Validation of behavioural indicators used to assess unconsciousness in sheep M.T.W. Verhoeven a,b, ⁎, M.A. Gerritzen a , M. Kluivers-Poodt a , L.J. Hellebrekers c , B. Kemp b a Wageningen University and Research Centre, Livestock Research, P.O. Box 338, 6700 AH Wageningen, The Netherlands b Adaptation Physiology Group, Department of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands c Wageningen University and Research Centre, Central Veterinary Institute, P.O. Box 65, 8200AB Lelystad, The Netherlands abstract article info Article history: Received 3 December 2014 Received in revised form 7 June 2015 Accepted 14 June 2015 Available online xxxx Keywords: EEG Non-stunned slaughter Propofol Reflexes Sheep (Un)consciousness The validity of behavioural indicators to assess unconsciousness under different slaughter conditions is under (inter)national debate. The aim of this study was to validate eyelid-, withdrawal-, threat reflex and rhythmic breathing as indicators to assess unconsciousness in sheep. Sheep were monitored during repeated propofol an- aesthesia (n = 12) and during non-stunned slaughter (n = 22). Changes in the EEG and behavioural indices of consciousness/unconsciousness were assessed and compared in sheep. Threat reflex and rhythmic breathing cor- related with EEG activity during propofol anaesthesia whilst absence of non-rhythmic breathing or threat reflex indicated unconsciousness. None of the behavioural indicators correlated with EEG activity during non-stunned slaughter. Absence of regular breathing and eyelid reflex was observed 00:27 ± 00:12 min and 00:59 ± 00:17 min (mean ± SD) respectively after animals were considered unconscious, indicating that absence of reg- ular breathing and eyelid reflex are distinctly conservative indicators of unconsciousness during non-stunned slaughter in sheep. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction European legislation describes laws, rules and procedures concerning slaughter of livestock. One of these laws, namely article 5 of the Council Regulation (EC) No (1099/2009, 2009), prescribes the need to determine unconsciousness before an animal is released from restraint. An animal is considered unconscious when there is “a state of unawareness (loss of consciousness) in which there is temporary or permanent disruption of brain function. As a consequence of the disruption, the animal is unable to respond to normal stimuli, including pain” (EFSA, 2006). Unconsciousness at slaughter is generally determined by assessing absence of reflexes originating from the brain stem (e.g., eye reflexes) or spinal cord (e.g., withdrawal reflex) in combination with in- dicators such as loss of posture, vocalisations and rhythmic breathing (Erasmus et al., 2010; Verhoeven et al., 2015). There is substantial (in- ter)national debate on what indicators most adequately assess uncon- sciousness at slaughter and which merit further investigation (EFSA, 2013). The use of recorded brain activity (as presented in an electroen- cephalogram or EEG) is considered to be the most objective method for assessing unconsciousness and is generally accepted as the current ‘gold standard’ (EFSA, 2012; Erasmus et al., 2010). The EEG reflects the sum of underlying electrical activity of populations of neurones supported by glia cells (Murrell and Johnson, 2006). The onset of unconsciousness can be determined by visual assessment of changes in patterns, ampli- tude and frequency of EEG activity. Generally, an increase in low fre- quency activity is accompanied by an increase in amplitude. When neurons depolarise at the same time or frequency, they fire in a synchronised fashion creating slow high amplitude waves as seen in un- conscious states suggesting a depression of the reticular formation (Lopes da Silva, 1982). Consciousness on the other hand is characterised by high frequency, low amplitude waves (Seth et al., 2005). The onset of unconsciousness can also be determined by calculating more standardised EEG spectral variables using a Fast Fourier Transformation (FFT). The output of a FFT represents the frequency composition of the signal, or alternatively formulated, how much power is presented in the different frequency bands. Spectral variables include: total spectral power (PTot), power in the different frequency bands and spectral edge frequency (F95). PTot is calculated as the total area under the power spectrum curve, which can also be broken down into the power per frequency band. The F95 is the frequency below which 95% of the total power of the EEG is located (Murrell and Johnson, 2006). Changes in these spectral variables are known to be related to anaes- thetic depth and clinical signs of unconsciousness (Martin-Cancho et al., 2003; Martín-Cancho et al., 2006; Schwender et al., 1996). In order to study absence of indicators that reflect unconsciousness, anaes- thetic agents can be used to induce different stages of unconsciousness and allow for recovery, where stunning and exsanguination will lead to rapid and irrecoverable death of the animal. The use of anaesthetic Research in Veterinary Science 101 (2015) 144–153 ⁎ Corresponding author at: Wageningen University and Research Centre, Livestock Research, P.O. Box 338, 6700 AH Wageningen, The Netherlands. Tel.: +31 317 480 162. E-mail address: merel.verhoeven@wur.nl (M.T.W. Verhoeven). http://dx.doi.org/10.1016/j.rvsc.2015.06.007 0034-5288/© 2015 Elsevier Ltd. All rights reserved. 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