Electroencephalographic spectrum power of sheep's brain after stunning

Abstract

The electroencephalographic spectrum power (ESP) shows the frequency band components of the brain electrical activity. The electroencephalogram (EEG) has a characteristic ESP during different behavioural states such as waking or sleeping and also during pathological states as epilepsy or brain death. Methods of slaughtering the farm animals should prevent needless suffering; all the animals are expected to be unconscious and insensible to pain before being hoisted, but there are no systematic studies of the animal's brain activity after stunning. This study evaluated the brain activity of sheep after stunning by means of percussion or electrical shock. Brain activity after electrical shock showed an epileptiform EEG, with decreasing delta power and increasing theta and gamma band power. After percussion stunning, the EEG showed a slightly decrease of high frequency power.

Keywords:

• electroencephalogram
• spectrum power
• sheep
• stunning

1. Introduction

The electroencephalogram (EEG) is a technique to record brain electrical activity (Delamonica 1987; Kay 1998; Pastor et al. 2006; Vilatuña & Yanchaliquin 2007; Cwynar & Zawadzki 2010). Neuronal groups fire in different frequency rates as following: delta (0.5–4.0 Hz), theta (4.0–8.0 Hz), alpha (8.1–12.0), beta (12.1–30.0 Hz) and gamma (30.1–80.0 Hz) (Riquelme 1995; Subasi & Ercelebi 2005; Bergamasco et al. 2006; Angel 2009). Frequency band power analysis of the EEG can be performed by means of the Fourier transformed formula (Riquelme 1995; Vilatuña & Yanchaliquin 2007; Angel 2009). Neurological disorders such as epilepsy have being described in animal models, sheep and rat. These EEG recordings show high-amplitude and high-frequency waves (Annegers 1997; Guerrero et al. 1997; Opdam et al. 2002).

Slaughtering of farm animals should induce unconsciousness and avoid pain (Caraves & Gallo 2007). However, undesirable stress, anxiety and pain could affect the animals during slaughtering (Grandin 1996; Jongman et al. 2000; Velarde et al. 2003; Gregory 2004, 2008). Several stunning methods have being described for slaughtering ruminants and monogastric animals, for instance, non-penetrating captive bolt (Lopez & Vanaclocha 2004), penetrating captive bolt (Gregory & Shaw 2000), electrical shock (Velarde et al. 2000) and carbon dioxide exposure (Lopez & Vanaclocha 2004; Bercerril-Herrera et al. 2009).

The captive bolt pistol method causes brain injury and an irreversible cerebral commotion dependent of the pistol power and the target on the skull (Grandin 1999; Lambooij et al. 2003; Caraves & Gallo 2007; Ríos & Acosta 2008). The electrical shock stunning method works by applying current through the brain or through the whole body, and it induces an epileptiform brain status, loss of consciousness and absence of pain (Lopez & Vanaclocha 2004), but it might also bring the animal into cardiac arrest (Lambooy 1982; Solis 2005). Electrical stunning efficacy is dependent on the electrodes placement and electricity characteristics (Grandin 1999; Digre et al. 2010). For electrical stunning is recommended a voltage between 70–250 volts, and a minimum of 1.2 amp from 2 to 8 sec (Grandin 1994; Velarde et al. 2000, 2003; Brancacci et al. 2006; Kun 2008). Some of the parameters used to evaluate the effectiveness of stunning are un-coordinated movements, clonic convulsions and dilated pupils (Blackmore & Newhook 1983; Grandin 2010). Recent studies are focused on the cortical brain activity and the quantitative analysis of the EEG as a measurement of pain perception during slaughtering (Hermsworth & Mellor 2009). This study attempts to evaluate the electroencephalographic status of sheep after stunning by the non-penetrating captive bolt pistol method and the electrical shock.

2. Materials and methods

2.1. Animals

Sixty 10-week-old Dorper-mixed lambs were used during this study. The experimental animals were divided into 2 groups of 30 animals each. Animals from group one were stunned by electrical shock and those from group two were stunned by penetrating captive bolt.

2.2. Stunning methods

2.2.1. Electrical shock

The electro narcosis was carried out by placing bipolar electrodes on the temporal area on each side of the skull at a point between the eyes and the ears, previously moistened with saline solution (Velarde et al. 2000). An electrical alternating current of 250 volts and 0.9 amp was administrated during 6 sec (HSA 2000; Velarde et al. 2003). The stunning was performed in a V-shaped box where the animals were standing and immobilised.

2.2.3. Penetrating captive bolt

A penetrating captive pistol bolt was used (Cash Special 22 calibre). The heads of the animals were immobilised and the pistol shot addressed on the midline of the skull, behind the ridge between the horns, pointing to the base of the tongue (HSA 2006).

2.3. EEG recording

The EEG was recorded in a physiograph (Power Lab® ADInstruments), at 1 KHz sample rate, 0.5 Hz high-pass digital filter. Two needle electrodes were placed subcutaneously over the skull. The recording electrodes were placed bilaterally over the frontotemporal area, and a reference electrode was placed on the parieto occipital boundary. The recording began 30 sec before the stunning and continued until the time of hoisting the animal (approximately 30 sec after stunning).

2.4. EEG analysis

The EEG recording from each animal was divided in two segments: baseline recording (prior to the stunning) and post-stunning recording. Spectrum power was obtained from the EEG data by means of the fast Fourier transform formula. The spectrum data were divided into six frequency bands: 1–4 Hz (delta), 4–8 Hz (theta), 8–13 Hz (alpha), 13–30 Hz (beta), 30–80 Hz (gamma), 80–500 Hz (higher frequencies). Data from each frequency band were normalised and reported as percentage values (Beyssen et al. 2004).

2.5. Statistical analysis

Multifactorial variance analysis ANOVA was carried out for statistical analysis (Statgraphics Centurion XV, Statistical Graphics Corp., USA). A multiple minimum range (LSD test by Fischer, P<0.05) was performed.

3. Results

The baseline EEG analysis from all the experimental animals showed an electroencephalographic spectrum power (ESP) coincident with those reported in the previous studies (Ong et al. 1997; Jongman et al. 2000) which showed high absolute power for delta, (beta 1–2), alpha (1–2) and theta (1–2) waves during handling and shearing.

There was no significant difference for the baseline ESP of the animals from group one and two (P<0.05). Frequency band power average was as following: delta 60%±1.5, beta 5.9%±1.4, alpha 3.4%±1.4 gamma 16.15%±1.4, theta 11%±1.4, and higher frequencies band 7%±1.4 (Figure 1).

Figure 1. Baseline ESP. This figure shows the frequency bands power of the EEG recorded from the experimental animals of group one and two before the stunning.

There was a significant decrease in delta power 23.2%±2.4, and a significant increase in theta power 43.22%±1.75 (P<0.05) after electrical shock stunning (Figure 2).

Figure 2. Electrical shock stunning. This graph shows a comparison of the ESP from experimental animals before and after the electrical shock stunning.

The spectrum power after penetrating captive bolt stunning was as follows: delta 60.4%±2.7, alpha 5.4%±2.1, beta 4.7%±2.7, theta 14.2%±2.4, gamma 17.1%±2.1, and higher frequencies band 34.5%±2.3. There was no significant difference for the spectrum power before and after penetrating captive bolt stunning (Figure 3).

Figure 3. Penetrating captive bolt stunning. Note in this figure a comparison of the ESP from the experimental animals before and after the penetrating captive bolt stunning.

Delta power was significantly higher for the animals stunned by a penetrating captive bolt than those animals from the electrical shock group [68.06%±2.71, 23.21%±2.54, respectively (P<0.05)]. Gamma power was significantly lower for the animals stunned by a penetrating captive bolt than those animals from the electrical shock group [8.65%±2.61, 19.93%±2.43, respectively (P<0.05); Figure 4].

Figure 4. Electrical shock vs. penetrating captive bolt stunning. This figure shows a comparison of the ESP from experimental animals after the electrical shock and the penetrating captive bolt stunning.

The amplitude of the EEG during baseline recordings was about 200 µV, as well as after the penetrating captive bolt stunning. Amplitude of the EEG after the electrical shock stunning was up to 600 µV. The EEG during basal recording and after the penetrating captive bolt stunning shows low-frequency waves prevalence, but very high-frequency waves after electrical shock stunning (Figure 5).

Figure 5. EEG recording. See here an example of the EEG traces from the experimental animals before and after the stunning by means of the two methods used in this study. Penetrating captive bolt stunning (trace on black), electrical shock (trace ongrey).

4. Discussion

Perception of pain in animals is associated with its description and the physiological responses in human beings (Stubsjøen et al. 2009); pain intensity is associated in turn with the level of molecular biomarkers of stress (Sattari et al. 2009). Additionally, EEG recordings can also be associated with this type of responses to painful stimulation. In humans, a perception of pain by immersion of the hands in cold water was related to an increasing of delta and beta waves in the EEG (Chen et al. 1989). On the other hand, inducing stress and pain to sheep by electrical stimulation caused a decreasing of the delta and alpha waves in the EEG (Ong et al. 1997); these results contrasted to the response to placing the hands into water at different temperatures (Egsgaard et al. 2009). Non-invasive measurements of pain have been evaluated in sheep, such as infrared thermography, ocular temperature and heart rate, where responses to painful stimuli are associated with pain perception in humans (Stubsjøen et al. 2009). In a study it was observed that pain was related to delta, alpha, and beta waves increasing, and, consequently, a reduction in pain by carbamazepine treatment was related to a decrease in those waves (Music et al. 2008). Even though there are divergent findings when attempting to show electrophysiological markers for pain perception, there are also coincidences between them. Delta power increasing in some brain areas is usually described as coincident with pain perception (Chen et al. 1989; Ong et al. 1997; Chen 2001). As shown in Figure 2, electrical shock stunning showed a decrease in delta waves, allowing us to infer that there was a reduction in pain perception.

Epilepsy could be a localised or a generalised condition (Subasi & Ercelebi 2005; Bennet et al. 2006; Nelson et al. 2006; Sakkalis et al. 2010). Pain is not perceived during generalised epilepsy crisis but could be present during a localised epilepsy crisis (Opdam et al. 2002; Loddenkemper & Kotagal 2005; Sun et al. 2008; Charlesworth et al. 2009; Machado & Solarte 2010). Based on these studies, we might speculate that there was no pain perception for the animals after the electrical shock stunning because an epileptic-like EEG state was induced.

By analysing the power spectrum of the EEG recordings, we evaluated the efficiency of the captive bolt method and the electric shock in sheep slaughtering. The EEG recordings after the electric shock resemble an epileptic crisis where there is no pain perception. On the other hand, the results for captive bolt stunning suggest no change in cortical activity and no alteration in the EEG spectrum power; in contrast there is a tendency to increase delta waves, which can be associated to pain perception. Although the penetrating captive bolt stunning leads to loss of mobility, our findings do not show evidence of absence of pain.

Finally, it is quite important to highlight that this study demonstrated that there are extreme differences between the stunning methods, thus, scientists must look for the best stunning method in terms of animal well-being.

Acknowledgement

The authors would like to thank Mr Erick Rodrigo Martínez for assistance in data recording.