Effect of electrical water bath stunning on physical reflexes of broilers: evaluation of stunning efficacy under field conditions

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1 Effect of electrical water bath stunning on physical reflexes of broilers: evaluation of stunning efficacy under field conditions M. Girasole,,1 R. Marrone, A. Anastasio, Antonio Chianese, R. Mercogliano, and M. L. Cortesi Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Via Federico Delpino, 1, Napoli, Italy; and ASL Napoli 2 Nord, Department of Prevention and Veterinary Service of Food Hygiene ABSTRACT The effects of different amounts and frequencies mals experiencing an abolition of corneal reflex at 20 s of stunning sine wave alternating current were investigated under field conditions. Seven hundred and fifty broilers were stunned in an electrical water bath with an average root mean square (RMS) current of 150, 200, and 250 ma and frequencies of 200, 400, 600, 800, and 1,200 Hz. The occurrence of corneal reflex, spontaneous eye blinking, and a positive response to a painful stimulus were monitored and recorded immediately after the stunning and at 20 s post-stun. Statistical analysis showed that the electrical stunning frequency (P = ), the stunning RMS current post-stun. At a current of 150 ma, the probability of a successful stun was over 90% at 200 Hz, approximately 40% at 400 Hz, and below 5% for frequencies greater than 600 Hz. So, stunning at frequencies greater than 600 Hz cannot be recommended when a RMS current of 150 ma is applied. The maximum probability of a successful stun was obtained for a current level of 200 ma at 400 Hz and for a current level of 250 ma at 400 and 600 Hz, whereas the stunning treatments at 1,200 Hz provided the lowest probability of a successful stun. (P < ) and the interaction between stunning Assessment of spontaneous eye blinking and responses frequency and stunning current (P < ) had a significant effect on the occurrence of ani- to comb pinching confirmed the indications coming from the analysis of corneal reflex. Key words: water bath stunning, physical reflex, broiler, welfare, alternating current 2016 Poultry Science 95: INTRODUCTION Broiler welfare, given the high number of animals slaughtered for human consumption, is a subject of concern. To protect poultry welfare, electrical water bath stunning is one of the methods most commonly used in commercial slaughterhouses. After shackling, the birds are immersed, generally up to the base of their wings (Schütt-Abraham et al., 1983; Gregory and Wotton, 1991; EFSA, 2004; Raj et al., 2006a,b; Prinz et al., 2010a,b), in an electrified water bath, inducing unconsciousness due to the passage of electric current through the central nervous system. Conventionally, a metal strip at the base of the water bath forms the positive electrode and the shackles form the negative electrode, so that the electric current flows through the bird from head to legs. Depending on the dimensions of the water bath, several birds are simultaneously treated. The presence of several birds at the same time creates parallel pathways of resistance and each bird acts as a sepa- C 2016 Poultry Science Association Inc. Received July 28, Accepted January 6, Corresponding author: mariagrazia.girasole@unina.it rate branch of the circuit. The conductive resistance of individual birds is highly variable and may depend on factors including body size, muscle and fat content, sex (Rawles et al., 1995; Prinz et al., 2010a,b), skull bone structure and thickness (Woolley et al., 1986a,b), and plumage condition (wet or dry) (Gregory and Wotton, 1992). These variations in resistance may strongly affect the stun efficacy when a constant voltage stunner is used, as birds may receive less or more current than planned (Berry et al., 2002). Different electrical setups can be chosen to achieve an effective, rapid, and long lasting stunning. The amount of current, the electrical frequency, the electrical waveform, and the stunning time are the most common parameters that can be optimized to improve the stunning effectiveness. In general, sine waves appear to be more effective than other waveforms and may even require lower amplitude currents (Raj et al., 2006a,b; Prinz et al., 2010a,b). For this reason, an alternating current (AC) is commonly used, even though some slaughter plants apply a pulsed direct current. The frequencies used in modern poultry electrical stunning systems range from 50 to 1500 or even 2000 Hertz. In general, higher frequencies are less effective at stunning, require greater current amplitudes, and may 1205

2 1206 GIRASOLE ET AL. produce a shorter stunning effect (Wilkins et al., 1998; Mouchonière et al., 1999). So, extra care is needed in checking that birds remain insensible throughout the bleeding period. On the other hand, low frequencies cause high muscle contractions and consequent rupture of skin and/or flesh small blood vessels, with consequent downgrading of meat quality (Wilkins et al., 1999). The use of high frequencies can reduce the occurrence of these adverse effects. Raj et al. (2001), for example, found a significantly lower occurrence of broken bones and breast meat hemorrhages in broilers stunned with a sine wave AC of 1,500 Hz compared to 50 Hz. A significant reduction of ventricular fibrillation and cardiac arrest was also observed with higher frequencies (Bilgili, 1999). Current European legislation establishes in the Annex I of the Regulation (EC) N.1099/2009 the minimum current levels at which the animals must be exposed in the electrified water bath to guarantee an effective stunning. In particular, it defines three different levels in correspondence of three different frequency ranges and suggests a minimum exposure time of four seconds for each bird. The unconsciousness induced by electrical stunning must last for at least 45 s (EFSA, 2004, 2012) to allow enough time for death to occur by bleeding out. Measuring the lack of consciousness (i.e., ability to perceive pain and fear) is complex and needs to be performed under a scientifically approved methodology. Use of the electroencephalogram (EEG) is considered the most objective method to assess the animal brain activity. The occurrence of an epileptiform activity characterized by high amplitude, low frequency polispikes, followed by a suppressed, isoelectric EEG with a profound reduction of electrical brainpower to less than 10% of the pre-stun level is thought to be the best available evidence of loss of brain stem function in chickens, whereas the same reduction in the 13 to 30 Hz frequency band of EEG signal has been used to indicate loss of sensitivity (Schütt-Abraham et al., 1983; Raj and O Callaghan, 2004a,b; Raj et al., 2006a,b). Several studies suggest that a reduction in correlation dimension value to 60% of the baseline value can be considered as an indicator of unconsciousness, when a correlation dimension analysis is applied to EEG traces of chickens during gas stunning (van den Broek et al., 2005; McKeegan et al., 2007, Lambooij et al., 2010). Due to different types of constraints such as line speed, difficulties in the observation, high number of birds slaughtered, time, and cost, use of EEG to ascertain poultry unconsciousness is not applicable in daily slaughterhouse practice. Other indicators are therefore to be used in field conditions. Changes in poultry behavior (e.g., spontaneous blinking and swallowing, wing flapping, head shaking), physical signs (e.g., onset of seizures, cessation of breathing, fixed eye), and physiological reflexes (e.g., response to external stimulus such as corneal reflex, response to pain stimulus such as comb or toe pinching) represent the common parameters used by the personnel to assess the efficiency of an electrical water bath stunning treatment. Among physical reflexes, the eye reflexes seem most reliable to evaluate unconsciousness (Prinz et al., 2010a,b; EFSA, 2013). The corneal reflex is elicited when the cornea is touched or stimulated, which results in blinking of the eyelids (blinking reflex) or movement of the nictitating membrane across the eye (Erasmus et al., 2010). The absence of corneal reflexes in a considerable number of broiler chickens is considered an effective indicator of deep unconsciousness or approaching brain death (Schütt-Abraham and Wormulh, 1988; Gregory, 1989; Prinz et al., 2010a,b). Moreover, an increase of corneal reflexes in many birds over time is considered as a sign of progressive recovery and may suggest the return of some brain function (von Wenzlawowicz and von Holleben. 2001). A positive response itself, however, does not necessarily indicate sensitivity in broilers and ability to perceive pain, as positive eye reflexes might occur on the basis of residual brain stem activity (Gregory, 1989). For this reason, it can be expected that a limited number of animals might still show a positive response for a short period post-stun. Prinz et al. (2010a,b) suggested that under practical field conditions a maximum of 30% of corneal reflexes can be used as an indicator to identify an acceptable stunning. Studies based on the comparison between EEG results and the occurrence of corneal reflex confirmed this assumption (Prinz et al., 2010a,b). Since the perception of pain is lost at the onset of insensibility, tests evaluating the pain reflex, such as toe pinching or comb pinching, are also practical means of assessing insensibility in field conditions (Erasmus et al., 2010). It was observed, however, that the comb pinch response may be absent also in inadequately stunned birds when an electrical water bath stunning is used (Richards and Sykes, 1964;Schütt-Abraham et al., 1983). A regular spontaneous eye blinking is also considered a reliable behavioral indicator of an ineffective stunning, especially if its frequency increases over time. In this case, it can be an expression of regaining consciousness when associated with other indicators suggestive of a rapid recovery of birds (Prinz et al., 2010a). The present work aims to investigate under field conditions the effect of different combinations of current amounts and sine wave AC frequencies on the occurrence of some behavioral and physical reflexes in broilers. The aim of the work was also to obtain indications, in relation to previous studies, about the probability of inducing an acceptable water bath stunning as a function of the electrical treatment applied. MATERIALS AND METHODS The experimental tests were carried out in a commercial poultry slaughter and processing plant, using an electrical stunner with a water bath able of

3 ELECTRICAL WATER BATH STUNNING OF BROILERS 1207 containing up to fourteen broilers. The birds were grouped on the basis of their live body weight in order to obtain groups of subjects as homogeneous as possible. Broilers were then individually hung upside down by the legs on the moving shackles of the slaughter line and stunned by immersion in the electrified water bath. The height of the water was adjusted to guarantee for all the birds immersion of their head and part of their neck. The shackle line speed was set at 3400 birds per hour, whereas the distance between two consecutive shackles was 20 cm. These values ensured for each bird a stunning time of about 15 s. No salt was added to the water. A digital ammeter on the stunner control panel allowed us to monitor and record the actual total current amount passing through the water bath. An additional stand-alone ammeter (HT9021 AC/DC TRMS Clamp meter 1000A, HT Italia srl, Faenza, Italy) was used to confirm the accuracy of current measurement provided by the stunner ammeter. A commercial constant voltage stunner (Water Stunner BA4, LINCO Food Systems A/S, Aarhus, Denmark) was used to apply a sine wave AC at frequencies of 200, 400, 600, 800, 1000, and 1,200 Hz. Voltage was adjusted on the stunner to obtain an estimated average root mean square (RMS) current for each broiler in the water bath of 150, 200, and 250 ma. At a frequency of 200 Hz, however, only a RMS current of 150 ma was tested, whereas at 1,200 Hz, only currents of 200 and 250 ma were tested. The average current passing through the individual birds was calculated dividing the total current amount passing through the water bath by the number of birds simultaneously immersed in the water. The effects of each combination of stunning electrical parameters were evaluated on 50 (consecutive on the slaughter line) commercial broiler chickens (genetic line Ross 708). So, a total of 750 birds were used. All the birds were six to seven weeks old and weighed on average 3.73 ± 0.26 kg. Some physical reflexes and behaviors of the birds were assessed for indications about stunning effectiveness. In particular, the presence of corneal reflex (blinking response elicited by touching the cornea), the occurrence of spontaneous eye blinking, and a positive response to comb pinching were used as indicators of an ineffective or poor stun. The corneal reflex was tested by touching the cornea with a feather, whereas the comb pinching was carried out by means of pointed tweezers. Use of more operators/observers allowed assessment of comb pinch and corneal reflex responses at two stages of the slaughter line: immediately after the stunning and before the neck was cut (approximately 20 s post-stun). The spontaneous blinking response was only assessed at 20 s post-stun in order to distinguish this response from mere muscular fibrillations that often occur after the stunning and that last a few seconds. For statistical analysis, data were analyzed using a least square regression on the number of broilers showing a suppression of corneal reflex after the stunning treatment. The factor effects were calculated by the maximum likelihood ratio test, with the stunning frequency, stunning current, and the interaction of frequency current as fixed factors. Stunning treatments that produced more than 65 to 70% of birds with a suppression of corneal reflex were considered as effective, whereas the remaining stunning treatments were considered as not effective. A nominal logistic regression on these data allowed to calculate the probability of an effective stunning as a function of the stunning treatment. All statistical procedures were performed by JMP 11 (JMP, 2014). The experiment was approved by the animal welfare regulation committee of the University of Naples Federico II. The institutional animal care and use guidelines have been followed. RESULTS AND DISCUSSION The number of birds showing a suppression of corneal reflex and the number of birds with a positive response to the comb pinching at each tested combination of frequency and current is presented in Table 1. In the model shown below and obtained by a least square regression on the number of animal with a suppression of corneal reflex following stunning treatments, P is the estimated percentage of broilers with a suppressed corneal reflex that was modeled as a function of the stunning frequency and stunning current; the standard error in the parameter estimates is shown within brackets. P =41.26 (6.04) (0.0048) F requency(hz) (0.033) Current(mA). The model fitted the experimental data with a root mean qquare error of 5% and a coefficient of multiple determination (R-Square value) of 0.8. In Figure 1, the predicted percentage of birds with negative corneal reflex is plotted against stunning frequency for different levels of RMS current. The graph clearly shows that an increase in the electrical stunning frequency at a given amplitude of current produces a decreasing number of birds with a suppression of corneal reflex. On the contrary, at each of the tested frequencies, an increase of RMS current amount results in an increasing number of broilers showing an abolition of corneal reflex. These results are in line with those reported by Raj et al. (2006a) and Prinz et al. (2010a). Statistical analysis showed that the electrical stunning frequency had a significant effect (P = ) on the occurrence of animals with suppressed corneal reflex. The effect of the stunning RMS current and the effect of the interaction of stunning frequency and stunning current were also significant (P < ).

4 1208 GIRASOLE ET AL. Table 1. Effect of different stunning treatments on the number of birds with suppressed corneal reflex, positive response to comb pinching, and spontaneous eye blinking. Responses were assessed at 20 s post-stun. Average body Estimated RMS No. of birds No. of birds No. of birds Frequency No. of weight (±SD) current/bird with suppressed positive to with spontaneous (Hz) birds (Kg) (ma) corneal reflex comb pinching eye blinking (±0.12) (±0.21) (±0.26) (±0.21) (±0.32) (±0.28) (±0.30) (±0.25) (±0.17) (±0.19) , (±0.21) (±0.23) (±0.24) , (±0.18) (±0.15) Figure 1. Estimated percentage of broilers with negative corneal reflex (%) as a function of electrical frequency (Hz) and stunning current level (ma). As described above, the absence of corneal reflex is considered a reliable indicator of deep unconsciousness in poultry (Gregory, 1989; Prinz et al., 2010a; Erasmus et al., 2010; EFSA, 2013), but a positive response does not necessarily mean that the animal is able to perceive pain. Prinz et al. (2010a,b) suggested that under practical field conditions a maximum amount of about 30% of birds with a positive corneal reflex can be accepted in an effective stunning treatment. If this criterion is assumed, the probability of inducing an effective stunning with a given current is limited to a narrow range of electrical frequencies (Figure 1). RMS current levels of 150 ma, for example, produce an acceptable stunning (i.e., with more than 65 to70% of birds experiencing an abolition of corneal reflex) only if frequencies up to about 200 Hz are applied. Similarly, effective stunning can be achieved with a minimum current of 200 ma and 250 ma only if frequencies up to 600 and 1,000 Hz, respectively, are delivered. Electrical frequencies above 1,200 Hz would have probably required a current amplitude greater than 250 ma to produce Figure 2. Probability of an effective stunning as a function of electrical frequency (Hz) and current level (ma). the suppression of corneal reflex in more than 65 to 70% of the broilers treated. On the basis of these considerations, it was possible to transfer the results of the experimental tests into a binomial scale. Combinations of stunning electrical parameters able to induce a suppression of the corneal reflex in more than 65 to 70% of birds were considered as effective, whereas the remaining combinations of electrical frequencies and RMS current levels were considered as not effective. A nominal logistic regression of these data allowed calculation of the probability to obtain an effective stun as function of the applied electrical frequency for different RMS current levels. Current level and frequency were treated as continuous variables. The results of this statistical model are presented in Figure 2. The model confirmed that, at a given current level, the likelihood of an effective stun tended to decrease when the electrical frequency was increased. At a current of 150 ma, for example, the probability of a successful stun was over 90% at 200 Hz, approximately 40%

5 ELECTRICAL WATER BATH STUNNING OF BROILERS 1209 Table 2. Effects of the stunning treatment on the number of birds with suppressed corneal reflex and a positive response to comb pinching assessed immediately after the stunning (T0) and before neck cut (T1: approximately 20 s post stun). Estimated RMS No. of birds with No. of birds with No. of birds No. of birds Frequency No. of current/bird suppressed corneal suppressed corneal positive to comb positive to comb (Hz) birds (ma) reflex at T0 reflex at T1 pinch at T0 pinch at T , , at 400 Hz, and below 5% for frequencies greater than 600 Hz. Therefore, stunning at frequencies greater than 600 Hz cannot be recommended when a RMS current of 150 ma is applied. The high incidence of birds with a positive response to comb pinch at these stunning frequencies and the high amount of spontaneous eye blinking confirmed this judgment. An increase in the amount of current delivered increased the probability of achieving an effective stun at the higher frequencies. The maximum probability of a successful stun was obtained for a current level of 200 ma at 400 Hz and for a current level of 250 ma at 400 and 600 Hz. These combinations of electrical parameters also produced a lower number of birds with a positive response to comb pinching. At a frequency of 400 Hz, a RMS current of 200 ma or 250 ma produced a similar effect on the probability of obtaining a successful stun. In the same way, at frequencies of 1,000 Hz and 1,200 Hz, the probability of a successful stun did not increase with increasing amount of current from 150 to 200 ma and from 200 to 250 ma, respectively. Similar results were reported by Hindle et al. (2010) who found, on the basis of a statistical model, that at high frequencies, as the current amplitude increased, there was a much lower rate of increase in the chance of a successful stun compared to lower frequencies. The stunning treatments at a frequency of 1,200 Hz provided the lowest probability of a successful stun as it was below 10% for all the tested current levels. For some combinations of frequency and current level, the number of broilers with a positive response to corneal reflex increased over time (Table 2). In particular, it happened in the groups of birds stunned with a RMS current of 150 ma at frequencies of 600 Hz, 800 Hz, and 1,000 Hz and in the birds stunned with a RMS current of 200 ma at 1,200 Hz, and it can be considered a sign of progressive recovery indicating a regain of consciousness and sensibility (Raj et al. 2006a; Prinz et al., 2010a). Therefore, when low current amounts are delivered, high frequencies (600 Hz or more) are not able to induce a sustained period of unconsciousness. So, they cannot be recommended even with a fast bleeding. The early corneal reflex test (assessed immediately after the stunning) showed a significant effect (P < ) for stunning frequency and for the interaction between stunning current and stunning frequency, but close to the level of significance for stunning current (P = 0.08). The stunning frequency was not significant for the number of birds with a positive response to comb pinching assessed both immediately after the stunning (P = 0.4) and 20 s post-stun (P = 0.5), whereas the stunning current level showed an effect on the number of birds with a positive response to this painful stimulus in both periods (P = and P < , respectively). Although, as mentioned above, a negative response to comb pinching is not always considered a reliable indicator of unconsciousness in electrical stunned poultry (Richards and Sykes, 1964; Schütt-Abraham et al., 1983), assessment of this reflex allowed confirmation of indications coming from the evaluation of the corneal reflex. In particular, the highest number of animals with a positive response to comb pinch was found for the stunning treatments that produced the lowest probability of an effective stun. Similarly, stunning treatments capable of suppressing the corneal reflex in a high number of animals resulted in a low number of birds with a positive response to comb pinching. Occurrence of a natural blinking response following the stunning can be considered a generic sign of consciousness and a behavioral indicator of sensibility in poultry(raj et al., 2006a; Prinz et al., 2010a; Erasmus et al., 2010). Statistical analysis showed that spontaneous eye blinking assessed at 20 s post-stun had a significant effect for stunning frequency (P < ), stunning current (P = 0.009), and the interaction between stunning current and frequency (P < ). The occurrence of spontaneous blinking response was

6 1210 GIRASOLE ET AL. high for those groups that showed a higher percentage of birds with positive corneal reflex and comb pinch responses (Table 1). REFERENCES Berry, P. S., I. R. Meeks, D. B. Tinker, and A. R. Frost Testing the performance of electrical stunning equipment for poultry. Vet. Rec. 151: Bilgili, S. F Recent advances in electrical stunning. Poult. Sci. 78: EFSA Scientific Opinion on the electrical requirements for water bath stunning equipment applicable for poultry. EFSA Panel on Animal Health and Welfare (AHAW). EFSA Journal. 10:2757. EFSA Scientific Opinion on monitoring procedures at slaughterhouses for poultry. EFSA Panel on Animal Health and Welfare (AHAW). EFSA Journal. 11:3521. 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/ Erasmus, M. A., P. V. Turner, and T. M. Widowski Measures of insensibility used to determine effective stunning and killing of poultry. J. Appl. Poult. Res. 19: Gregory, N. G Stunning and slaughter. Pages in Processing of Poultry. G. C. Mead, ed. Elsevier Applied Science, London, UK. Gregory, N. G., and S. B. Wotton Effect of depth of immersion in the waterbath on the effectiveness of electrical stunning in chickens. Res. Vet. Sci. 51: Gregory, N. G., and S. B. Wotton Effect of wetting a chicken s feathers on the effectiveness of electrical stunning. Res. Vet. Sci. 53: 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: Lambooij, E., H. G. 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: McKeegan,D.E.F.,J.A.McIntyre,T.G.M.Demmers,J.C. Lowe, C. M. Wathes, P. L. C. van den Broek, A. M. L. Coenen, and M. J. Gentle Physiological and behavioural responses of broilers to controlled atmosphere stunning: Implications for welfare. Anim. Welf. 16: Mouchonière, M., G. Le Pottier, and X. Fernandez The effect of current frequency during water bath stunning on the physical recovery and rate and extent of bleed out in turkeys. Poult. Sci. 77: Prinz, S., G. Van Oijen, F. Ehinger, A. Coenen, and W. Bessei. 2010a. Electroencephalograms and physical reflexes of broilers after electrical water bath stunning using an alternating current. Poult. Sci. 89: Prinz, S., G. Van Oijen, F. Ehinger, W. Bessei, and A. Coenen. 2010b. Effects of waterbath stunning on the electroencephalograms and physical reflexes of broilers using a pulsed direct current. Poult. Sci. 89: Raj, A. B. M., and M. O Callaghan. 2004a. Effects of amount and frequency of head-only stunning currents on the electroencephalogram and somatosensory evoked potentials in broilers. Anim. Welf. 13: Raj, A. B. M., and M. O Callaghan. 2004b. Effects of electrical waterbath stunning current frequencies on the spontaneous electroencephalogram and somatosensory evoked potentials in hens. Br. Poult. Sci. 45: Raj, A. B. M., M. O Callaghan, and T. G. Knowles. 2006a. The effects of amount and frequency of alternating current used in water bath stunning and of slaughter methods on electroencephalograms in broilers. Anim. Welf. 15:7 18. Raj, A. B. M., M. O Callaghan, and S. I. Hughes. 2006b. The effects of amount and frequency of pulsed direct current used in water bath stunning and of slaughter methods on spontaneous electroencephalograms in broilers. Anim. Welf. 15: Raj, A. B., L. J. Wilkins, M. O Callaghan, and A. J. Phillips Effect of electrical stun/kill method, interval between killing and neck cutting and blood vessels cut on blood loss and meat quality in broilers. Br. Poult. Sci. 42: Rawles, D., J. Marcy, and M. Hulet Constant-current stunning ofmarket-weightbroilers.j.appl.poult.res.4: Richards, S. A., and A. H. Sykes Observations on the electrical stunning and slaughter of poultry. Vet. Rec. 76: Schütt-Abraham, I., H. J. Wormuth, and J. Fessel Electrical stunning of poultry in view of animal welfare and meat production. Pages in Stunning of Animals for Slaughter. G. Eikelenboom, ed. Martinus Nijhoff, The Hague, the Netherlands. Schütt-Abraham, I., and H. J. Wormuth, Cardiac arrest stunning in poultry. Pages Proc. 34th Int. Cong. of Meat Sci. &Tech., Brisbane, Australia. 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 real time calculation of correlation integral derived statistics applied to EEG time series. Physica D. 203: von Wenzlawowicz, M., and K. von Holleben Assessment of stunning effectiveness according to present scientific knowledge on electrical stunning of poultry in a waterbath. Arch. Geflugelkd. 65: Wilkins, L. J., N. G. Gregory, S. B. Wotton, and I. D. Parkman Effectiveness of electrical stunning applied using a variety of waveform-frequency combinations and consequences for carcass quality in broiler chickens. Br. Poult. Sci. 39: Wilkins, L. J., S. B. Wotton, I. D. Parkman, P. J. Kettlewell, and P. Griffiths Constant current stunning effects on bird welfare and carcass quality. J. Appl. Poult. Res. 8: Woolley, S. C., F. J. W. Borthwick, and M. J. Gentle. 1986a. Tissue resistivities and current pathways and their importance in preslaughter stunning of chickens. Br. Poult. Sci. 27: Woolley,S.C.,F.J.W.Borthwick,andM.J.Gentle.1986b.Flow routes of electric currents in domestic hens during pre-slaughter stunning. Br. Poult. Sci. 27: