after stunning. Prior to stunning, a normal EEG and ECG rhythm can be observed (left part of the figure). After stunning (middle part of the figure), a tonic phase is followed by minimal brain activity and heart fibrillation is followed by malfunction of the heart on the ECG (right part of the figure). An artifact is seen (middle part of the figure) caused by cutting 11 s after stunning. Color version available in the online PDF.

5 516 Lambooij et al. Table 1. Results (mean ± SD) of the meat quality parameters (temperature, ph, and color) measured in the fillets after chilling, 1 and 4 d postmortem (p.m.) in carcasses of broilers stunned in a conventional water bath (140 ma, 399 Hz for 12 s) and head only in a cone-shaped restrainer (0.5 s, 130 V; 1 s, 110 V followed by 1.5 s, 90 V and 50 Hz) (~271 ± 81, 358 ± 72, 311 ± 70 ma) Item Water bath Head only P-value Carcasses (n) Temperature after chilling ( C) 16.8 ± ± ph after chilling 6.47 ± ± 0.15 <0.001 ph 1 d p.m ± ± L* after chilling 54.3 ± ± a* after chilling 4.5 ± ± b* after chilling 11.1 ± ± 1.6 <0.001 L* 1 d p.m ± ± a* 1 d p.m. 5.1 ± ± b* 1 d p.m ± ± L* 2 d p.m ± ± a* 2 d p.m. 5.1 ± ± b* 2 d p.m ± ± L* 3 d p.m ± ± a* 3 d p.m. 5.6 ± ± b* 3 d p.m ± ± L* 6 d p.m ± ± a* 6 d p.m. 5.4 ± ± b* 6 d p.m ± ± The L* value is a measure for lightness. The a* and b* are the chromacity coordinates. 262 ± 65 beats/min. On the ECG, the heart was observed to malfunction (abnormal PQRST characteristic) after cutting. Experiment 2: Meat Quality After a brief learning period, the operators were capable of easily positioning the broilers in the cone at a line speed of 7,000 broilers/h (Figure 1). Mal-stunning occurred in <0.03% of the stuns, which was detected by the equipment by low current measurement (<240 ma). When mal-stunning occurred, the broilers were taken from the line before cutting. During stunning almost all broilers displayed wing flapping, which was restricted by the cone. The results of the meat quality parameters are presented in Tables 1 and 2. Breast meat ph and L*a*b* values after chilling were lower (P < 0.05) in the headonly group (Table 1). The percentage of fillets showing no blood splashes was 60% in head-only stunned broilers compared with 20% in the conventional water bath. The percentage of fillets showing severe blood splashes was 3% in head-only stunned broilers compared with 27% in broilers stunned in the conventional water bath. Blood splashing in the legs was more severe in broilers stunned in the conventional water bath (Table 2). DISCUSSION It has long been recognized that the practice of hanging live broilers upside down in shackles before stunning causes unnecessary pain and suffering (Sparrey and Kettlewell, 1994; Debut et al., 2005; Bedanova et al., 2007). Others have accentuated the undesirable and stressful aspects of handling practices with poultry that often result in physical injury, mortalities, and downgrading of carcasses (Knowles and Broom, 1990; Nicol and Scott, 1990). Shackling is considered to be a traumatic experience for broilers, and its effect is dependent upon duration of shackling (Bedanova et al., 2007). It was concluded that the optimum shackling period lay between 12 to 60 s to reduce the risk of a major stress response in broilers with adverse consequences for meat quality. According to Bedanova et al. (2007), a time lapse between hanging in the shackles and stunning is unavoidable. Average time between hanging and stunning ranges from 3 min (broilers) to 6 min (turkeys) according to Sparrey and Kettlewell (1994). European Council directive 1099/2009 (European Council, 2009) on the protection of animals at the time of slaughter or killing requires that poultry are sufficiently relaxed in the shackles to facilitate an effective stun. In our experiments, it was monitored that the broilers displayed calm behavior when placed in the cone. After stunning and sticking, they were automatically shackled while unconscious. The procedure of cone restraining may re- Table 2. Percentage of blood splashes in the fillets and legs according to a scale of 1 to 5 (1 = none; 2 3 = minor; 4 5 = severe) in carcasses of broilers stunned in a water bath (n = 30) or head only in a cone (n = 30) Item None (%) Minor (%) Severe (%) Water bath Fillets Left legs Right legs Head only Fillets Left legs Right legs

6 RESTRAINING AND STUNNING OF BROILERS 517 duce the traumatic experience and its effect on meat quality (Table 1). When a cone restrainer prototype using head-only stunning was placed in a commercial slaughterhouse, 6 operators were trained and found capable of placing the broilers in the cones at a line speed of 7,000 birds per hour. The operators informed us that this required less effort than putting the birds on conventional shackles at a similar speed. The procedure of positioning the birds correctly in the cone-shaped restrainer, however, requires a separate handling method and motivation to perform correctly. Alternative methods to the water bath stunner include head-only stunning (Hillebrand et al., 1996; Lambooij et al., 2010; Lines et al., 2011a,b) and head-cloaca stunning (Lambooij et al., 2012) where proper restraint of the bird s head remains a challenge. When the broilers were placed in the cone during our experiments, they presented their head immediately in the right position to stun. Therefore, the upside-down position in the cone is maintained for as short a time as possible in the given situation in the slaughterhouse. None of these alternative methods, including the present cone restrainer, have as yet resolved the need to handle and invert the birds. Only controlled atmosphere stunning systems avoid this problem, because gas stunning can be applied while the birds remain in the transport crates (Gerritzen et al., 2013), but controlled atmosphere stunning systems are relatively expensive and not always feasible to the slaughterhouse facility. In a previous experiment on the basis of a visual EEG analysis the duration of unconsciousness was scored from 30 to 65 s after stunning, moreover only 2 broilers responded at 30 s after a 0.5 electrical stun to a noxious stimulus and none after a 3 or 5 s stun (Lambooij et al., 2012). Combining head-only stunning with exsanguination has a synergistic effect on the release of glutamate and aspartate, which increases the duration of unconsciousness. Sticking following a stun should be carried out as promptly as possible when using head-only stunning because the duration to loss of brain responsiveness varies for different species following sticking (Cook et al., 1996). It can be recommended to start exsanguination immediately after stunning; Gregory and Wotton (1990) suggested neck cutting by severing all the major blood vessels in the neck within 10 to 15 s from the end of the stun to prevent return to consciousness. In our laboratory study, the neck cutting was performed within 15 s. On the basis of CD analyses (Figure 3), early neck cutting may prevent recovery because high CD values are taken to indicate awareness and low values indicate a state of unconsciousness. Studies with chickens suggested that they were asleep at a score of 6 (Coenen and van den Broek, 2005). Early lower ph and L*a*b* values were seen after cone restraining and head-only stunning in our experiment (Table 1), compared with water bath stunning, which might be due to the longer time between stunning and sticking. The same phenomenon was observed after head-only stunning and was suggested to be due to wing flapping (Lambooij et al., 2010). Altered rates of postmortem glycolysis, induced by other stunning procedures such as gas stunning, have been found to result in changes in meat quality traits such as color and texture (Raj, 1994). Compared with high frequency electrical stunning, argon killing resulted in less bleed-out and this may contribute to increased carcass downgrading. Probably due to the convulsions that occur during the stun/kill operation with argon, meat ph is already lower and lactate higher than with the stunning operations at which no or fewer convulsions occur. After 24 h of chilling, no differences were observed between stunning methods. A comparable process might be the case in the present experiment under commercial conditions, where the broilers were observed to wing flap in the cone during stunning. Hemorrhaging results in a decrease in quantity (trimming) and quality of poultry products, and hence causes financial loss to the poultry industry. Therefore, hemorrhaging, particularly regarding valuable breast meat, is considered a major quality defect. Some hemorrhages are associated with hypercontracted and disrupted muscle fibers, indicating severe muscular strain. In our study, approximately 80% of the fillets showed no blood splashes and the remaining 20% did not show any severe blood splashes. This was an improvement on the level of blood splashing observed compared with the control birds stunned in the conventional water bath. In earlier laboratory experiments, more than 67% of the broiler carcasses stunned at 50 Hz displayed blood spots, 85% of which were from broilers that died during the stunning procedure. Stunning at 400 Hz produced 35% of carcasses with blood spots of which 81% were from broilers that died during the stunning procedure (Hindle et al., 2010). Stunning at 1,000 Hz resulted in 18% of carcasses with blood spots of which 88% were from broilers that died during the stunning procedure. Although there appears to be a tendency toward fewer incidences of blood splashing as frequency increases, the stun to kill policy does not appear to improve the situation (Hindle et al., 2010). During head-only stunning (Lambooij et al., 2012) broilers showed wing flapping, which was sometimes intensive. Wing flapping may cause broken wings and induce hemorrhaging. This is a disadvantage of head-only stunning. However, the present study showed that the cone may prevent excessive wing flapping. In conclusion, both in a laboratory setting and in a commercial slaughterhouse, operators could restrain broilers in a cone for correct head-only stunning, followed by neck-cutting and shackling of unconscious broilers. The applied current should be 264 ± 29 ma (~130 V; sinusoidal AC) and neck cutting should be performed as soon as possible. In addition, using the combined procedure of restraining broilers in a cone followed by head-only stunning and neck cutting in a com-

7 518 Lambooij et al. mercial slaughterhouse, 60% of the breast fillets were free of blood splashes after slaughter, whereas after water bath stunning the percentage was 20%. ACKNOWLEDGMENTS This research was performed for and sponsored by the Dutch Ministry for Economic Affairs. Thanks are extended to managers of Tiel Engineering and the poultry slaughterhouse for technical support. REFERENCES Bedanova, I., E. Voslarova, P. Chloupek, V. Pistekova, P. Suchy, J. Blahova, R. Dobsikova, and V. Vecerek Stress in broilers resulting from shackling. University of Veterinary and Pharmaceutical Sciences, Brno 612:42. Coenen, A. M. L., and P. L. C. van den Broek Correlation dimension of the chicken EEG during sleeping, waking and drowsiness. Nederlandse Vereniging voor Slaap-Waak Onderzoek. Heeze Nederland 16: Cook, C. J., S. A. Maasland, C. E. Devine, K. V. Gilbert, and D. K. Blackmore Changes in the release of amino acid neurotransmitters in the brains of calves and sheep after head-only electrical stunning and throat cutting. Res. Vet. Sci. 60: Debut, M., C. Berri, C. Arnould, D. Guemené, V. Santé-Lhoutellier, N. Sellier, E. Baéza, N. Jehl, Y. Jégo, and C. Beaumont Behavioural and physiological responses of three chicken breeds to pre-slaughter shackling and acute heat stress. Br. Poult. Sci. 46: EFSA (European Food Safety Authority) AHAW/ Welfare aspects of stunning and killing methods. Scientific Report of the Scientific Panel for Animal Health and Welfare on a request from the Commission related to welfare aspects of animal stunning and killing methods (Question N EFSA-Q ). Report AHAW/ EFSA Scientific Opinion on the electrical requirements for water bath stunning equipment applicable for poultry 1 EFSA Panel on Animal Health and Welfare (AHAW) 2, 3 EFSA Journal 10:2757. European Council European Council Regulation No. 1099/2009 on the protection of animals at the time of killing. Official Journal of the European Union L303/1 30. Gerritzen, M.A., H.G.M. Reimert, V.A. Hindle, M.T.W. Verhoeven, and W. B. Veerkamp Multistage carbon dioxide gas stunning of broilers. Poult. Sci. 92: Gregory, N. G., and S. B. Wotton Effect of stunning on spontaneous physical activity and evoked activity in the brain. Br. Poult. Sci. 31: Hillebrand, S. J. W The sensory quality of turkey meat. Thesis, University of Utrecht, Utrecht, the Netherlands. Hillebrand, S. J. W., E. Lambooy, and C. H. Veerkamp The effects of alternative electrical and mechanical stunning methods on hemorrhaging and meat quality of broiler breast and thigh muscles. Poult. Sci. 75: Hindle, V. A., E. Lambooij, H. G. M. Reimert, L. D. Workel, and M. A. Gerritzen Animal welfare concerns during the use of the water bath for stunning broilers, hens, and ducks. Poult. Sci. 89: Johnson, N. L., and S. Kotz Distributions in Statistics. Discrete Distributions. Houghton Mifflin, Boston, MA. Kettlewell, P. J., and R. N. Hallworth Electrical stunning of chickens. J. Agric. Eng. Res. 47: Knowles, T. G., and D. M. Broom The handling and transport of broilers and spent hens. Appl. Anim. Behav. Sci. 28: Lambooij, E., C. Pieterse, S. J. W. Hillebrand, and G. B. Dijksterhuis The effects of captive bolt and electrical stunning and restraining methods on broiler meat quality. Poult. Sci. 78: Lambooij, E., H. R. M. Reimert, and V. A. Hindle Evaluation of head-only electrical stunning for practical application: Assessment of neural and meat quality parameters. Poult. Sci. 89: Lambooij, E., H. R. M. Reimert, L. D. Workel, and V. A. Hindle Head-cloaca controlled current stunning: assessment of brain and heart activity and meat quality. Br. Poult. Sci. 53: Lines, J. A., A. B. M. Raj, S. B. Wotton, M. O Callaghan, and T. G. Knowles. 2011a. Head-only electrical stunning of poultry using a water bath: A feasibility study. Br. Poult. Sci. 52: Lines, J. A., S. B. Wotton, R. Barker, J. Spence, L. Wilkins, and T. G. Knowles. 2011b. Broiler carcass quality using head-only electrical stunning in a water bath. Br. Poult. Sci. 52: Monin, G., and A. Ouali Muscle differentiation and meat quality. Pages in Developments in Meat Science. R. A. Lawrie, ed. Elsevier Appl. Sci., London, UK. Nicol, C. J., and G. B. Scott Pre-slaughter handling and transport of broiler chickens. Appl. Anim. Behav. Sci. 28: Nijdam, E., P. Arens, E. Lambooij, E. Decuypere, and J. A. Stegeman Factors influencing bruises and mortality of broilers during catching, transport, and lairage. Poult. Sci. 83: Nijdam, E., E. Delezie, E. Lambooij, M. J. A. Nabuurs, E. Decuypere, and J. A. Stegeman Comparison of bruises and mortality, stress parameters and meat quality in manually and mechanically caught broilers. Poult. Sci. 84: Raj, A. B. M Effect of stunning method, carcase chilling temperature and filleting time on the texture of turkey breast meat. Br. Poult. Sci. 35: Shields, S., and M. Raj A critical review of electrical waterbath stun systems for poultry slaughter and recent developments in alternative technologies. J. Appl. Anim. Welf. Sci. 13: Sparrey, J. M., and P. J. Kettlewell Shackling of poultry: Is it a welfare problem? World s Poult. Sci. J. 50: van den Broek, P. L. C Monitoring anaesthetic depth. Modification, evaluation and application of the correlation dimension. PhD Thesis. Nijmegen University Press, the Netherlands. van den Broek, P. L. C., J. van Egmond, C. M. van Rijn, F. Takens, A. M. L. Coenen, and L. H. D. J. Booij Feasibility of realtime calculation of correlation integral derived statistics applied to EEG time series. Physica D 203: Veerkamp, C., C. Pieterse, and I. Hulsegge Broilermeat quality. Poultry Int. February:40 46.