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Meat Science 121 (2016) 148–155

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Blood parameters and electroencephalographic responses of goats to slaughter without stunning A.B. Sabow a,g, Y.M. Goh b,c,⁎, I. Zulkifli a,b, A.Q. Sazili a,b,e, U. Kaka f,h, M.Z.A. Ab Kadi d, M. Ebrahimi c, K. Nakyinsige i, K.D. Adeyemi a,j a

Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d Department of Electrical and Electronic Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia e Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia f Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia g Department of Animal Resource, University of Salahaddin, Erbil, Kurdistan Region, Iraq h Department of Veterinary Surgery and Obstetrics, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tandojam, Sindh, Pakistan i Department of Food Science and Nutrition, Islamic University in Uganda, P.O.Box 2555, Mbale, Uganda j Department of Animal Production, University of Ilorin, Ilorin, Nigeria b c

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 11 May 2016 Accepted 16 May 2016 Available online 18 May 2016 Keywords: Blood biochemistry Electroencephalogram Goats Minimal anaesthesia Slaughter

a b s t r a c t The study compared changes in blood biochemistry, hormonal and electroencephalographic indices associated with possible noxious stimuli following neck cut slaughter in conscious, non-anaesthetized versus minimallyanaesthetized goats. Ten male Boer crossbreed goats were assigned into two groups and subjected to either slaughter conscious without stunning (SWS) or slaughter following minimal anaesthesia (SMA). Hormonal responses and changes in electroencephalographic (EEG) parameters were not influenced by slaughter method. The SWS goats had higher glucose and lactate than did SMA goats. It can be concluded that the noxious stimulus from the neck cut is present in both conscious and minimally anaesthetized goats. The application of slaughter without stunning causes changes in the EEG activities that are consistent with the presence of post slaughter noxious sensory input associated with tissue damage and would be expected to be experienced as pain in goats. © 2016 Published by Elsevier Ltd.

1. Introduction Goat meat production has been increasing over the last few years from 4.90 million tons in 2008 to 4.99 million tons in 2011 (FAO, 2010). This has been driven by its perceived healthiness compared with other red meat (Argüello, Castro, Capote, & Solomon, 2005; Webb, Casey, & Simela, 2005). The gradual expansion of goat meat industries is as a result of increased consumer demand (Dhanda, Taylor, Murray, Pegg, & Shand, 2003). This development has stimulated researches to improve the availability and quality of goat meat (Adeyemi, Sabow, Shittu, Karim, & Sazili, 2015; Sabow et al., 2016). The last step of the meat production chain (slaughter) has received much debate as regards to its humaneness (Sabow et al., 2015; Salwani et al., 2015). Fear and pain are important elements of stress that have profound effects on meat quality (Heinz & FAO, 2001). ⁎ Corresponding author at: Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. E-mail addresses: [email protected], [email protected] (Y.M. Goh).

http://dx.doi.org/10.1016/j.meatsci.2016.05.009 0309-1740/© 2016 Published by Elsevier Ltd.

Slaughter procedures are usually regulated by legislation, codes of practice and species-specific recommendations but the suitability of commonly used methods depends as well on availability of facilities, consumer demands and economic considerations (Anil, 2012). Religion is one of the most influential factors determining choice and subsequent selection or purchase of foods. Due to religious requirements, it has become evident that the market for meat from slaughtered animals without stunning is an important proportion of the global production and supply (van der Spiegel et al., 2012). The global value of trade in halal meat is huge with Muslim countries alone consuming meat estimated to be worth USD 57.2 billion in 2008 (Farouk et al., 2014). The value of halal red meat and co-products imported into countries and regions with sizeable population of Muslims (Indonesia, Maghreb, Malaysia, Middle East, Saudi Arabia and United Arab Emirates) in 2011 was USD 28.5 billion (Farouk, 2013). According to the British Veterinary Association (2013), the number of slaughtered sheep and goats by ventral-neck incision without prior stunning in the UK increased 70% between 2003 and 2011. A recent survey on animal welfare carried out in abattoirs across the UK published by the Food Standards Agency (2015) indicated that the number of goats and sheep slaughtered

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without pre-stunning is 15.2% in 2013, which is higher than the 10% of the population that were slaughtered without stunning in 2011. Slaughtering of animals without stunning prior to ventral neck incision and exsanguination is recognized as the appropriate method for slaughtering animals intended for meat consumption by a number of religious faiths, such as Islam and Judaism (Sabow et al., 2015). This practice is allowed in many countries and recognized by the World Organization for Animal Health (OIE) as a slaughter method used by certain faiths (Farouk et al., 2014; Velarde et al., 2014). Meanwhile the European Union (EU) legislation grants exceptions from stunning for religious groups, but it is extremely controversial with regards to animal welfare. Both sides agree that animal welfare is important in the production of meat. Areas of concern include possible pain and distress during and immediately following the neck cut and the time to onset of insensibility (Grandin, 2010; Gregory, 2005). Pain caused by neck cutting has been the subject of much debate. It has been suggested that the use of an exquisitely sharp knife produces minimal behavioral reactions in animals and as a result, the neck cut is not perceived as painful by the animal (Regenstein, 2012; Rosen, 2004). This in itself is the main perception by advocates of religious slaughter of many faiths, as it is known that both fear and pain affects eventual meat quality (Grandin & Regenstein, 1994). However, there are little neurophysiological and physiological evidences to support this suggestion. Until recently it was not clear whether slaughter of conscious animals by neck cutting causes pain or distress (Gibson et al., 2009; Zulkifli et al., 2014). This was due to the complexities of measuring pain in animals and limitations on the interpretation of behavioral and physiological responses to slaughter by neck cut alone. The phylogenetic similarities in structure and function of the central nervous systems (CNS) between humans and other mammals leave little doubt that farm animals can indeed experience pain (Barnett, 1997). Moreover, there is little doubt that these animals are conscious pre-slaughter, during slaughter, and for a period post-slaughter, without stunning (Gibson, Dadios, & Gregory, 2015). Therefore, it is possible that animals experience nociception/pain during slaughter prior to the onset of insensibility (Gibson et al., 2009; Zulkifli et al., 2014). The electroencephalogram (EEG) is the recording of electrical activity from electrodes placed in various positions on the scalp (Kaka et al., 2015). Electroencephalogram spectrum changes have been used as an indicator of the experience of pain in red deer (Johnson, Wilson, Woodbury, & Caulkett, 2005), sheep (Otto & Gerich, 2001), pigs (Haga & Ranheim, 2005), cattle (Zulkifli et al., 2014; Gibson, Johnson, Stafford, Mitchinson, & Mellor, 2007) and horses (Murrell et al., 2003). In addition to that, plasma levels of adrenaline and noradrenaline are commonly used to measure stress or distress before and during slaughter in animals (Bórnez, Linares, & Vergara, 2010; Ndlovu, Chimonyo, Okoh, & Muchenje, 2008). It is well known that animals exposed to stressful situations respond through activation of both the sympathetic and hypothalamic-pituitary-adrenal axes. The activation of the first axis determines the release of adrenaline (epinephrine) and noradrenaline (norepinephrine) into the blood stream as a preparatory event in which the animal perceives a problem and prepares its immediate reactions (Micera, Dimatteo, Grimaldi, Marsico, & Zarrilli, 2010). Slaughter procedures need to maintain product quality as well as protect animal welfare. Although there has been some research in this area, most information originates from work in conventional slaughter methods with limited comparison to religious slaughter. This was due to the limited access to religious slaughter without stunning in most developed countries because of legal and welfare reasons. Animals subjected to minimal anaesthesia have been accepted as a humane model to study noxious stimuli associated with the neck cut slaughter in cattle and sheep (Johnson, Mellor, Hemsworth, & Fisher, 2015), but has not been reported in goats, particularly in countries where pre-slaughter stunning is mandatory. The minimal anaesthesia model is a proven model to evaluate the presence of noxious stimuli, especially when used in conjunction with electroencephalography (Gibson et al., 2009;

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Mellor, Gibson, & Johnson, 2009). Under the minimal anaesthesia model, animals have been shown to be able to demonstrate EEG responses from the cerebral cortex, as well as normal physiological cardiovascular responses to nociceptive stimulation, that are consistent with that of fully awake animals (Johnson, Murrell, Gibson, & Mellor, 2007). However, as emotion and conscious awareness also contributes significantly to the perception of pain in animals, there could be differences in response between awake and minimally anaesthetized animals, particularly on parameters that are under the influence of the autonomic nervous system. As a consequence, there is a need to study and compare electroencephalographic response and changes in blood parameters following the neck cut in minimally anaesthetized versus fully awake animals. Apart from offering scientific insights into stress and noxious stimuli, responses which have not been conducted until now in goats, this study strives to evaluate the utility of the minimally anaesthetized model in animal slaughter research. In particular its use in studying the controversial topic associated with religious slaughter. Therefore, the objective of the present study was to compare the changes in blood biochemistry, hormonal and electroencephalographic changes associated with possible noxious stimuli following neck cut slaughter in conscious, non-anaesthetized halal-slaughtered goats (SWS) versus minimally-anaesthetized goats (SMA). 2. Materials and methods 2.1. Ethical note This study was conducted following the animal ethics guidelines of the Research Policy of Universiti Putra Malaysia. 2.2. Animals and slaughtering procedure A total of 10 male Boer crossbreed goats weighing 23.15 ± 1.42 kg were obtained from the same farm and of the same age (approximately 7 months old). The goats were allotted into two groups consisting of 5 animals each and subjected to either conscious slaughter without stunning (SWS) or slaughter following minimal anaesthesia (SMA). Slaughtering was carried out at the Department of Animal Science Research Abattoir, Faculty of Agriculture, Universiti Putra Malaysia. In the first group (SWS), the goats were slaughtered according to halal slaughtering procedure as outlined in the MS1500: 2009 (Department of Standards Malaysia, 2009). The process involved severing the carotid arteries, jugular veins, trachea and esophagus. The neck cut position was performed at the level of first cervical vertebra (C1) based on the requirements of OIE (2007). Another group of goats (SMA) were allocated to a similar slaughter designed to mimic the action of the neck cut under minimal anaesthesia. 2.3. Anaesthesia The protocol for minimal anaesthesia induction was performed as per the methods of Johnson et al. (2009) and Kaka et al. (2015). Under the minimal anaesthesia model, animals are maintained at end-tidal halothane (ETHal) of 0.85–0.95%, while under conventional general anaesthesia animals are maintained at ETHal higher than 1.5%. Anaesthesia was induced using 5 mg/kg propofol administered through rapid injection into cephalic vein and maintained with halothane in 100% oxygen. Vaporizer was adjusted to maintain ETHal between 0.9% and 1.0%. A blood pressure cuff of 40–60% circumference of the ante-brachium was used to measure blood pressure noninvasively. Mean blood pressure was maintained above 60 mm Hg throughout the anaesthetic period. The temperature was monitored by using an esophageal thermistor probe and was maintained between 37 and 38 °C by a heating pad and a warm blanket. All parameters were monitored using Datex-Ohmeda monitor (GE healthcare, Helsinki, Finland).

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2.4. Blood sampling and analyses For easier blood sampling, hair on the goats' necks were shaved a day before the slaughter. For animals in the SMA group (subjected to minimal anaesthesia), baseline blood samples (5 ml) (T1) were collected through jugular venipuncture using 21-gauge needles while observing all aseptic precautions. During the experiment, further blood samples were taken immediately after the point of anaesthesia induction (from jugular vein) (T2), and immediately post neck cut (from blood flow) (T3). However, from the SWS group, only two blood samples were collected – baseline (T1) and immediately post neck cut (T3). The blood samples were collected, processed and subjected to biochemical and hormonal analyses. The blood samples for biochemical analysis were collected into a Vacutainer (BD Franklin Lakes, NJ, USA) serum tubes, temporarily stored at 0–4 °C on crushed ice and transported to the Clinical Pathology and Haematology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia within 2 h after collection. Lactate dehydrogenase (LDH), creatine kinase (CK), glucose, lactate, creatinine, urea, total protein and calcium were determined using an automatic analyzer (Hitachi 902, Tokyo, Japan). The blood samples for catecholamines (adrenaline and noradrenaline) hormonal analysis were collected into Vacutainer K3 ethylene diaminetetraacetic acid (EDTA) tubes and kept slant in crushed ice for 1 h, followed by centrifugation at 3000 g, 4 °C for 10 min. The recovered plasma fraction was divided into 1.5 ml aliquots and stored at − 80 °C until analysis. The quantitative analysis of adrenaline (epinephrine) content in blood was carried out using Adrenaline Plasma Enzyme-Linked Immuno Sorbent Assay (ELISA) High Sensitive kit # BA E-4100 (LDN®GmbH & Co, KG, Nordhorn, Germany) while noradrenalin (norepinephrine) quantification was carried out using Noradrenaline Plasma ELISA High Sensitive kit # BA E-4200 (LDN®GmbH & Co, KG, Nordhorn, Germany) following the manufacturer's instructions.

2.5. Electroencephalography (EEG) and electrocardiographic (ECG) recording For each animal, prior to neck cut, baseline electroencephalogram activities and immediately post neck cut were recorded telemetrically using a personal computer installed with Chart 5.5.5 recording software and connected to Powerlab 4/20 data recording system (Powerlab data acquisition system, AD Instruments Ltd. Sydney, Australia). Three stainless steel sterile disposable acupuncture needles (Wuxi Jiajian Medical Instrument Co., Ltd. Wuxi, Jiangsu, China) were placed subcutaneously with the inverting (reference) electrode over the zygomatic process of left frontal bone, the non-inverting (active) electrode over the left mastoid process, and the ground electrode caudal to the occipital process (Kongara, Chambers, & Johnson, 2010). The electroencephalogram was recorded at a sampling rate of 1 kHz and raw EEG was re-sampled with low pass filter of 200 Hz into delta frequency (0.1 to 4 Hz), theta frequency (4.1 to 8 Hz), alpha frequency (8.1 to 12 Hz) and beta frequency (12.1 to 20 Hz) as reported earlier by Zulkifli et al. (2014). Electroencephalogram data were collected for 10–15 min after slaughter (once the animal is dead based on ECG). Electrocardiogram was recorded continuously in the standard lead II configuration, with the negative electrode on the right forelimb and positive electrode on the left hind limb (Kaka et al., 2015). Analysis of the EEG was carried out off line after the completion of experiments. The median frequency (F50) and total EEG power (Ptot) were calculated for consecutive non-overlapping 5-second epochs. The root mean square (RMS) for each of the waveform was calculated. EEG data from 90-second blocks before to 90-second blocks after the neck cut were taken for statistical analysis based on the time to loss of pupillary reflex. The EEG results were expressed as the percentage of baseline value (Kongara et al., 2010). Heart rate in beats per minute was derived manually from the ECG data. Pre-slaughter mean heart rate for each animal was calculated for a period of 10 s.

Post slaughter mean heart rate was calculated at intervals of 15 s (after 15, after 30 up to after 300) until 5 min. 2.6. Data analysis The experiment was a complete randomized design (CRD). Data were analyzed using the general linear model (GLM) procedure of the Statistical Analysis System package (SAS) Version 9.2 software (Statistical Analysis System, SAS Institute Inc., Cary, NC, USA). Data were subjected to one-way analysis of variance (ANOVA) using a model that included slaughter method as a possible source of variation with sampling time as a repeated measure. Duncan multiple range test was used to test the significance of variance between the means of the studied parameters. Statistical significance is considered as p b 0.05 throughout the paper. 3. Results and discussion 3.1. Blood biochemical parameters The blood parameters provide invaluable insights into the physiological changes associated with stress and noxious stimuli following slaughter (Nowak, Mueffling, & Hartung, 2007). Blood biochemistry changes are as influenced by slaughter method are depicted in Table 1. Generally, blood parameters measured at T1 were not significantly different (p b 0.05) between SWS and SMA. Within the SMA goats, the mean changes in the level of all blood parameters at T2 were not significantly different from that of T1. For both slaughter groups, biochemical blood parameters except glucose and lactate concentration attained at T3 were not significantly different. Blood glucose levels in SWS goats

Table 1 Changes in blood biochemical parameters in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia and blood sampling time (mean ± SE, n = 5). Parameter

Sampling time Treatment groups SWS

Glucose (mmol/l)

T1 T2 T3 Lactate T1 T2 (mmol/l) T3 Total protein T1 (g/l) T2 T3 Calcium T1 (mmol/l) T2 T3 Urea T1 (μmol/l) T2 T3 Creatinine T1 (μmol/l) T2 T3 Creatine kinase T1 T2 (U/l) T3 Lactate dehydrogenase (U/l) T1 T2 T3

y

3.74 ± 0.11 NA 6.02a,x ± 0.37 1.73y ± 0.08 NA 2.71a,x ± 0.20 65.88 ± 2.31 NA 67.38 ± 3.17 2.12 ± 0.09 NA 2.09 ± 0.02 6.62 ± 0.55 NA 8.24 ± 0.71 68.00 ± 2.93 NA 76.80 ± 2.57 172.20y ± 11.26 NA 264.60x ± 63.09 534.20y ± 17.96 NA 607.80x ± 34.21

SMA 3.77y ± 0.23 4.32xy ± 0.54 5.22b,x ± 0.36 1.77 ± 0.06 1.63 ± 0.04 1.85b ± 0.04 66.80 ± 1.23 60.68 ± 1.41 61.57 ± 3.04 2.16 ± 0.07 2.09 ± 0.15 2.05 ± 0.07 6.93 ± 0.21 6.62 ± 0.32 6.45 ± 0.47 67.83 ± 1.96 63.50 ± 2.70 68.50 ± 3.54 174.50y ± 10.26 175.00y ± 16.12 250.00x ± 12.61 543.00y ± 21.32 505.00y ± 16.13 583.00x ± 27.45

SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. T1 = Prior to slaughter; T2 = At the point of anaesthesia induction; T3 = Post slaughter. NA = not available. a,b Means within the same row with different superscripts are significantly different at p b 0.05. x,y Means within the same column with different superscripts are significantly different at p b 0.05.

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were significantly (p b 0.05) higher than that of the SMA. In farm animals, increased sympatho-adrenal activity stimulated by physical and psychological stress leads to hyperglycemia due to the increased breakdown of glycogen in the liver (Adenkola & Ayo, 2010; Anil et al., 2006). However, this result did not indicate a higher stress response in SWS group, since the glucose values were within normal range. The difference could be attributed to the fact that the SWS animals were conscious during the slaughter process (Opara, Udevi, & Okoli, 2010; Plumb, 1999). de México, Izcalli, and de México (2011) mentioned that during slaughter, animals are exposed to new experiences that inevitably causes fear which in turn cause evident physical and emotional stress in the animal. In the present study, blood lactate concentration was significantly higher (p N 0.05) in the SWS than the SMA. Similarly, Grandin (1998) indicated that slaughter without prior stunning in cattle resulted in increased blood lactate as a result of rapid anaerobic glycolysis. However, Nakyinsige et al. (2014) observed that non-stunned rabbits had significantly (p b 0.05) lower levels of glucose and lactate at post slaughter compared to those gas stunned. Irrespective of the slaughter methods, the mean changes in glucose and lactate concentration were significantly higher at T3 when compared to T1. Glucose concentration increases through increased rate of glycogenolysis and gluconeogenesis associated with the increase in catecholamine and glucocorticoids (Nakyinsige et al., 2013). The changes in blood values related to energy metabolism in goats during slaughter suggest the need to cope with slaughter stress. Similar to the present observation, Ekiz and Yalcintan (2013) also reported that the concentration of glucose was higher at exsanguination than their respective basal values, a clear effect that could be associated with post slaughter stresses (Bórnez et al., 2010; Grandin, 2013; Pollard et al., 2002). Elevated levels of creatine kinase (CK) and lactate dehydrogenase (LDH) in serum are indicative of stress, muscle damage and muscle fatigue (Nakyinsige et al., 2013; Nakyinsige et al., 2014). In the present study, the level of CK and LDH activity at T3 were significantly higher (p b 0.05) when compared to T1, although there was no difference (p N 0.05) between the SWS and SMA. This indicates that slaughtering with or without anaesthesia did not affect the activities of liver enzymes. These findings are in agreement with those of Ekiz and Yalcintan (2013) in goats and Ekiz, Ergul Ekiz, Kocak, Yalcintan, and Yilmaz (2012) in lambs who found significant differences in LDH and CK activities between before and after neck cut. Also, it was noted that all blood biochemical values of the pre and the post slaughter values differed from each other for SWS and SMA goats (Table 1). Therefore, the data merely pointed to the fact that the slaughter of conscious animals led to a much higher change in the blood biochemistry. Therefore, the act of slaughter, be it under anaesthesia or not, inflicts a significant degree of change in most biochemical parameters. It is generally accepted that biochemical blood changes are produced in response to physical stress situations like that induced by slaughter procedures and this could involve a reduction of welfare. 3.2. Catecholamine levels It is well known that both the sympathetic and the hypothalamic-pituitary-adrenal axis react when animals are exposed to stressful situations (Schaefer, Chen, Tsao, Nouri, & Rabkin, 2001). The activation of the first axis responds to short-term stress through the release of catecholamines (adrenaline and noradrenaline) into the blood stream (Anil, 2012; O’Neill, Webb, Frylinck, & Strydom, 2006). Results of analyses of changes in the amount of catecholamines (adrenaline and noradrenaline) are presented in Table 2. The changes in blood plasma adrenaline and noradrenaline concentration of animals, whether slaughtered with or without anaesthesia did not differ significantly at T3. Regardless of the treatment methods, there was a significant increase (p b 0.05) in the amount of both adrenaline and noradrenaline levels at T3 compared to T1. A similar trend was also reported by

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Table 2 Changes in catecholamine levels in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia (mean ± SE, n = 5). Parameter

Sampling time

Treatment groups SWS

Adrenaline (ng/ml) Noradrenaline (ng/ml)

T1 T2 T3 T1 T2 T3

SMA y

97.03 ± 7.46 NA 267.92x ± 8.22 113.49y ± 6.15 NA 395.68x ± 6.50

98.51y ± 7.42 108.51y ± 9.14 231.19x ± 10.43 112.40y ± 8.20 127.40y ± 6.56 365.61x ± 6.42

SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. T1 = Prior to slaughter; T2 = At the point of anaesthesia induction; T3 = Post slaughter. NA = Not available. x,y Means within the same column with different superscripts are significantly different at p b 0.05.

Nakyinsige et al. (2014) in rabbits, in which values of adrenaline and noradrenaline were significantly different following the slaughter procedure. According to Gregory (1998), when an animal bleeds out, there is a fall in pressure, which activates the sympathetic adrenal medullary nervous system resulting in the release of adrenaline and noradrenaline from the sympathetic endings. Shaw and Tume (1992) explained that catecholamines under normal physiological conditions are released from the adrenal medulla to regulate certain body functions such as maintenance of blood pressure. However, under stressful situations, high concentrations of catecholamines are discharged into the bloodstream in preparation for the possibility of rapid energy expenditure (Shaw & Tume, 1992). With regard to the SMA goats, the mean changes in plasma concentration of adrenaline and noradrenaline were not significantly different at T1 and T2. In this study, the values of adrenaline and noradrenaline were 2.5 and 3.5 times higher in the T3 than T1, respectively, for SWS goats, while the values were 2.3 and 3.3 times higher in SMA goats. Hormonal data in goats at the point of slaughter indicated a stressful response from animals in order to cope with the situation even though it may not necessarily translate into pain sensation (Nakyinsige et al., 2014). 3.3. Brain activity The examination of electroencephalographic changes provides important evidence to the extent of nociception and stress experienced by the animals at the point of slaughter (Gibson, Johnson, Murrell, Chambers et al., 2009). Electroencephalogram activities for the two slaughter methods are presented in Figs. 1, 2 and 3. The percentage change of post-slaughter RMS for alpha, beta, delta and theta wave activities did not differ between SWS and SMA. The RMS for each of the waveform was significantly (p b 0.05) increased after neck cut compared to that of baseline in goats of both treatment groups (Fig. 1). These results indicate that the act of slaughtering in conscious, nonanaesthetized and anaesthetized animals is associated with noxious stimulation that would be expected to be painful as demonstrated by Murrell and Johnson (2006). Murrell and Johnson (2006) reported that minimal anaesthesia model is a technical process subjected to each animal to maintain unconsciousness but still able to demonstrate EEG responses to noxious stimulation. Johnson, Gibson, Stafford, and Mellor (2012) summarized the outcomes of a number of studies in which the minimal anaesthesia model was used to determine the effect of slaughter in calves without stunning and found that the act of slaughter by ventral-neck incision without stunning is associated with pain in the period between the slaughter and subsequent loss of consciousness. According to Gregory, Fielding, von Wenzlawowicz, and von Holleben (2010), the durations between completing the cut and the onset of unconsciousness plays a vital role and the increase the risk of pain and distress in livestock. The current observation is in line with those of Zulkifli

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Fig. 1. Electroencephalogram root mean square (RMS) for each of the (a) alpha, (b) beta, (c) delta and (d) theta waveform expressed as percent of baseline values in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia. SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. Values are means ±1 standard error bar. The horizontal line indicates baseline value. *Indicate significant differences (p b 0.05) between baseline and post neck cut in the same slaughter group.

et al. (2014) who reported higher levels of RMS for alpha and beta wave activities at post slaughter in non-stunned cattle as compared to prior to slaughter. Changes in F50 and Ptot have been previously associated with arousal and nociception in horses, dogs, pigs and calves as reviewed by Gibson et al. (2007) and Murrell and Johnson (2006). In the present study, the percentage change of F50 was significantly (p b 0.05) increased from that of baseline after neck cut in both treatment groups. However, the differences in F50 of goats between the treatment groups were non-significant post slaughter (Fig. 2). Similar to the current observation, Zulkifli et al. (2014) reported that the EEG median frequency for non-stunned cattle increased significantly at post slaughter time. Gibson, Johnson, Murell, Chambers, Stafford et al. (2009) and Gibson, Johnson, Murrell, Hulls, Mitchinson, et al. (2009) reported that the F50 values in calves during the 30 s following ventral-neck incision changed significantly (p b 0.05) compared to the pre-treatment values and the influence of ventral-neck incision was associated with significant noxious sensory input that most probably was perceived as pain in conscious animals. There were no significant differences in Ptot values amongst treatments following slaughter. However, there was a significant increase

in Ptot as percent change of baseline after the neck cut in SWS and SMA goats (Fig. 3). This suggests that the increase observed after neck cut may be partially caused by movement of the animal which is associated with tissue damage during the neck cutting. Post-treatment changes in the total power of the EEG values may not directly related to nociception as in F50.·This explanation is supported by Gibson, Johnson, Murrell, Chambers et al. (2009) and Gibson, Johnson, Murrell, Hulls, et al. (2009) who stated that changes in Ptot following slaughter should be interpreted with caution as the variations in the Ptot have been previously associated with noxious stimulation (Gibson et al., 2007) and with reductions in cortical function during stunning (Gibson et al., 2009).The findings in current experiment is in agreement with those of Gibson, Johnson, Murrell, Chambers et al. (2009) and Gibson, Johnson, Murrell, Hulls, et al. (2009), who reported increase in Ptot following slaughter by ventral-neck incision without prior stunning. It is heartening to note that the minimal anaesthesia model in goats provided EEG results that mirrored those of conscious animals. Huozha, Rastogi, Korde, and Madan (2011) studied electroencephalographic changes during experimental pain induction in conscious and locally anaesthetized goats using a rubber tourniquet applied at the base of the tail and fastened. The authors found that

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Table 3 Changes in heart rate (beats per minute) and time pupillary reflex loss in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia (mean ± SE, n = 5).

Fig. 2. Electroencephalogram median frequency (F50) expressed as percent of baseline values in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia. SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. Values are means ±1 standard error bar. The horizontal line indicates baseline value. *Indicate significant differences (p b 0.05) between baseline and post neck cut in the same slaughter group.

even under local anaesthesia, pain persisted since the amplitudes and frequencies were in a similar order. 3.4. Heart activity Table 3 shows the results of heart activity obtained from goats subjected to conscious slaughter without stunning (SWS) and slaughter following minimal anaesthesia (SMA) followed by bleeding. The heart rate (heart auto-rhythmicity) was detected and recorded using the low impedance electrodes connected to an electrocardiograph. The heart rate can be used as an indicator for autonomic physiological responses.

Time after neck cut (seconds)

Treatment groups SWS

SMA

−101 02 15 30 45 60 75 90 105 120 135 150 165 180 240 300 Time pupillary reflex loss (min)

86.66w ± 11.19 85.667w ± 7.56 111.00a,w ± 5.02 182.33a,x ± 14.55 184.00a,x ± 19.16 186.67a,x ± 20.69 171.17a,x ± 6.73 165.33a,x ± 10.46 164.67x ± 11.19 166.33x ± 7.56 170.33x ± 5.02 162.33x ± 3.81 147.17y ± 6.48 148.00y ± 10.38 130.50z ± 7.91 115.25w ± 9.33 2.14 ± 0.19

88.33w ± 1.50 87.33w ± 6.40 78.33b,w ± 3.20 93.00b,wx ± 5.60 103.00bx ± 7.55 107.00bx ± 4.84 126.67b,y ± 9.67 131.00b,y ± 5.70 149.33y ± 6.23 154.67z ± 7.77 156.83z ± 7.91 157.33z ± 9.94 154.33z ± 8.42 149.33z ± 5.81 133.33y ± 6.40 109.25x ± 5.33 2.44 ± 0.13

SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. a,b Means within the same row with different superscripts are significantly different at p b 0.05. w-z Means within the same column with different superscripts are significantly different at p b 0.05. 1 Pre-treatment value. 2 Neck cut.

Generally, heart rates increased immediately after the neck cut in both groups which were associated with noxious input. Slaughter methods influenced (p b 0.05) heart rate. Between 10 and 90 s, SWS goats had higher rate of heartbeat compared with SMA goats. These variations may be attributed to reflex increases in vagal activity or mediated indirectly by alterations in respiratory function or other responses. Increase in heart rate is mainly attributed to the increase in sympathetic stimulation (Grøndahl-Nielsen, Simonsen, Damkjer Lund, & Hesselholt, 1999; Peers, Mellor, Wintour, & Dodic, 2002). This is consistent with the findings of Lambooij, van der Werf, Reimert, and Hindle (2012) who reported increased heart rate following neck cut with or without preslaughter stunning in veal calves. Nevertheless, the observed increase in heart activity after neck cut contrasts the findings of Gibson, Johnson, Murrell, Chambers et al. (2009) and Gibson, Johnson, Murrell, Hulls, et al. (2009) which showed decrease in heart rate after ventral-neck incision. The changes in heart rate observed in this study occurred within a few seconds of the slaughter and continued for only a short period (about 60 s) and declining gradually thereafter. Similar findings have been reported in other studies (Gibson et al., 2007; Johnson et al., 2009). As presented in Table 3, no difference (p N 0.05) was observed for the time lapse from the point of slaughter to the loss of pupillary reflex between SWS and SMA goats. The absence of a pupillary reflex in livestock that have been slaughtered is generally used as an indicator of insensibility or unconsciousness (Zulkifli et al., 2014). 4. Conclusions

Fig. 3. Electroencephalogram total power (Ptot) expressed as percent of baseline values in goat subjected to slaughter fully conscious and slaughter following minimal anaesthesia. SWS = Animal slaughtered conscious without stunning; SMA = Animal slaughtered following minimal anaesthesia. Values are means ±1 standard error bar. The horizontal line indicates baseline value. *Indicate significant differences (p b 0.05) between baseline and post neck cut in the same slaughter group.

The result of the present findings showed that the noxious stimuli from the neck cut are evident in both conscious and minimally anaesthetized goats. This confirmed that both groups of animals experienced nociception. Responses for most blood parameters, except glucose and lactate were not affected by slaughter methods. In addition, slaughter method had no effect on electroencephalographic responses in goats. This study affirm that while the minimally anaesthetized animal is a good model to study animals subjected to neck cut slaughter, blood changes associated with stress and nociception should be interpreted

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